FORSCHUNGSBERICHT

SCIENCE REPORT2017/2018 ST. ANNA KINDERKREBSFORSCHUNG ST. ANNA CHILDREN’S CANCER RESEARCH INSTITUTE

TUMOR BIOLOGY Understanding tumor heterogeneity and relapse in neuroblastoma patients

to allow adequate treatment options. MOLECULAR MICROBIOLOGY

LCH BIOLOGY Clinically important Targeted resistant mutations in Philadelphia inhibition chromosome-positive leukemias.

of the MAPK pathway leads to a STUDIES & STATISTICS rapid and sustained clinical response Busulfan and in severe multisystem LCH. melphalan is considered standard high-dose chemotherapy for high-risk neuroblastoma.

GENETICS OF LEUKEMIAS A novel genetic MOLECULAR BIOLOGY OF SOLID TUMORS subtype of leukemia. Understanding mechanisms EPIGENOME-BASED PRECISION MEDICINE of tumor cell plasticity EPIGENETIC and its role in diversity in Ewing sarcoma metastasis. Ewing sarcoma. MORE SCIENCE REPORTS INSIDE >>> [ 06 ] Forschungsbericht St. Anna Kinderkrebsforschung | Science Report St. Anna Children’s Cancer Research Institute Inhalt

Einleitung Vorwort des Institutsleiters 10 Vorwort des wissenschaftlichen Direktors 12 30 Jahre – unseren Spendern sei Dank! 14

Daten & Fakten Kompetitive Drittmittel 2018 20 Zuweisung der Geldmittel 2018 20 Finanzierung 2018 20 Nationen 21 Personelle Zusammensetzung 21 PatientInnenaufkommen im S2IRP 22 2-Jahres-Überlebensrate krebskranker Kinder 23 5-Jahres-Überlebensrate krebskranker Kinder 24 Forschungsnetzwerke 26

Inhalt Forschungsbericht I St. Anna Kinderkrebsforschung | Science Report St. Anna Cancer Research Institute [ 07 ]

Science Reports ■ Clinically important resistant mutations in Philadelphia Introduction 30 ■  chromosome-positive leukemias 84 Science Reports 2017 ■ Allogeneic Stem Cell Transplantation for ■ Targeted inhibition of the MAPK pathway children with Acute Lymphoblastic Leukemia: leads to a rapid and sustained clinical response Outcome after mismatched-donor in severe multisystem LCH 38 transplantation is influenced by Disease Risk 90

■ Understanding mechanisms of tumor cell plasticity and its role in Career Ewing sarcoma metastasis 42 Working at CCRI 98 ■ Busulfan and melphalan is considered standard high-dose chemotherapy for If you want to apply for a position 99 high-risk neuroblastoma. 46 Scientific Staff 100 ■ A novel fusion gene in treatment-resistant pediatric myeloid neoplasia 50 Finanzbericht ■ A novel genetic subtype of leukemia 56 Richtlinien zur Spendenverwendung 104 ■ Epigenetic diversity in Ewing sarcoma 60 Mittelherkunft 106 Science Reports 2018 Mittelverwendung 107

■ Understanding tumor heterogeneity and relapse in neuroblastoma patients to Anhang allow adequate treatment options 64 International Grants 110 ■ Investigating bone marrow dissemination in metastatic neuroblastoma 68 National Grants 112

■ Establishing diagnoses in children with rare Acknowledgments 114 hematological cytopenias via modern genomic Bachelor-, Master/Diploma- and PhD-Theses 116 approaches 72 Publications 2017 118 ■ No evidence that addition of subcutaneous IL-2 to immunotherapy with dinutuximab Publications 2018 126 beta, given as an 8 h infusion, improved Impressum 136 outcomes in patients with high-risk neuroblastoma, hence, dinutuximab beta Fundraising 137 alone should thus be considered the standard of care 76

■ Path to overcoming the threat of adenovirus infections in pediatric stem cell transplant recipients 78

Inhalt

Einleitung [ 10 ] Forschungsbericht St. Anna Kinderkrebsforschung | Science Report St. Anna Children’s Cancer Research Institute

Vorwort des Institutsleiters

Zulassung einer Immuntherapie beim Hochrisiko- Neuroblastom, die von der St. Anna Kinderkrebsfor- schung über viele Jahre maßgeblich mitentwickelt wurde. Darüber hinaus sind uns viele weitere wichtige Publikationen und wissenschaftliche Meilensteine gelungen, wie Sie im vorliegenden ann man auf wissenschaftliche Erfolge Zweijahresbericht im Detail sehen. stolz sein? Ja, umso mehr, wenn sie dazu beitragen, junges Leben zu retten. Als weiteres Highlight ist zu erwähnen, dass der Fortschritte in der Grundlagenwissen- St. Anna Kinderkrebsforschung 2017 gemeinsam schaft, der klinischen Wissenschaft mit dem St. Anna Kinderspital von der und der Diagnostik bringen uns dabei voran, das Europäischen Union als einer von nur zwei österrei- Ksehen wir als unsere Aufgabe und Pflicht und als chischen Institutionen die Auszeichnung Expertise- unser Ziel. zentrum verliehen und sie zur Koordinatorin eines Ich glaube, in unserem Fall sind sowohl Stolz als europäischen Referenznetzwerks gegen Kinderkrebs auch Freude und Dankbarkeit zulässig. Denn im (ERN PaedCan) ernannt wurde. Die Auszeichnung Jahr nach dem 30. Jubiläum der St. Anna Kinder- ist auch die Basis für viele assoziierte Folgeprojekte, krebsforschung dürfen wir mit diesem Jahresbericht die im Jahr 2018 anliefen und alle zum Ziel haben, auch auf die Erfolge zurückblicken, die in den die Kräfte gegen Kinderkrebs zu bündeln und zu letzten beiden Jahren von unserem Institut erbracht optimieren. wurden. Entsprechend konnten wir uns auch wieder über 2017 veröffentlichte eine unserer jungen Fördergelder freuen. Die Zuwendungen der Forscherinnen gemeinsam mit ihren wissenschaftli- Europäischen Union und nationale Forschungsför- chen Partnern eine wichtige und weithin beachtete derungen machten 2018 in puncto Förderungen Publikation in der renommierten Fachzeitschrift zum erfolgreichsten Jahr in der Geschichte der St. „Nature Medicine“, und zwar zum Thema Epigenetik Anna Kinderkrebsforschung und stärken unseren beim Ewing-Sarkom. Im gleichen Jahr erfolgte die Ehrgeiz, auch in Zukunft ganz vorne in der Liga der

Einleitung Forschungsbericht St. Anna Kinderkrebsforschung | Science Report St. Anna Children’s Cancer Research Institute [ 11 ]

Univ.-Prof. Dr. Wolfgang Holter

internationalen Spitzenforschung zum Wohle krebskranker Kinder mitzumischen.

Unser Institut wurde vor über 30 Jahren begründet, da liegt ein Generationenwechsel in der Natur der Sache. Gründungsmitglieder gingen in den wohlverdienten Ruhestand, junge, aufstrebende krebskranker Kinder vertraut sind. Es ist eine große Forscherinnen und Forscher bereicherten unser Freude, einen so wertvollen Zugewinn zu unserem Institut. So danken wir besonders Herrn Doz. Dr. wissenschaftlichen Team verzeichnen zu können. Gerhard Fritsch (Gruppe „Klinische Zellbiologie“) und Herrn Univ.-Doz. Dr. Peter Ambros und Frau Mit dem vorliegenden wissenschaftlichen Dr. Inge Ambros (Gruppe „Tumorbiologie“) für Jahresbericht möchte ich mich, auch im Namen aller ihren langjährigen Einsatz rund um das Forschungs- Mitarbeiterinnen und Mitarbeiter, sehr herzlich bei institut. Vor allem danken wir ihnen dafür, dass Sie den vielen Unterstützerinnen und Unterstützern, weiterhin den jüngeren Mitarbeiterinnen und den Mitgliedern des Ehrenkomitees, den Vorstands- Mitarbeitern mit Rat und Tat zur Seite stehen. mitgliedern, dem wissenschaftlichen Beirat und unseren zahlreichen Spenderinnen und Spendern, Eine wichtige Entwicklung für die weitere die uns schon viele Jahre treu begleiten, bedanken. ■ Verstärkung unseres Instituts war die Ernennung eines neuen wissenschaftlichen Direktors. Mit Assoc.-Prof. Dr. Kaan Boztug gewinnt die St. Anna Univ.-Prof. Dr. Wolfgang Holter, Kinderkrebsforschung nicht nur einen führenden Institutsleiter Experten für angeborene seltene Erkrankungen der Blutbildung und des Immunsystems, sondern auch einen Kollegen, dem als Oberarzt in der pädiatrischen Onkologie und als Leiter der Immunologie der Klinikalltag im St. Anna Kinderspital sowie die Praxis in der Behandlung

Einleitung [ 12 ] Forschungsbericht St. Anna Kinderkrebsforschung | Science Report St. Anna Children’s Cancer Research Institute

Vorwort des wissenschaftlichen Direktors

St. Anna Kinderkrebsforschung tatsächlich eine Herzensangelegenheit. Hier kann ich mein Wissen gezielt einsetzen und meinen Fokus auf das Wohl der Kinder richten. Am Institut arbeitet eine Gruppe von äußerst enthusiastischen Forscherinnen und ls wissenschaftlicher Direktor des Forschern, deren wichtige wissenschaftliche Instituts ist es mir eine besondere Arbeiten ich in diesem Bericht vorstellen möchte. Freude, den Forschungsbericht der St. Anna Kinderkrebsforschung mit Dank neuer Technologien wissen wir heute, dass eröffnen zu dürfen. Obwohl meine Krebserkrankungen, beispielsweise solche, die unter Ernennung erst am Ende dieser Berichtsperiode dem Überbegriff Leukämie zusammengefasst Aerfolgte, verbindet mich mit der St. Anna Kinder- werden, tatsächlich sehr unterschiedlich sind. krebsforschung bereits eine mehrjährige Geschichte: Unsere molekularbiologischen Erkenntnisse Als Oberarzt in der pädiatrischen Onkologie und erlauben heute eine zunehmend vollständigere Cha- Leiter der Immunologie am St. Anna Kinderspital rakterisierung jedes einzelnen Patienten; der Krebs sehe ich im Klinikalltag die Möglichkeiten und wird in immer feinere Subgruppen unterteilt und Grenzen in der Behandlung krebskranker Kinder, zerfällt schlussendlich in genetisch und/oder die mich stets zu noch tiefer greifenden Forschungs- epigenetisch klassifizierbare „seltene“ aktivitäten motivieren. Seit 2016 leite ich das von Erkrankungen“. Das erklärt wahrscheinlich die mir konzipierte und von der Ludwig Boltzmann schon seit langem bekannte Beobachtung, dass Gesellschaft gegründete Ludwig Boltzmann Institute Therapien bei einzelnen Patientinnen und Patienten for Rare and Undiagnosed Diseases, an dem die St. mehr oder weniger gut wirken und wir für weitere Anna Kinderkrebsforschung als Partnerinstitution Fortschritte in der Kinderonkologie teilweise dem- von Anfang an beteiligt war. So ist für mich als lei- entsprechend individualisierte Therapieansätze denschaftlichen Wissenschaftler, aber auch als Kin- benötigen. Allerdings stellt uns die vollständige deronkologen, der die Grenzen des heute Erfassung und Berücksichtigung der enormen Machbaren aus der Klinik kennt, die Berufung zur Datenmengen vor neue Herausforderungen. Zur

Einleitung Forschungsbericht St. Anna Kinderkrebsforschung | Science Report St. Anna Children’s Cancer Research Institute [ 13 ]

Assoc.-Prof. Dr. Kaan Boztug

verlässlichen Interpretation und Analyse aller Patientendaten werden wir daher zunehmend auf die Möglichkeiten der Bioinformatik und der künstlichen Intelligenz zurückgreifen müssen.

Unerlässlich ist dabei auch die internationale geheilt werden können. Dieser Anspruch prägt auch Zusammenarbeit. Ebenso wie bei anderen Gruppen meine Arbeit und ist gleichzeitig der Antrieb für die von seltenen Erkrankungen profitiert die leidenschaftliche und konsequente Weiterentwick- Erforschung von Kinderkrebs von interdisziplinärer, lung unserer Forschungstätigkeit. multizentrischer und internationaler Kooperation. Gemeinsam werden sichere Datenbanken aufgebaut, Ich wünsche Ihnen viel Freude beim Lesen und die sämtliche Merkmale eines Krankheitsbildes Durchblättern des Forschungsberichts 2017/2018 standardisiert erfassen und den weltweiten und danke für Ihre Unterstützung und das Austausch zwischen Expertinnen und Experten Vertrauen in unsere Arbeit. erlauben. Nur so ist es möglich, bei seltenen Erkrankungen wie Kinderkrebs eine ausreichend Assoc.-Prof. Dr. Kaan Boztug, große Fallzahl zu erreichen, um die besten Behand- Wissenschaftlicher Leiter lungsmöglichkeiten zu ermitteln.

Mein Ziel ist es, die Stellung des Instituts als eines der weltweit führenden Forschungszentren im Bereich Kinderkrebs weiter auszubauen und For- schungskooperationen gezielt zu intensivieren. Die Vision unserer Forscherteams und des gesamten Instituts ist, in naher Zukunft auch jenen Kindern dauerhaft zu helfen, die mit den vorhandenen Be- handlungsmöglichkeiten noch nicht nachhaltig

Einleitung [ 14 ] Forschungsbericht St. Anna Kinderkrebsforschung | Science Report St. Anna Children’s Cancer Research Institute

30 Jahre – unseren Spenderinnen und Spendern sei Dank! Bunte Einfälle – spannende Aktionen – großzügige Spenden Das Spektrum der Spendenmöglichkeiten ist vielfältig und die Unterstützung der Spenderinnen und Spender grenzenlos – wie einige großartige Spenderideen zeigen:

nsere Spenderfamilie ist groß und ■ Mit viel Applaus wurden Peter Matić, Jasminka großartig! Wir sind dankbar für die Stancul, Gertrud Weinmeister, Christian langjährige, treue Unterstützung – Altenburger, Franz Bartolomey und Herbert Mayr 2018 konnten wir bereits das 30- anlässlich des exklusiven musikalisch-literarischen Jahr-Jubiläum unseres Forschungs- Benefizkonzerts zum 30-jährigen Bestehen der instituts feiern – und wir freuen uns jedes Mal, ein St. Anna Kinderkrebsforschung gefeiert. Das Uneues Familienmitglied herzlich willkommen zu Publikum im ausverkauften Gläsernen Saal des heißen! Das Faszinierende an der Arbeit im Wiener Musikvereins erlebte einen Kunstgenuss Spendenbüro der St. Anna Kinderkrebsforschung der Extraklasse mit heiteren Texten und sind die Menschen, ihre Hilfsbereitschaft, ihre musikalischen Stücken, unter anderem von wunderbaren Ideen und ihr großartiges Spende- Ephraim Kishon, Alfred Brendel und Franz nengagement. Dank der konsequenten Forschung Schuberts „Forellenquintett“. Die wunderbaren können heute bereits vier von fünf Kindern und Künstlerinnen und Künstler verzichteten für den Jugendlichen, die vor 40 Jahren noch als unheilbar guten Zweck auf ihre Gage. Dieses einmalige galten, gerettet werden. Finanziert wird die Kulturerlebnis war vor allem Eva Angyan, der St. Anna Kinderkrebsforschung, die seit 2002 Präsidentin des St. Anna Kinderkrebsforschung- das Österreichische Spendengütesiegel führt und Unterstützungskomitees, und Erste-Bank-Vor- zum steuerlich begünstigten Empfängerkreis standsmitglied Willibald Cernko zu verdanken. gehört, seit Anfang an hauptsächlich durch Beiden liegt die Unterstützung der Arbeit der Spenden. Dank gebührt daher unseren St. Anna Kinderkrebsforschung sehr am Herzen. Spenderinnen und Spendern – denn sie alle schenken krebskranken Kindern eine Chance auf ■ Die Aschermittwochfeier der Künstlerinnen und eine gesunde Zukunft. Künstler in der Wiener Hofburgkapelle gilt als

Einleitung Forschungsbericht St. Anna Kinderkrebsforschung | Science Report St. Anna Children’s Cancer Research Institute [ 15 ]

besondere Einstimmung auf die Fastenzeit. Die Wiener Hofmusikkapelle unter der Leitung von Direktor Prof. Dr. Walter Dobner und dem künstlerischen Leiter Prof. Erwin Ortner und alle Mitwirkenden agierten im Dienste der guten Sache und unterstützten mit den Einnahmen ebenfalls unsere Forschungsarbeit.

■ Der offizielle Startschuss zur Eisschlecksaison bereitet der St. Anna Kinderkrebsforschung jedes Jahr doppelte Freude: Eis schlecken und eine großzügige Spende leisten! So unterstützten auch 2018 wieder mehr als 300 Eissalons in ganz Österreich unser Forschungsinstitut mit einer namhaften Summe.

■ Für ein spezielles Benefiz-Kinoerlebnis mit vielen sammenzubringen und ein Zeichen gegen den lachenden Gesichtern und einer Spendensamm- Krebs zu setzen. Und das passiert seit damals lung zugunsten der St. Anna Kinderkrebsfor- jährlich auf beeindruckende Weise! Rund 30 schung sorgte der Verein Österreichische Journa- Kinder und Jugendliche sorgen am Konzertabend listenwerkstätte mit dem Film „Fünf Freunde und für Gänsehautfeeling beim Publikum – und für das Tal der Dinosaurier“. einen hervorragenden Spendenbetrag.

■ Anlässlich seines 80. Geburtstags besuchte ■ Die Unterstützung durch die von Marianne Komm.-Rat Alfred Umdasch in Begleitung seiner Brandtner geleitete Bastelrunde Hirtenberg, die Gattin Elisabeth die St. Anna Kinderkrebsfor- der St. Anna Kinderkrebsforschung die Erlöse schung. Das Forschungsinstitut durfte sich über ihrer jährlichen Oster- und Adventmärkte zur eine großzügige Spende freuen. Verfügung stellt, hat bereits eine langjährige Tradition – wie auch das Benefiz-Sautrogrennen in ■ Zu einem Benefiz-Adventkonzert im Schloss Scharndorf. Die Spendenhöhen sind jedes Mal Rosenau zugunsten der St. Anna Kinderkrebsfor- beeindruckend. schung lud Regina Sturm ins winterliche Waldviertel. Im gemütlichen Rahmen trug sie ■ Tim Tom Nordens Pop-Art-„Sky Painting“ für den stimmungsvolle Adventlieder vor, umrahmt von guten Zweck sorgte bei der Benefizauktion für Anekdoten zum Schmunzeln und Weihnachtsge- einen sensationellen Spendenerfolg. Seine schichten, rezitiert von Stargast Erni Mangold. Pop-Art-Werke entstehen spektakulär im freien Fall vom Bungee-Kran aus 65 Meter Höhe. Mit ■ Im Jahr 2017 gründete die damals 19-jährige seiner Technik des „Sky Paintings“ hat sich der ös- Felicia Bösenkopf die Organisation „Stimmen terreichische Performancekünstler über die gegen Krebs“ mit der Vision, junge Menschen zu- Grenzen hinaus einen Namen gemacht.

Einleitung [ 16 ] Forschungsbericht St. Anna Kinderkrebsforschung | Science Report St. Anna Children’s Cancer Research Institute

St. Anna Kinderkrebsforschung- Unterstützungskomitee Schon seit einiger Zeit begleitet ein prominent besetztes Ehrenkomitee die St. Anna Kinderkrebsfor- schung. Die Mentorinnen und Mentoren – Personen aus Politik, Wirtschaft und Kultur – zeichnen sich durch eine besondere Position aus und sind ehrenamtlich tätig. Durch ihre Präsenz in der Öffentlichkeit können sie viel zugunsten der St. Anna ■ Auf dem Rücken der Pferde liegt nicht nur das Kinderkrebsforschung bewegen. Seit einigen Jahren Glück dieser Erde, sondern manchmal auch ein unterstützt Eva Angyan, Gattin des Intendanten der hervorragender Spendenbetrag. Beim internatio- Gesellschaft der Musikfreunde in Wien, Dr. Thomas nalen Dressurturnier in Achleiten wurde ein Angyan, als Komiteepräsidentin das Forschungsinsti- Betrag in beeindruckender Höhe gesammelt. Die tut. Die Unterstützung der Kinderkrebsforschung ist Präsidentin des Österreichischen Pferdesportver- ihr ein Herzensanliegen und deshalb bittet Frau bandes Elisabeth Max-Theurer freute sich, die Angyan regelmäßig eine ausgesuchte Gästeschar aus Spende persönlich übergeben zu können. Politik, Wirtschaft und Kultur zu einem Treffen, um gemeinsame Projekte zugunsten der St. Anna Kinder- krebsforschung zu planen. Lange Nacht der (Kinderkrebs-)Forschung Ein Unterstützungskomitee mit klingenden Forschung auf reizvolle Weise entdecken und Namen: Dr. Charlotte Rothensteiner, Mag. Maria Zukunft erleben! Dazu lädt die St. Anna Kinder- Polsterer-Kattus, Erste-Bank-Vorstand Willibald krebsforschung alljährlich ein. 2018 war im Rahmen Cernko, Komm.-Rat Karl Javurek, Isabella Kapsch, von Österreichs größtem Forschungsfest, der Komm.-Rätin Dipl.-Kffr. Elisabeth Gürtler, „Langen Nacht der Forschung“, ein spielerisches Komm.-Rätin Brigitte Jank, Prof. Erwin Ortner, Kennenlernen des Laboralltags möglich und es Bäckermeister Senator Komm.-Rat Kurt Mann, der wurde jede Menge „Wissenschaft zum Anfassen“ ehemalige Wiener Bürgermeister und Präsident des geboten. Interaktiv, fesselnd und leicht verständlich WWTF Dr. Michael Häupl, Meinungsforscher Prof. wird Wissen über die Bausteine des Lebens und die Dr. Rudolf Bretschneider, Interspot-Chefin Ingrid molekularen Grundlagen der Krebsentstehung Klingohr, Concordia-Vorstand Mag. Ulla Konrad und vermittelt. Vorführungen in den Labors und Maestro Franz Welser-Möst freuen sich, die spannende wissenschaftliche Vorträge verdeutlichen, St. Anna Kinderkrebsforschung als Mentorinnen und was unsere Forscherinnen und Forscher zur Mentoren zu begleiten und tatkräftig zu unterstützen. Verbesserung der Behandlungsqualität und zur Erhöhung der Heilungschancen an Krebs erkrankter Kinder und Jugendlicher leisten. Unsere Kuscheltiere: kleine Lebensretter, Viele Besucherinnen und Besucher interessierten die Freude schenken sich für unsere Arbeit, staunten über unsere Erfolge Bereits seit 26 Jahren sind die Kuscheltiere der und nützten die Gelegenheit, sich zu informieren und St. Anna Kinderkrebsforschung bei Jung und Alt einen Blick hinter die Forschungskulissen zu werfen. sehr beliebt und als Sammelobjekte auch heiß

Einleitung Forschungsbericht St. Anna Kinderkrebsforschung | Science Report St. Anna Children’s Cancer Research Institute [ 17 ]

Vielfältige Spendenmöglichkeiten Onlinespende, Barspende, Spende mit Zahlschein, Spende mit Einziehungs- oder Dauerauftrag, Benefizveranstaltungen, Sammelaktionen, Spenden statt Geburtstags- oder Weihnachtsgeschenken, Erwerb unserer Kuscheltiere, Kranzablösespenden, Legat/ Testament etc.

begehrt! Jedes Jahr im Oktober heißt die Maskott- Das Team der St. Anna Kinderkrebsforschung ist chenfamilie einen Neuzugang willkommen. 2018 dankbar für die langjährige spendenfreudige war es „Benni, der brave Beagle“. Die kleinen Unterstützung und ich persönlich für die Begegnung Lebensretter freuen sich auf ein nettes Zuhause und mit so vielen großzügigen, warmherzigen und jede Menge Spielgefährten. Für eine Spende schenkt hilfsbereiten Menschen. Herzlichen Dank! man nicht nur krebskranken Kindern eine Chance, sondern auch sich selbst oder seinen Lieben Freude. Bank Austria, Erste Bank und einige Sparkassen Mag. Andrea Prantl unterstützten die St. Anna Kinderkrebsforschung Verantwortliche wieder beim Vertrieb. Sogar an der Rezeption des Spenderbetreuung renommierten Wiener Innenstadt-Hotels Imperial Senior Executive ist das jeweils aktuelle Maskottchen erhältlich. Fundraising Manager Welche Stofftiere angeboten werden und wie sie zu bestellen sind, steht auf unserer Internetseite.

Jede Spende hilft! Wollen Sie Informationen oder Unterlagen, Ob mit einer persönlichen Spende oder mit einer oder haben Sie Fragen? gemeinsamen Sammelaktion, ob per Zahlschein, Das Spendenteam und ich freuen Kreditkarte oder Onlinespende – jede finanzielle uns auf Ihre Kontaktaufnahme: Zuwendung ermöglicht die Fortsetzung unserer For- schungsarbeit im Kampf gegen Kinderkrebs. Firmen ▶ +43 (0)1 40 470-4000 spenden immer häufiger den für Kunden-Weih- ▶ [email protected] nachtsgeschenke vorgesehenen Betrag. Statt um ▶ www.kinderkrebsforschung.at Geschenke wird bei Geburtstagen, Jubiläen oder anderen Feiern gerne um Spenden gebeten. Auch ▶ Bank Austria der Verzicht auf Blumen und Kränze bei IBAN: AT79 1200 0006 5616 6600 Begräbnissen, um stattdessen zu spenden, hilft BIC: BKAUATWW krebskranken Kindern. Ein Testament oder ein ▶ Erste Bank Legat zugunsten der St. Anna Kinderkrebsforschung IBAN: AT66 2011 1000 0318 3777 schenkt ebenfalls langfristig eine gesunde Zukunft. ■ BIC: GIBAATWW

Einleitung

Daten und Fakten [ 20 ] Forschungsbericht St. Anna Kinderkrebsforschung | Science Report St. Anna Children’s Cancer Research Institute

Quelle FWF 25,38% der EU 49,28% kompetitiven FFG 9,25% Drittmittel im Jahr 2018 OeNB 2,62% Sonstige Drittmittelgeber 13,46%

Forschung 89,45% Zuweisung der Geldmittel Spendenwerbung 6,43%

im Jahr 2018 Verwaltungsaufwand 3,52%

Sonstiger Aufwand 0,61%

Spenden und Verlassenschaften 86,80% Finanzierung

im Jahr 2018 Kompetitive Drittmittel 13,20%

Daten und Fakten NATIONEN ÖSTERREICH

MitarbeiterInnen BOSNIEN-HERZEGOWINA der St. Anna DEUTSCHLAND Kinderkrebsforschung in den Jahren FRANKREICH 2017 und 2018 GRIECHENLAND IRAN

ISLAND

ITALIEN

MONTENEGRO

POLEN

PORTUGAL

RUSSISCHE FÖDERATION

SERBIEN

SLOWAKEI

TÜRKEI

UNGARN

VEREINIGTE STAATEN VON AMERIKA Personelle Zusammensetzung

Frauen 68% Männer 32%

Daten und Fakten [ 22 ] Forschungsbericht St. Anna Kinderkrebsforschung | Science Report St. Anna Children’s Cancer Research Institute

PatientInnenaufkommen im S²IRP als Koordinierungszentrum für klinische Studien

16.000 PatientInnen gesamt 15.413 14.000 PatientInnen international 8.000 9.690

4.000 PatientInnen Österreich 5.723

0 1981 1990 2000 2010 2018

Die St. Anna Kinderkrebsforschung fungiert als nationales und im Bereich des Neuroblastoms, der Langerhans-Zell- Histiozytose, und der Stammzell- transplantation für akute Leukämie als internationales Koordinierungszentrum für klinische Studien. Das Studienmanagement wird von der Abteilung für Studien und Statistik (S²IRP) durchgeführt.

Daten und Fakten Forschungsbericht St. Anna Kinderkrebsforschung | Science Report St. Anna Children’s Cancer Research Institute [ 23 ]

Anstieg der 2-Jahres- Überlebensraten von krebskranken Kindern und Jugendlichen Morbus Hodgkin Maligne Keimzelltumoren Wilms-Tumor 100% Akute lymphoblastische Leukämie Non-Hodgkin-Lymphom 80% Neuroblastom und Ganglioneuroblastom 60% Osteosarkom 40% Rhabdomyosarkom Hirntumoren 20% Ewing-Sarkom Akute myeloische Leukämie 0% 1950 1960 1970 1980 1990 2000 Quelle: GPOH, Kinderkrebsregister Mainz

Daten und Fakten [ 24 ] Forschungsbericht St. Anna Kinderkrebsforschung | Science Report St. Anna Children’s Cancer Research Institute

5-Jahres-Überleben von krebskranken Kindern in Europa zwischen 2000 und 2007

Daten und Fakten Forschungsbericht St. Anna Kinderkrebsforschung | Science Report St. Anna Children’s Cancer Research Institute [ 25 ]

5-JAHRES-ÜBERLEBENDE in %

NORDEUROPA Dänemark Finnland Island Norwegen Schweden

UK UND IRLAND Irland England und Wales Nordirland Schottland

ZENTRALEUROPA Österreich Belgien Frankreich Deutschland Schweiz Niederlande OSTEUROPA Bulgarien Estland Ungarn Lettland Litauen Polen Slowakei SÜDEUROPA Kroatien Italien Malta Portugal Slowenien Spanien

Europa gesamt 0 10 20 30 40 50 60 70 80 90 100

Quelle: Gatta et al., Lancet Oncology 2014 Daten und Fakten [ 26 ] Forschungsbericht St. Anna Kinderkrebsforschung | Science Report St. Anna Children’s Cancer Research Institute

Forschungsnetzwerke national und international

ALBANIEN ARGENTINIEN AUSTRALIEN BELGIEN BOLIVIEN BOSNIEN-HERZEGOWINA BRASILIEN BULGARIEN CHILE CHINA DÄNEMARK DEUTSCHLAND FINNLAND FRANKREICH GRIECHENLAND GROSSBRITANNIEN HONGKONG INDIEN IRLAND ISLAND ISRAEL ITALIEN JAPAN KANADA KROATIEN

Daten und Fakten Forschungsbericht St. Anna Kinderkrebsforschung | Science Report St. Anna Children’s Cancer Research Institute [ 27 ]

NEUSEELAND NIEDERLANDE NORWEGEN ÖSTERREICH POLEN PORTUGAL RUMÄNIEN RUSSLAND SCHWEDEN SCHWEIZ SERBIEN SINGAPUR SLOWAKEI SLOWENIEN SPANIEN SÜDKOREA TANSANIA TSCHECHIEN TÜRKEI UKRAINE UNGARN URUGUAY USA WEISSRUSSLAND

Daten und Fakten

Science Reports [ 30 ] Forschungsbericht St. Anna Kinderkrebsforschung | Science Report St. Anna Children’s Cancer Research Institute

Introduction Scientific Director Assoc.-Prof. Kaan Boztug, MD

n 2018, Assoc.-Prof. Dr. Kaan Boztug, leading expert for congenital rare diseases of haematopoiesis and the immune system, was selected as the top candidate for the position as Scientific Director in a competitive international call for tenders and appointed by a Itop-level hiring committee. In March 2019, he took over the agendas of the Scientific Director and thus Since 2016, Kaan Boztug has been leading the the scientific responsibility for the dedicated research Ludwig Boltzmann Institute for Rare and projects of approximately 140 employees at CCRI Undiagnosed Diseases, an institute founded by the working in diagnostics, clinical studies and trials, and Ludwig Boltzmann Society. CCRI is one of its experimental research in paediatric oncology. partner institutions, as are CeMM - Research Center Our new Scientific Director has a long and for Molecular Medicine of the Austrian Academy of successful relationship with the CCRI: As Sciences and the Medical University of Vienna. Kaan Consultant in Paediatric Oncology and Head of Boztug’s overall aim is to further expand the Immunology at the St. Anna Children‘s Hospital, he institute’s position as one of the world‘s leading sees both challenges but also opportunities in the research centres in the field of childhood cancer and day-to-day treatment of children suffering from to intensify interdisciplinary and international cancer in the clinic, driving his enthusiasm for research cooperation, bridging basic science and in-depth mechanistic research investigations. translational oncology.

Science Reports Forschungsbericht St. Anna Kinderkrebsforschung | Science Report St. Anna Children’s Cancer Research Institute [ 31 ]

About Kaan Boztug After his medical studies in Düsseldorf, Freiburg and London and his doctorate with Iain Campbell at The Scripps Research Institute in La Jolla/San Diego in the USA, Kaan Boztug completed his clinical training and postdoctoral research work with Christoph Klein at the Hannover Medical School. In 2011 he took over a position as Principal Investigator at the CeMM Research Center for Molecular Medicine of the Austrian Academy of Sciences and was appointed Associate Professor in the Department of Paediatrics and Adolescent Medicine of the Medical University of Vienna. In addition, he is Director of the CeRUD Vienna Scientific Objectives: Center for Rare and Undiagnosed Diseases and ■ Molecular dissection of inborn errors of Head of the Jeffrey Modell Center for Primary Im- immunity with predominant immune dysregulation mundeficiencies at the St. Anna Children‘s Hospital and investigation of novel targets for cancer and at the Medical University of Vienna. Since 2016 immunotherapy. Kaan Boztug has headed the LBI-RUD Ludwig Boltzmann Institute for Rare and Undiagnosed ■ Genetics, molecular pathophysiology of Diseases. Numerous national and international inherited bone marrow failure syndromes and awards confirm his scientific excellence, among other cancer predisposition syndromes. them an FWF START Grant, an ERC Starting and an ERC Consolidator Grant as well as the Clemens von ■ Installment of a systematic pediatric precision Pirquet-Award as the most cited scientist in oncology program to identify molecular targets Paediatrics and Adolescent Medicine 2018. for tailored treatment of childhood cancer.

Science Reports [ 32 ] Forschungsbericht St. Anna Kinderkrebsforschung | Science Report St. Anna Children’s Cancer Research Institute

Kaan Boztug’s reflections on the institute’s strategy: challenges and prospects for the future

Q: What major advances do you see in childhood cancer research? Where is the current trend in childhood cancer research heading? A: Childhood cancer is different from adult cancer. While adults often suffer from cancers influenced by environmental factors (e.g. smoking), genetic causes that occur during organ development play a much more important role in children. In addition, low mutation rates limit the number of suitable targets We want to dive ever deeper into molecular for new drugs and the efficacy of immunotherapies. processes to decipher the highly complex signalling Therefore, despite great advances in key concepts of pathways of the human body that are relevant for cancer therapy such as aggressive chemotherapy, childhood cancer. This requires state-of-the-art international standardisation and monitoring of the experimental research and bioinformatics. In recent minimal residual disease for childhood cancer, there years in particular, we have increasingly been able to are currently only a few targeted therapies available. see and understand the connections between the So while the classical methods of surgery, immune system and the possibility of improving chemotherapy and radiation are and will of course cancer treatment. Cancer immunotherapy, for remain the most important treatment options for example, is based on targeted intervention in cellular childhood cancer, new innovative approaches are signalling pathways that control tumour growth. I urgently needed. Hence, traditional therapy expect this field to make a significant contribution to regimens will have to be supplemented with state-of- the therapies of the future. the-art (epi)genome-based personalised treatment The directive for the future will be: Treat the strategies. patient, not just the cancer. And every cancer is CCRI has a special position here in Vienna due different. This corresponds to the definition of to the close interaction between research and clinic. precision medicine, according to which precise treatment is based on a deep molecular understanding of the disease. And so, in close partnership with the St. Anna Children‘s Hospital, we at CCRI will set up a programme for precision medicine for childhood cancer.

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Q: What challenges do you see for CCRI? studies. It is becoming increasingly clear that the A: Providing highly specialised treatment and care concept of classical clinical study designs with ever for patients with rare diseases and complex clinical more precise subdivision of molecularly defined pictures such as childhood cancer is still a challenge. subgroups of diseases does not function adequately Some forms of childhood cancer are so rare that in when dealing with small numbers of cases, like in Austria they occur only once or twice a year. peadiatric oncology. Thus, a paradigm shift will take Without international cooperation, it is simply place in the coming decades: Treatment will impossible to collect enough cases to assess their increasingly be based on an empirical, scientific-mo- clinical significance. Cross-border and interdiscipli- lecular understanding of disease. And CCRI is the nary cooperation and collaboration are therefore of ideal place to implement such innovative new central importance for progress in childhood cancer concepts. Because of the wide-ranging research in research and for the future of adequate, nationwide the field of childhood cancer and the direct treatment options. Initiatives such as the European connection to the St. Anna Children‘s Hospital, the reference network ERN-PaedCan which CCRI and rapid transfer of new research results from the the St. Anna Children´s Hospital are coordinating, laboratory to the hospital bed is possible. and other international cooperations such as the In addition to my activities in the field of framework of LBI-RUD must therefore be further precision oncology, my own research group will have expanded. a major focus on research into the genetic and immunological causes of childhood cancer. Excitingly, we and our worldwide cooperation Q: What are your plans for the future of partners have seen more and more clearly in recent the institute? Which path will CCRI take? years, that cell signalling cascades affected in A: Based on decades of biomedical research, we now patients with severe congenital immunodeficiency have data and bioinformatics tools at our disposal to allow conclusions to be drawn about targeted investigate more precisely than ever before the therapy in cancer patients. These and other research molecular characteristics of cancer development, priorities will form strong synergies with the other disease progression and healing processes. research groups at CCRI. Deciphering the human genome, epigenome, As with other groups of rare diseases, childhood proteins, etc., provides crucial information to cancer research benefits from interdisciplinary, combat rare diseases such as childhood cancer. The multicentre and international cooperation. The challenge, however, is to extract the relevant vision for the future of our research team and the information from the huge amount of data and entire institute will be to provide long-term help to combine it with other results. To expand the those children who cannot yet be cured sustainably bioinformatics team at St. Anna‘s Children‘s Cancer with the available treatment options. This claim also Research Institute is therefore an important step on shapes my work and at the same time drives the the road to a synergized precision oncology program passionate and consistent further development of for tailored treatment of paediatric cancer patients. our research activities. ■ Another focus will be on the area of clinical Kaan Boztug, July 2019

Science Reports [ 34 ] Forschungsbericht St. Anna Kinderkrebsforschung | Science Report St. Anna Children’s Cancer Research Institute

Introduction Science Report 2017/2018

he mission of the St. Anna Children’s Cancer Research Institute is to prevent childhood cancer, diagnose it earlier, develop new, and optimise existing therapies. The institute strives to provide the best possible scientific environment to carry out Tresearch ranging from fundamental laboratory studies to large-scale clinical trials. Thanks to the support of our donors and the top class work of our scientific team, children suffering from cancer in Austria have the highest chance of survival in Europe. Using cutting-edge technologies to develop new approaches and make groundbreaking discoveries, our goal is to push survival rates ever higher, and to share our knowledge so that all children with cancer have equal access to quality care, no matter where they live. The years 2017 and 2018 proved eventful and outstanding for the St. Anna Children’s Cancer Research Institute. The institute´s 30th anniversary was approaching and celebrated, personnel restructuring took place, including my appointment as scientific director, a new record was set in the institutional revenue from third-party funding and last but not least scientific results were published, which will contribute significantly to the healing of children suffering from cancer. In the field of solid tumours, important papers were published unravelling mechanisms of tumour heterogeneity and dissemination of neuroblastoma. This resulted in cooperative research projects in

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order to further optimize the diagnostics and and type of mutations, TKI dose reductions should therapy of this deadly disease. therefore be considered very carefully. Our research groups specializing in Ewing To fight childhood cancer as effectively as sarcoma also advanced several hypotheses, both in possible, our research also focuses on cancer basic and translational research. This included a environment and associated risks such as infectious better understanding of tumour cell plasticity and its diseases. This includes the development of strategies role in Ewing sarcoma metastasis. Another study against dangerous virus infections, which can be published in Nature Medicine provided a life-threatening for immunocompromised children comprehensive assessment of the epigenetic with cancer. Thus, it could be shown that the heterogeneity of Ewing‘s sarcoma, underlining the detection of human adenoviruses in the intestine importance of non-genetic aspects in the context of prior to stem cell transplantation poses a cancer biology and personalized medicine. significantly higher risk of an invasive, life-threate- New discoveries have also been made in ning infection after treatment. leukaemia research. Thus, a novel genetic subtype In clinical research, this reporting period also of B-progenitor acute lymphoblastic leukaemia saw the publication of two major Lancet Oncology with a poor outcome could be described. It is papers on first-line therapy for high-risk characterized by intragenic PAX5 amplification and neuroblastoma patients, as well as a relevant may be an important driver of this type of publication on the outcome of relapse after leukaemia. allogeneic haematopoietic stem-cell transplantation An accurate diagnosis of haematological in children with acute lymphoblastic leukaemia. malignancies is essential in order to make an Furthermore, the first successful treatment of a appropriate treatment decision and avoid secondary high-risk patient with rare Langerhans cell organ damage. This is also the case for congenital histiocytosis with a tyrosine kinase inhibitor was haematological cytopenias which often occur as reported. precursor states of such diseases. Both the diversity With this impressive scientific performance, the and phenotypic overlap of different diseases can institute has once again come a great deal closer to make it difficult to identify the underlying genetic its goal: Namely, to understand and ultimately cure aetiologies. In a translational project with the St. of childhood cancer. ■ Anna Children´s Hospital, targeted mutation screening in affected children could identify new pathogenic mutations. Assoc.-Prof. Kaan Boztug, MD In paediatric myeloid neoplasia it was found that the novel oncogenic fusion gene, NDEL1-PDGFRB, alters tumour responsiveness to tyrosine kinase inhibitors (TKI), causing treatment-resistance. Further clinically important resistant mutations in Philadelphia chromosome-positive leukaemia were detected, that may even overcome highly effective third generation TKI. Depending on the presence

Science Reports All reports of this non-commercial publication describe achievements published in high-ranking scientific journals, as indicated by the respective citations. Most of the figures used in this report are taken from the publications and in these cases copyright belongs to the respective journals. Science Reports 2017 [ 38 ] Forschungsbericht St. Anna Kinderkrebsforschung | Science Report St. Anna Children’s Cancer Research Institute

Targeted inhibition of the MAPK pathway leads to a rapid and sustained clinical response in severe multisystem LCH

The disease Langerhans cell histiocytosis (LCH) has features of both cancer and inflammatory disease. Under the microscope one can observe an accumulation of cells that share similarities with tissue resident immune cells, but their exact origin is still unclear. LCH encompasses an extraordinary clinical diversity. Almost any organ can be involved and, accordingly, the clinical picture is heterogeneous and can present as skin rash, bone tumor, or mimic leukemia, among others. The course of the disease also has a unique behavior: single lesions often resolve spontaneously, whereas once there is more than one lesion, LCH tends to be more aggressive and requires treatment with chemotherapy or

Science Reports Forschungsbericht St. Anna Kinderkrebsforschung | Science Report St. Anna Children’s Cancer Research Institute [ 39 ]

hematopoietic stem cell transplantation. In children toxicity and require extensive supportive care. with multi-system LCH involving the so-called risk Targeted treatment with a RAF-inhibitor might be a organs (bone marrow, liver, and spleen), the new, promising option because around 60 % of one-year mortality risk is 16%, exceeding the current patients with LCH harbour the activating mutation mortality of patients with acute lymphoblastic of the B-RAF serine/threonine protein kinase leukaemia. Optimal therapy for this patient group (BRAFV600E). still needs to be established. The two most effective treatment options to date are a combination of high-dose cytarabine with cladribine or A child with high-risk LCH hematopoietic stem cell transplantation. However, When we were confronted with a critically ill both treatment regimens are associated with severe child that failed conventional chemotherapy, we

Figure 1. Vemurafenib treatment resolves skin lesions and reduces the number of bone marrow- infiltrating cells; skin lesions and bone marrow biopsies before and during treatment with vemurafenib.

Science Reports [ 40 ] Forschungsbericht St. Anna Kinderkrebsforschung | Science Report St. Anna Children’s Cancer Research Institute

decided to attempt treatment with the RAF-inhibitor Blood test vemurafenib. At presentation, the child had fever, In patients with multisystemic LCH, the BRAF hepatosplenomegaly, was pancytopenic and had mutation can be detected in the blood, similar to extensive skin lesions due to infiltration with LCH other diseases, where ‚liquid biopsies‘ are used to cells. It had not responded to conventional detect cell-free tumour DNA in patient blood. LCH chemotherapy and was due for haematopoietic stem is very different from all other cancers in that the cell transplantation, but the clinical condition of the mutation that is found in LCH cells can also be patient was too unstable to safely start with the che- detected in different, seemingly normal blood cells mo-conditioning for the transplantation. like monocytes and dendritic cells. This is only The effect of the RAF-inhibitor was almost found in patients with multisystem LCH, but it is immediate: Three days after treatment was initiated unknown how this correlates with LCH. We the patient improved clinically, fever decreased, and established a test for the amount of mutated BRAF laboratory values started to stabilize. After a short in the blood of patients, because we wanted to treatment period the child seemed clinically in investigate these cells further and to find out remission and had no signs of toxicity during whether we could use BRAF levels as a marker for treatment with the exception of a maculopapular disease severity during treatment. exanthema that quickly resolved after tapering of the When we then followed the levels of mutated vemurafenib dosage. BRAF in the blood of this patient during treatment We stopped the treatment with vemurafenib after with vemurafenib, we found that the child still had eight months because the patient was well and high levels of BRAFV600E in the blood despite not without any clinical signs of disease. However, one having clinical signs of the disease. This was a week later the child developed high fever and the surprising finding because it showed that clinical laboratory values worsened again, reflecting rapid remission is possible despite high levels of mutated reactivation of LCH. Consequently, we restarted BRAF. vemurafenib, which led to rapid resolution of the In contrast, after the conditioning for the trans- fever and normalization the laboratory values. plantation (which is done by the combination of Since there is only very limited information on different chemotherapies), levels of BRAF were long-term effects of RAF inhibition, we did not want below our detection limit, suggesting that to treat our patient for an extended time period with chemotherapy can more efficiently get rid of vemurafenib. We therefore decided to perform a mutated cells. haematopoietic stem cell transplantation. The child was transplanted from a matched sibling donor. HSCT was well tolerated and one month after Implications for future protocols for the transplant the patient was clinically well and showed treatment of children with LCH no signs of disease. Our work showed that treatment with a RAF-inhibitor leads to a rapid and sustained clinical response in severe multisystem LCH, with minimal side effects. However, our observations also suggest that monotherapy with vemurafenib might not be sufficient to cure LCH. This is evidenced by the

Science Reports Forschungsbericht St. Anna Kinderkrebsforschung | Science Report St. Anna Children’s Cancer Research Institute [ 41 ]

Figure 2. Laboratory values, dosage with vemurafenib, and disease activity score during treat- ment with the RAF-inhibitor vemurafenib. Upper panel shows the laboratory values (coloured lines) and vemurafenib dosage (blue area) during treatment. The disease activity score is a unified system to measure LCH activity (from 0-22). The lower panel shows the % of mutated BRAF in the blood of the patient.

rapid relapse after stopping the inhibitor and supported by the sustained high levels of mutant cells in the blood of the patient during vemurafenib treatment. What does this mean for future (much-needed) studies? We think vemurafenib should either be used in combination with a chemo- therapeutic agent or regarded as a bridge to a more intensive treatment. Furthermore, together with colleagues worldwide we are trying to implement the assessment of BRAF as potential biomarker for LCH. This patient also taught us something about the Publications: Kolenová A, Schwentner R, Jug G, nature of LCH: clinical remission (but very likely not Simonitsch-Klupp I, Kornauth C, Plank L, Horáková J, cure) is possible, even when there are still high Bodová I, Sýkora T, Geczová L, Holter W, Minkov M, amounts of BRAF present in the blood. This is Hutter C. Targeted inhibition of the MAPK pathway: puzzling, and resolving this conundrum will likely emerging salvage option for progressive life-threatening be the key to better understand this disease. ■ multisystem LCH. Blood Adv. 2017 Feb 2;1(6):352-356.

Science Reports [ 42 ] Forschungsbericht St. Anna Kinderkrebsforschung | Science Report St. Anna Children’s Cancer Research Institute

Understanding mechanisms of tumor cell plasticity and its role in Ewing sarcoma metastasis

Metastasis as a consequence of tumor cell plasticity step to the other requires a high level of intrinsic Metastasis is the leading cause of cancer cellular plasticity. We therefore hypothesize that mortality. This holds also true for Ewing sarcoma, cancer specific mechanisms of cellular plasticity a malignant tumor of bone and soft tissue in provide attractive therapeutic targets to prevent adolescents and young adults. In the pre-chemothe- tumor dissemination. Therefore, it is our goal to rapy era, when primary tumors were eradicated with unravel the molecular underpinnings of Ewing surgery and radiotherapy alone, almost all Ewing sarcoma plasticity. sarcoma patients died from distant metastases suggesting presence of disseminated tumor cells already at the time of surgery. Today, patients, who Ewing sarcoma cell plasticity as a present with clinically overt metastatic disease at consequence of oncogene fluctuations diagnosis (approx. 20%) and patients, who relapse Ewing sarcoma pathogenesis is driven by the early, still have a poor prognosis (<30% long-term oncogenic fusion protein EWS-FLI1 as a result of a survival) despite intensive multimodal therapy. chromosomal rearrangement. EWS-FLI1 acts as an Metastasis is a complex multi-step process that is aberrant gene regulatory molecule that orchestrates still not fully understood. It comprises many perturbed expression of a wide variety of genes different steps including local invasion into adjacent involved in multiple cellular processes including tissue, migration, intra- and extravasation, and cell cycle, metabolism, and differentiation. It is the colonization of distant sites. Transition from one major driver of Ewing sarcoma proliferation and

Science Reports Forschungsbericht St. Anna Kinderkrebsforschung | Science Report St. Anna Children’s Cancer Research Institute [ 43 ]

Figure 1. Tumor cell plasticity in response to various intrinsic and extrinsic inputs results in different phenotypic and functional outputs.

Figure 2. Boyden chamber assay reveals increased Ewing sarcoma cell migration upon EWS-FLI1 depletion.

survival. However, recent studies indicated that from the primary site and growth at a secondary, experimental modulation of EWS-FLI1 expression metastatic site. Based on surrogate marker by RNA interference resulted in a reversible expression, the proportion of tumor cells in an phenotypic switch from a highly proliferative, EWS-FLI1-low, potentially migratory state in a poorly migratory to a poorly proliferative but primary Ewing sarcoma has been estimated to be highly migratory phenotype. These results 1 to 2% (Franzetti et al. 2017, Oncogene suggested that EWS-FLI1 fluctuations might result 36:3505-3514). In a parallel study, we investigated in oscillations between proliferative and migratory on the molecular level signaling pathways affected cell states required for dissemination of tumor cells by EWS-FLI1 modulation.

Science Reports [ 44 ] Forschungsbericht St. Anna Kinderkrebsforschung | Science Report St. Anna Children’s Cancer Research Institute

Cytoskeletal reprograming as a consequence MRTFB. Consistent with lack of physical SRF of EWS-FLI1 modulation binding, SRF binding motifs were found to be absent Studying six different Ewing sarcoma cell lines in MRTFB binding regions. Instead, we found a we found that downregulation of EWS-FLI1 striking enrichment of the binding motifs for ETS consistently activated a set of genes associated with (consistent with the observed EWS-FLI1 binding) the activation of a signaling pathway (RhoA) and, surprisingly, TEA-domain (TEAD) involved in cytoskeletal re-organization and tumor transcription factors. TEADs are the transcriptional cell migration suggesting an inhibitory role of effectors of the mechano-sensitive Hippo signaling EWS-FLI1 on RhoA signaling. RhoA integrates a pathway that controls tissue homeostasis and organ multitude of extracellular and cell intrinsic signals growth during normal animal development. This involved in cell-to-cell and cell-extracellular matrix result suggested that EWS-FLI1 interferes with communication. However, further studies indicated TEAD activity. In fact, when experimentally that the road block conferred by the fusion oncogene depleting TEADs from EWS-FLI1-high Ewing occurred at a step further downstream in the sarcoma cells, we were able to mimic the genotypic pathway in the cell nucleus. We found that and phenotypic consequences of EWS-FLI1 EWS-FLI1 occupied the majority of target genes modulation on tumor cell plasticity. These results regulated by the RhoA effector protein myocardin established a novel link between EWS-FLI1 and an related transcription factor MRTFB. These genes important developmental pathway and uncovered mainly annotate to the regulation and architecture of the molecular basis for EWS-FLI1 dependent the actin cytoskeleton. Upon depletion of EWS-FLI1 cytoskeletal reprograming leading to Ewing sarcoma by RNA interference, MRTFB occupancy on these metastasis. genes greatly increased resulting in release from their expression block and, in consequence, cytoskeletal reprograming of Ewing sarcoma cells to The Hippo/YAP/TAZ pathway as a potential a more mesenchymal, migratory phenotype. therapeutic target to prevent metastasis TEAD transcription factors require co-activation by YAP and TAZ transcription factors for activity. A novel link between EWS-FLI1 and an established Consistent with previous results (Hsu & Lawlor, mechano-sensitive developmental signaling pathway 2010, Oncogene 30:2077–2085) suggesting orchestrates Ewing sarcoma plasticity stabilization of YAP in Ewing sarcoma by interaction The RhoA regulated effector MRTFs act as with an epigenetic protein (BMI), we found YAP to transcriptional co-activators for nuclear be constitutively expressed in the nucleus of Ewing transcription factors, of which so far serum response sarcoma cells. In addition, however, we observed a factor (SRF) was considered to be the main target. strong increase in TAZ expression upon EWS-FLI1 However, in our studies testing genome-wide knockdown. In fact, we demonstrated increased chromatin occupancy of EWS-FLI1, MRTFB and complex formation between MRTFB, YAP, TAZ and SRF we did not see any significant overlap between TEAD in EWS-FLI1-low migratory cells. As YAP, MRTFB and SRF. Therefore, using a bioinformatics TAZ and TEADs are frequently overexpressed in approach we performed motif enrichment analysis many cancers, there is a strong industrial interest in of chromatin regions bound by EWS-FLI1 and the development of small molecule inhibitors of

Science Reports Forschungsbericht St. Anna Kinderkrebsforschung | Science Report St. Anna Children’s Cancer Research Institute [ 45 ]

Figure 3. A molecular mechanism of phenotypic plasticity in Ewing sarcoma.

their complex formation as potential cancer This work has been supported by grants from the therapeutic compounds. Preliminary results from Austrian Science Fund (I1125-B19), the Liddy our group indicate that pharmacologic inhibition of Shriver Initiative, and the European Commission YAP/TAZ/TEAD complex formation inhibits the Framework Program 7 grants 259348 (ASSET) and migratory ability of Ewing sarcoma cells in vitro and 602856 (EEC). ■ the formation of lung metastasis in a mouse xenograft model. Therefore, our ongoing research, in cooperation with Georgetown University Publications: Katschnig AM, Kauer MO, Schwentner R, Washington DC, and the Department of Pathology, Tomazou EM, Mutz CN, Linder M, Sibilia M, Alonso J, Medical University Vienna, aims at validating these Aryee DNT, Kovar H. EWS-FLI1 perturbs MRTFB/YAP-1/ promising in vivo results and at identifying further TEAD target gene regulation inhibiting cytoskeletal targetable hubs in the signaling pathways involved in autoregulatory feedback in Ewing sarcoma. Oncogene. Ewing sarcoma plasticity. 2017 Oct 26;36(43):5995-6005.

Science Reports [ 46 ] Forschungsbericht St. Anna Kinderkrebsforschung | Science Report St. Anna Children’s Cancer Research Institute

Busulfan and melphalan is considered standard high- dose chemotherapy for high-risk neuroblastoma

Background High-dose chemotherapy with haemopoietic stem-cell rescue improves event-free survival in patients with high-risk neuroblastoma; however, which regimen has the greatest patient benefit has not been established. We aimed to assess event-free survival after high-dose chemotherapy with busulfan and melphalan compared with carboplatin, etoposide, and melphalan.

Methods We did an international, randomised, multi-arm, open-label, phase 3 cooperative group clinical trial of patients with high-risk neuroblastoma at 128 institutions in 18 countries that included an open-label randomised arm in which high-dose chemotherapy regimens were compared. Patients (age 1–20 years) with neuroblastoma were eligible to be randomly assigned if they had completed a multidrug induction regimen (cisplatin, carboplatin, cyclophosphamide, vincristine, and etoposide with or without topotecan, vincristine, and doxorubicin) and achieved an adequate disease response. Patients were randomly assigned (1:1) to busulfan and

Science Reports Forschungsbericht St. Anna Kinderkrebsforschung | Science Report St. Anna Children’s Cancer Research Institute [ 47 ]

Treatment schematic. See main text for details of all treatment regimens. COJEC=cisplatin, carboplatin, cyclophosphamide, vincristine, and etoposide. HDT=high-dose chemotherapy. SCR=stem-cell rescue.

Number at risk (number censored) Busulfan and melphalan Carboplatin, etoposide, and melphalan

melphalan or to carboplatin, etoposide, and busulfan was given (0·8–1·2 mg/kg per dose for 16 melphalan by minimisation, balancing age at doses according to patient weight). After 24 h, an diagnosis, stage, MYCN amplification, and national intravenous melphalan dose (140 mg/m²) was given. cooperative clinical group between groups. The Doses of busulfan and melphalan were modified busulfan and melphalan regimen comprised oral according to bodyweight. The carboplatin, busulfan (150 mg/m² given on 4 days consecutively etoposide, and melphalan regimen consisted of in four equal doses); after Nov 8, 2007, intravenous carboplatin continuous infusion of area under the

Science Reports [ 48 ] Forschungsbericht St. Anna Kinderkrebsforschung | Science Report St. Anna Children’s Cancer Research Institute

Number at risk (number censored) Busulfan and melphalan Carboplatin, etoposide, and melphalan

Number at risk (number censored) Busulfan and melphalan Carboplatin, etoposide, and melphalan

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plasma concentration–time curve 4·1 mg/mL per group), infection (55 [19%] of 283 vs 74 [27%] of min per day for 4 days, etoposide continuous 271), and stomatitis (138 [49%] of 284 vs 162 [59%] infusion of 338 mg/m² per day for 4 days, and of 273); 60 (22%) of 267 patients in the busulfan and melphalan 70 mg/m² per day for 3 days, with doses melphalan group had Bearman grades 1–3 for all three drugs modified according to bodyweight veno-occlusive disease versus 21 (9%) of 239 in the and glomerular filtration rate. Stem-cell rescue was carboplatin, etoposide, and melphalan group. given after the last dose of high-dose chemotherapy, at least 24 h after melphalan in patients who received busulfan and melphalan and at least 72 h after Interpretation carboplatin etoposide, and melphalan. All patients Busulfan and melphalan improved event-free received subsequent local radiotherapy to the survival in children with high-risk neuroblastoma primary tumour site followed by maintenance with an adequate response to induction treatment therapy. and caused fewer severe adverse events than did The primary endpoint was 3-year event-free carboplatin, etoposide, and melphalan. Busulfan and survival, analysed by intention to treat. This trial is melphalan should thus be considered standard registered with EudraCT, number 2006-001489-17. high-dose chemotherapy and ongoing randomised studies will continue to aim to optimise treatment for high-risk neuroblastoma. Findings Between June 24, 2002, and Oct 8, 2010, 1347 patients were enrolled and 676 were eligible for Funding random allocation, 598 (88%) of whom were European Commission 5th Framework Grant randomly assigned: 296 to busulfan and melphalan and the St Anna Kinderkrebsforschung. ■ and 302 to carboplatin, etoposide, and melphalan. Median follow-up was 7·2 years (IQR 5·3–9·2). At 3 years, 146 of 296 patients in the busulfan and melphalan group and 188 of 302 in the carboplatin, etoposide, and melphalan group had an event; 3-year event-free survival was 50% (95% CI 45–56) versus Publications: Ladenstein R, Pötschger U, Pearson ADJ, 38% (32–43; p=0·0005). Nine patients in the Brock P, Luksch R, Castel V, Yaniv I, Papadakis V, Laureys busulfan and melphalan group and 11 in the G, Malis J, Balwierz W, Ruud E, Kogner P, Schroeder H, carboplatin, etoposide, and melphalan group had de Lacerda AF, Beck-Popovic M, Bician P, Garami M, died without relapse by 5 years. Trahair T, Canete A, Ambros PF, Holmes K, Gaze M, Severe life-threatening toxicities occurred in 13 Schreier G, Garaventa A, Vassal G, Michon J, Valteau- (4%) patients who received busulfan and melphalan Couanet D; SIOP Europe Neuroblastoma Group (SIOPEN). and 29 (10%) who received carboplatin, etoposide, Busulfan and melphalan versus carboplatin, etoposide, and and melphalan. The most frequent grade 3–4 adverse melphalan as high-dose chemotherapy for high-risk events were general condition (74 [26%] of 281 in neuroblastoma (HR-NBL1/SIOPEN): an international, the busulfan and melphalan group vs 103 [38%] of randomised, multi-arm, open-label, phase 3 trial. Lancet 270 in the carboplatin, etoposide, and melphalan Oncol. 2017 Apr;18(4):500-514.

Science Reports [ 50 ] Forschungsbericht St. Anna Kinderkrebsforschung | Science Report St. Anna Children’s Cancer Research Institute

A novel fusion gene in treatment-resistant pediatric myeloid neoplasia

Detection of a new fusion gene involving the platelet-deriver growth factor beta (PDGFRB) We have identified a novel fusion gene, NDEL1-PDGFRB, in an 18-month old child diagnosed with juvenile myelomonocytic leukemia (JMML). This is a rare myelodysplastic/myeloproli- ferative neoplasia occurring in young children, characterized by excessive proliferation of monocytic and granulocytic cells infiltrating different organs. Based on cytogenetic analysis of the diagnostic sample, the presence of the translocation t(5;17)(q33;p11.2) raised the possibility of PDGFRB involvement which was subsequently confirmed by FISH analysis. Sequencing of 5’-RACE-PCR products revealed an in-frame fusion between NDEL1 and PDGFRB, where exons 1-5 of NDEL1 encoding the coiled-coil domain were fused to PDGFRB exons 10-22 including the transmembrane (TM) and tyrosine kinase domains (TKD) (Fig.1). The reciprocal fusion transcript PDGFRB-NDEL1 could not be identified by RT-PCR. Analysis of the genomic breakpoints revealed breaks in intron 5 of NDEL1 and intron 9 of PDGFRB. An adequate amount of good quality DNA and RNA was isolated from a dried blood spot obtained at the time of birth (Guthrie card) of the patient, as documented by

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Figure 1. Structure of the NDEL1-PDGFRB fusion gene

successful control gene amplification (ABL1), but tion TKI. The observed development of resistance to the fusion gene was not detectable at the DNA or two different type-II TKIs including imatinib and RNA levels by different nested PCR approaches. nilotinib prompted screening of the entire TKD of PDGFRB for the presence of mutations. Sanger Mutational analysis of the PDGFRB sequencing revealed the missense point mutation tyrosine kinase domain C2550G in the activation loop of the TKD converting In contrast to earlier data on fusion genes the aspartate residue at position 850 into glutamate involving PDGRFB in myeloid malignancies, which (D850E). This mutation was identified in peripheral were generally responsive to treatment with the blood (PB) and bone marrow (BM) specimens tyrosine kinase inhibitor (TKI) imatinib, the patient obtained at the time of both relapses in virtually all experienced relapses of the disease refractory to both cells belonging to the leukemic clone but was imatinib and nilotinib, a more potent second-genera- undetectable in the diagnostic PB or BM samples.

Science Reports [ 52 ] Forschungsbericht St. Anna Kinderkrebsforschung | Science Report St. Anna Children’s Cancer Research Institute

Structural modelling of the PDGFRß tyrosine kinase domain In order to elucidate the structural effects mediated by the D850E mutation in the PDGFRβ TKD, we have generated structure models of the kinase domain both in active (DFG-in) and inactive (DFG-out) conformations, which interact preferentially with type-I and type-II TKIs, respectively (Fig.2). Since the structure of PDGFRβ TKD at the level of atomic resolution is not yet available, we have modelled the kinase domain on the basis of crystallographic structures of closely related homologous proteins including c-KIT, CSF-1R and VEGFR2. All structural models indicated that the observed type-II TKI resistance of cells expressing the D850E mutation in NDEL1-PDGFRβ was conceivably related to stabilization of the A-loop in the active conformation. In this conformation, the DFG triad which is expected to stabilize the A-loop in the serving as a hypomochlion for the A-loop, adopts active conformation. This interaction can be further the so-called DFG-in position. The modelled enhanced by the D850E mutation, because the structure of the inactive DFG-out conformation longer side chain of glutamate in comparison to (Fig.2;orange) revealed the typical auto-inhibitory aspartate brings the negatively charged carboxylic interaction between D850 and the amino acid at the group 1.1 Å closer to the positively charged +3 position, R853, which is commonly observed in histidine. This increases the stability of the A-loop in inactive TKDs of other receptor tyrosine kinases the active conformation (Fig.2), because the forces (RTKs) from the PDGFR family, and is believed to of electrostatic interaction between opposite charges stabilize the A-loop in the inactive conformation increase with the second power of decreasing (Fig.2). However, modelling of the mutant PDGFRβ distance, and become largely ineffective at distances TKD in the inactive conformation could not explain exceeding 4.5 Å. The structural model also suggested the resistance to type-II TKIs and the enhanced that the mutation H657K would have an effect kinase activity addressed below, because the similar to the mutation D850E in terms of stabilizing negatively charged E850 is also able to form a salt the active conformation (Fig.2). The other bridge with the positively charged side chain of interaction involved R853 and E946 in the C-lobe of R853. By contrast, the DFG-in model suggested the the TKD. The +3 position to D850 is one of the least occurrence of two intriguing amino acid interactions conserved positions in the A-loop of RTKs from the upon transition of the A-loop from inactive to the PDGFR family, and the arginine at this position in active state (Fig.2; green). One interaction PDGFRβ (R853) has the longest side chain among implicated the negatively charged D850 and the all members. The DFG-in model suggested that the positively charged, conserved H657 in the αC-helix, positively charged side chain of R853 can reach a

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Figure 2. Protein models of the PDGFRß TKD structure. The modelled DFG-out (orange) and DFG-in (green) conformations of PDGFRß TKD are displayed. The zoom-in windows show the relevant stabilizing electrostatic interactions with the corresponding distances between charges: D850-R853 in the DFG-out model; H657-D850, H657-E850, K657-D850, R853- E946 and H853-E946 in the DFG-in model. Oxygen atoms carrying negative charge are marked in red, nitrogen atoms carrying positive charge in blue. The gray arrow in the center indicates rotation of the A-loop upon transition from inactive to active state.

Science Reports [ 54 ] Forschungsbericht St. Anna Kinderkrebsforschung | Science Report St. Anna Children’s Cancer Research Institute

Assessment of TKI-sensitivity in vitro To assess the oncogenic potential of the newly identified fusion gene, the IL3-dependent murine cell line Ba/F3 was stably transduced with wildtype or mutant NDEL1-PDGFRB constructs by employing a transposon-based system. The in vitro responsiveness of Ba/F3-NDEL1-PDGFRB cells to different TKIs of type-I (dasatinib, midostaurin, pacritinib) and type-II (imatinib, nilotinib, sorafenib) was determined by MTT assays. Cells expressing wildtype NDEL1-PDGFRB were sensitive to all TKIs tested (Table 1). By contrast, proliferation of Ba/F3 cells carrying the D850E mutation in the NDEL1-PDGFRB gene could only be inhibited by the indicated type-I TKIs at sub-micromolar concentrations (Table 1). The observation of TKI resistance apparently induced by the D850E mutation in the kinase domain of PDGFRβ was in contrast to the same amino acid exchange at the corresponding site in PDGFRα (D842E). This finding raised questions regarding important structural differences between the two highly homologous RTKs. While PDGFRβ displays an arginine in the +3 position to the mutation site distance of approximately 2.7 Å to the negatively (R853), PDGFRα has the much shorter and less charged carboxyl group of E946, which may facilitate basic histidine in the corresponding position electrostatic bonds and provide additional (H845). stabilization of the DFG-in It appeared conceivable therefore that interaction conformation of the PDGFRβ TKD. The structural between the side chains of R853 and E946 in the model therefore suggested resistance of mutant PDGFRβ TKD could stabilize the active NDEL1-PDGFRB with the D850E mutation to conformation in the presence of H657K and D850E type-II TKIs (including imatinib and nilotinib), mutations, thus mediating resistance to type-II TKIs. which can only bind to the inactive conformation of To address this notion, the mutation R853H was the PDGFRβ TKD, but indicated sensitivity to type-I introduced into NDEL1-PDGFRB constructs, thus TKIs (e.g. dasatinib, midostaurin) binding to the mimicking the sequence of the A-loop in the active conformation. To address the predictions PDGFRα TKD. In line with the properties of the provided by the protein model, we have introduced D842E mutation in the FIP1L1-PDGFRα fusion, this several mutations affecting the aforementioned change restored the TKI type-II sensitivity of cells interactions, and tested the sensitivity of generated carrying one of the activating mutations, H657K or constructs against a panel of TKIs (Table 1). D850E, in NDEL1-PDGFRβ (Fig.2).

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Table 1. TKI responsiveness of wild-type and mutant NDEL1-PDGFRB genes. (a) Displayed are IC50 values of different TKIs against Ba/F3 cells expressing wild-type (wt) or mutant NDEL1-PDGFRß fusion proteins. The corresponding IC50 values for Ba/F3 expressing FIP1L1-PDGFRß wt and D842E are given for comparison. Red background indicates resistance and green background indicates sensitivity to the TKIs tested.

TKI WT D850E H657K R853H D850E/R853H H657K/R853H Imatinib 80 ± 10 >5000 >5000 50 ± 4 15 ± 3 15 ± 3 Nilotinib 90 ± 10 2200 ± 100 1500 ± 70 40 ± 3 12 ± 3 10 ± 3 Sorafenib 40 ± 5 2100 ± 70 1600 ± 70 30 ± 2 15 ± 2 10 ± 2 Dasatinib 21 ± 3 25 ± 3 22 ± 4 10 ± 1 12 ± 2 10 ± 2 Midostaurin 25 ± 4 13 ± 2 11 ± 3 20 ± 2 15 ± 3 12 ± 3

Our observations provide the first evidence for a major alteration of responsiveness to TKI treatment mediated by an exchange between two negatively charged amino acids in a tyrosine kinase. The Publications: findings not only confirm the important role of R853 Byrgazov K, Kastner R, Gorna M, Hoermann G, Koenig M, in establishing the resistant phenotype of the mutant Lucini CB, Ulreich R, Benesch M, Strenger V, Lackner H, NDEL1-PDGFRB, but also underline the potential of Schwinger W, Sovinz P, Haas OA, van den Heuvel-Eibrink protein modelling for prediction of sensitivity and M, Niemeyer CM, Hantschel O, Valent P, Superti-Furga G, resistance to TKI treatment. The data provide new Urban C, Dworzak MN, Lion T. NDEL1-PDGFRB fusion insights into specific amino acid interactions in gene in a myeloid malignancy with eosinophilia associated mutant RTKs which are of clinical relevance for with resistance to tyrosine kinase inhibitors. Leukemia. improved selection of appropriate TKI treatment. ■ 2017;31(1):237-240

Science Reports [ 56 ] Forschungsbericht St. Anna Kinderkrebsforschung | Sience Report St. Anna Children’s Cancer Research Institute

A novel genetic subtype of leukemia

Acute lymphoblastic leukemia is a heterogeneous disease Acute lymphoblastic leukemia (ALL) is the most common cancer in children and young adults and consists of genetically, biologically and clinically highly heterogeneous disease entities. Owing to the concepts of risk-adapted or targeted therapies, the accurate genetic classification of ALL is becoming increasingly important. The genome-wide comprehensive analysis of ALL has led to the delineation of a rising number of genetic subtypes characterized by aneuploidy, copy number alterations, chromosomal rearrangements, structural variants, and sequence mutations, which in general perturb cellular pathways such as tumor suppression, cell cycle control, kinase and cytokine receptor signaling, epigenetic regulation, and lymphoid development. Regarding lymphopoiesis in B-cell precursor ALL, PAX5 – a transcription factor pivotal for orchestrating B-cell lineage commitment and maintenance – is one of the most frequent targets of somatic mutations, comprising deletions, fusion genes, point mutations, and amplifications. In contrast to PAX5 deletions, which are secondary events, PAX5 fusion genes, certain point mutations, and intragenic amplifications are thought to represent initiating genetic alterations, which promote leukemia development. While we have

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data from 15 study centers, in which more than 5.500 patients had been screened for copy number alterations using a multiplex ligation-dependent probe amplification (MLPA) approach, which, amongst other gene loci, individually targets PAX5 exons 1, 2, 5, 6, 8, and 10, we identified seventy-seven patients with PAX5 amplifications. PAX5 amplifications are commonly associated with other copy number alterations such as loss of CDKN2A/B and IKZF1 as well as gain of EBF1. previously focused on the detection of PAX5 fusion However, apart from P2RY8-CRLF2 fusions, which genes (e.g. Nebral et al. 2007, 2009; Denk et al. 2014) occur only in a minor proportion of the cases and and the biochemical and functional characterization are known to arise either as early or late events of the encoded potentially oncogenic chimeric during leukemogenesis, PAX5 amplifications are proteins (Fortschegger et al. 2014, Schinnerl et al. mutually exclusive of other fusion genes, strongly 2015), recently, PAX5 amplifications caught our suggesting that they indeed represent leukemia- attention. initiating alterations, and in support of this notion they are consistently preserved at relapse. While we first excluded the presence of other rearrangements Leukemia with PAX5 amplification is a rare and by targeted fluorescence in situ hybridization (FISH) high-risk disease entity against a panel of known genes recurrently involved Although sporadic cases with PAX5 in translocations, we then subjected a number of amplifications have been reported earlier, neither samples with PAX5 amplifications to transcriptome their overall incidence nor their prognostic sequencing (RNA-seq) and did not detect any fusion relevance have so far been determined. To answer genes in any single PAX5 amplified patient these questions, together with long-standing (unpublished data). collaboration partners from the United Kingdom, Overall, PAX5 amplifications occur in roughly we have initiated a study within the framework of 1% of pediatric B-cell precursor ALL and the 5-year the international BFM study group. By collecting event-free and overall survival rates of the patients

Science Reports [ 58 ] Forschungsbericht St. Anna Kinderkrebsforschung | Sience Report St. Anna Children’s Cancer Research Institute

Figure 1. Detection of intragenic PAX5 amplifications. Copy number profiles (weighted log2 ratios) determined by CytoScan HD arrays of the PAX5 locus of five patients with PAX5 amplifications showing that only PAX5 exons 2-5 are amplified.

are only 49% and 67%, respectively – clearly may result in the expression of in-frame internally indicating that PAX5 amplified leukemia is a tandem amplified transcripts and this is indeed the high-risk disease. case in patients with amplifications of PAX5 exons 2-5 (unpublished data). The predicted mutated PAX5 protein encoded by these transcripts consists Intragenic PAX5 amplifications represent of multiple tandem repeats of the PAX5 a unique type of mutation DNA-binding domain, the octapeptide motif, Since the employed MLPA approach did not which participates in protein-protein interactions target each of the PAX5 exons, we assessed the actual and the nuclear localization signal, followed by the extent of the amplifications by single nucleotide unaltered PAX5 C-terminal regulatory module polymorphism (SNP) array analysis, which showed being comprised of activating and inhibitory that, with a few exceptions, typically the internal moieties. This protein domain structure indicates PAX5 exons 2-5 are amplified (Figure 1), and that, that mutated internally amplified PAX5 has as determined by MLPA, the copy numbers of these distinctive DNA-binding characteristics and exons range from 4-10 (median 6). The amplified transcriptional regulatory effects. As wild-type exons are located at the original PAX5 locus (Figure murine PAX5 induces chromatin changes by 2), indicating an intragenic tandem amplification, recruiting chromatin-remodeling, histone- representing a unique type of genetic lesion which modifying and basal transcription factor complexes has so far not been described for any other cancer- to its target sites and is a critical epigenetic associated gene. regulator of gene transcription in early B-cell Theoretically, intragenic tandem amplifications development, we hypothesize that internally of certain PAX5 exon combinations or single exons amplified PAX5 may alter the epigenetic landscape

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Figure 2. Fluorescence in situ hybridization (FISH) analysis of PAX5 amplified cases. FISH analysis was conducted using PAX5 exon-specific cosmids cos-hPAX5-1 (spanning exons 2-5; red signals) and cos-hPAX5-3 (span- ning exons 9-10; green signals). (A) Metaphase showing that the extra PAX5 copies are located on the short arm of chromosome 9 at the PAX5 locus and (B-C) interphase nuclei showing a clus- tering of PAX5 exons 2-5-specific probe signals. Arrowheads indicate the amplified allele.

and interfere with proper B-lymphopoiesis or other key cell signaling pathways. To verify this notion and to assess the regulatory properties of internally tandem amplified PAX5, we are currently generating model cell lines expressing the mutated protein and intend to conduct ChIP-, ATAC- and RNA-sequencing. Our preliminary data show that the mutated PAX5 protein via its multiple DNA-binding domains binds to roughly twice as many target loci as compared to wild-type PAX5 and that both common and distinct sites are bound. However, Publications: Schwab C, Nebral K, Chilton L, Leschi C, consistent with the finding that in mice the Waanders E, Boer JM, Zaliova M, Sutton R, Öfverholm II, transcriptional regulation of only a fraction of the Ohki K, Yamashita Y, Groeneveld-Krentz S, Fronkova E, genes targeted by wild-type PAX5 is in fact Bakkus M, Tchinda J, Barbosa TDC, Fazio G, Mlynarski W, PAX5-dependent, only a minor subset of the genes Pastorczak A, Cazzaniga G, Pombo-de-Oliveira MS, Trka J, bound by the mutated protein undergoes Kirschner-Schwabe R, Imamura T, Barbany G, Stanulla M, transcriptional changes. Since – as expected – Attarbaschi A, Panzer-Grümayer R, Kuiper RP, den Boer binding alone is not sufficient to drive ML, Cave H, Moorman AV, Harrison CJ, Strehl S. Intragenic transcription, we now aim to elucidate the amplification of PAX5: a novel subgroup in B-cell precursor genome-wide impact on chromatin modifications acute lymphoblastic leukemia? Blood Adv. 2017 elicited by the internally amplified PAX5 protein. ■ Aug 14;1(19):1473-1477.

Science Reports [ 60 ] Forschungsbericht St. Anna Kinderkrebsforschung | Science Report St. Anna Children’s Cancer Research Institute

Epigenetic diversity in Ewing sarcoma

scientists from Austria, France, Germany and Spain led by Eleni Tomazou from the St. Anna Children’s Cancer Research Institute (CCRI) profiled many Ewing tumors. They found that the disease’s clinical diversity is reflected by epigenetic heterogeneity. Using novel bioinformatic methods, the team Cancer is caused by genetic programs running studied the tumors’ DNA methylation patterns – astray. Tumors of the elderly carry many DNA one of the most important facets of the human mutations – but much fewer genetic variants exist epigenome. Ewing sarcoma showed unique in childhood cancers, leaving their clinical diversity characteristics that differ markedly from others unexplained. This conundrum has been addressed cancers, and the DNA methylation patterns also for Ewing sarcoma, one of the most aggressive varied between patients. Moreover, the researchers childhood cancers. found that Ewing sarcoma tumors appear to retain Tumors of the elderly, such as breast cancer and part of the characteristic DNA methylation patterns colon cancer, accumulate thousands of DNA of their cell-of-origin. mutations. These genetic defects contribute to can- Thus, the diverse clinical courses observed cer-specific properties such as uncontrolled growth, among Ewing sarcoma patients may be explained invasion in neighboring tissues, and evasion from epigenetically: As DNA methylation influences gene the immune system. Similar properties are also activity, the combination of Ewing sarcoma specific found in childhood cancers, although those tumors and cell-of-origin specific patterns can lead to carry much fewer genetic defects, making it difficult different outcomes. The epigenetic diversity also to explain their clinical heterogeneity. appears to correlate with the tumors’ aggressiveness This is true for Ewing sarcoma, a very aggressive and metastatic state. bone cancer in children and adolescents. A single These insights into the biology of Ewing sarcoma genetic defect – the EWS-ETS fusion – is present in provide the basis for developing epigenetic all tumors, initiating cancer development and biomarkers that can reliably predict disease course defining Ewing sarcoma as a disease. But there are and therapy response. After two decades of very few DNA mutations that could cause the stagnation in the therapy for patients with Ewing observed difference in the disease course of Ewing sarcoma, new impulses for personalized treatment of sarcoma patients. Tackling this question, a team of this difficult to treat disease are needed.

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Figure 1. DNA methylation profiling reveals a characteristic epigeno- mic signature of Ewing sarcoma: Epigenetic heterogeneity in Ewing sarcoma (EwS) analyzed at three levels: between cancer types (inter-cancer), between EwS tumors (inter-individual), and within EwS tumors (intra-tumor). Adapted from Sheffield et al., Nature Medicine 2017.

These findings in Ewing sarcoma also provide an interesting concept for other cancers with low genetic complexity. In the era of precision medicine, understanding the causes and consequences of tumor heterogeneity will be crucial to develop personalized therapies. Indeed, the group led by CCRI PI Eleni Tomazou Publications: Sheffield NC, Pierron G, Klughammer J, continues to study epigenetic heterogeniety and its Datlinger P, Schönegger A, Schuster M, Hadler J, Guillemot impact on tumor developement, evolution and D, Lapouble E, Freneaux P, Champigneulle J, Bouvier R, resistance to therapy. This work is expected to Walder D, Ambros IM, Hutter C, Sorz E, Amaral AT, Álava establish an epigenome-based precision medicine Ed, Schallmoser K, Strunk D, Rinner B, Liegl-Atzwanger B, program for pediatric sarcomas. Huppertz B, Leithner A, Pinieux Gd, Terrier P, Laurence V, Funding: The Austrian National Bank (OeNB), a Michon J, Ladenstein R, Holter W, Windhager R, Dirksen charitable donation from Kapsch Group (www. U, Ambros PF, Delattre O, Kovar H, Bock C*, Tomazou kapsch.net/ kapschgroup), the Austrian Science EM* DNA methylation heterogeneity defines a disease Fund (FWF), the Human Frontier Science Program spectrum in Ewing sarcoma. Nature Medicine. 2017 and the Austrian Academy of Sciences supported Mar;23(3):386-395; DOI:10.1038/nm.4273 this study. ■ *corresponding author

Science Reports All reports of this non-commercial publication describe achievements published in high-ranking scientific journals, as indicated by the respective citations. Most of the figures used in this report are taken from the publications and in these cases copyright belongs to the respective journals. Science Reports 2018 [ 64 ] Forschungsbericht St. Anna Kinderkrebsforschung | Science Report St. Anna Children’s Cancer Research Institute

Understanding tumor heterogeneity and relapse in neuroblastoma patients to allow adequate treatment options

Neuroblastoma is a pediatric solid tumor of the sympatho-adrenal lineage and accounts for 15% of all cancer-related deaths in children. The disease is characterized by tremendous clinical heterogeneity ranging from spontaneous regression and differenti- ation towards highly aggressive metastatic disease (Ambros et al., 1996, recent review e.g. Matthay et al., 2016). Drivers of biological diversity are inter- and intratumoral genetic heterogeneity, and clonal evolution as well as tumor cell plasticity (Bogen et al., 2016, Berbegall et al., 2018, Abbasi et al., 2018). Despite intensive multi-modal treatment, the 5-year overall survival of stage M (metastatic) patients is still below 50% due to therapy refractory or relapsing disease (Ladenstein et al., 2017). In pediatric oncology and especially in neuroblastoma clinical trials, genomic aberrations are used as decision support to allow the most adequate therapeutic procedure. Existing molecular classifiers stem from single biopsies of bulky tumors, but this simplified view of neuroblastoma

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homogeneity is inappropriate. Intratumor Figure 1. heterogeneity must be taken into account for Segmental chromosomal prognostic/predictive biomarker identification. aberrations detected by single Applying cutting-edge omics and liquid biopsy nucleotide polymorphism technologies helped to elucidate different aspects of (SNP) array analysis in intratumor heterogeneity, both spatially and hetMNA neuroblastoma temporally, including intratumor, primary tumor/ samples. Samples are sorted distant metastasis and diagnosis/relapse according to patient age at comparisons. diagnosis and the red dashed line marks the 18-month age Spatial and temporal genomic heterogeneity cutoff. Horizontal lines depict and relapse identification larger chromosomal Genomic data on tumor and corresponding bone aberrations and vertical lines marrow samples from 155 neuroblastoma patients amplifications or smaller revealed aberrations unique to certain tissues and deletions (red indicate losses time points besides concordant aberrations shared and blue indicate gains or amplifications e.g. MYCN amplification on chromosome 2p black line).

Science Reports [ 66 ] Forschungsbericht St. Anna Kinderkrebsforschung | Science Report St. Anna Children’s Cancer Research Institute

by all samples. The relatively high frequency of deletions in 1q and 19q at relapse and their higher Figure 2. frequency in disseminated tumor cells at both time Hypothetical model of proposed tumor points as compared to corresponding tumor samples evolution of hetMNA tumors. This model support the hypothesis of a branched clonal is based on the comparison of clinical and evolution and a parallel progression of primary and biological parameters of hetMNA metastatic tumor cells. The different frequency of neuroblastoma tumors and homogenously these aberrations in tumor samples and MYCN amplified (homMNA) tumors with disseminated tumor cells, combined with their evaluable SNP array data. Green cells strong prognostic/predictive power indicate the depict favorable MYCN non-amplified relevance of analyzing diagnostic disseminated tumor cells in tumors of <18m patients tumor cells. Therefore, searching for biomarkers to whereas blue cells represent non-MNA identify the relapse-seeding clone should involve NB cells with a highly rearranged genome diagnostic disseminated tumor cells alongside the and a multitude of segmental tumor tissue (Abbasi et al. 2017). chromosomal aberrations (SCAs) in tumors of older patients. Red illustrates MNA cells. Arrows depict a possible Prognostic power of heterogeneous transition of aneuploid hetMNA tumors MYCN amplification into aneuploid homMNA tumors which The presence of super-numerical copies of the can be suspected based on similarities in MYCN oncogene (MYCN amplification) is the the genetic background. The fact that strongest predictor for an aggressive tumor course in most aneuploid homMNA tumors were neuroblastoma patients. Thus, the information on found in >18m patients would suggest a the presence or absence of MYCN amplification time-dependent clonal takeover of (MNA) is integral for different clinical studies as homMNA cells. decisive element to enable ‘personalized treatment’ options. However, in case this aberration is present only in a sub-population of tumor cells, the clinical/ biological impact had so far not been known. To therapy. For these tumors, we hypothesize the lack of elucidate this enigma, we analyzed the genomic full-blown malignancy of the MYCN-amplified clone background of MYCN heterogeneously amplified at the time of diagnosis. In most patients over 18 (hetMNA) tumors by a combination of a single cell months, on the other hand, MYCN-amplification analysis technique and pan genomic analysis. The evolved in a genetically already highly malignant integration of genomic and clinical data of 99 tumor background, which possibly stifled a clonal neuroblastoma patients led to new insights into outgrowth. Thereby, we could provide clear these diagnostically challenging tumors. Infants/ definitions concerning the biologic/prognostic toddlers with heterogeneous MYCN amplified implications of age and genomic background (Bogen tumors, especially when developing in a favorable et al 2016, Berbegall et al. 2018). These data will genetic background, had an excellent outcome - in allow a more precise classification of these patients localized, resectable stages even without cytotoxic into genetically defined treatment groups. ■

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Publications: Berbegall AP, Bogen D, Pötschger U, Beiske Abbasi MR, Rifatbegovic F, Brunner C, Mann G, Ziegler A, K, Bown N, Combaret V, Defferrari R, Jeison M, Mazzocco Pötschger U, Crazzolara R, Ussowicz M, Benesch M, K, Varesio L, Vicha A, Ash S, Castel V, Coze C, Ladenstein Ebetsberger-Dachs G, Chan GCF, Jones N, Ladenstein R, R, Owens C, Papadakis V, Ruud E, Amann G, Sementa AR, Ambros IM, Ambros PF. Impact of Disseminated Navarro S, Ambros PF, Noguera R, Ambros IM. Neuroblastoma Cells on the Identification of the Relapse- Heterogeneous MYCN amplification in neuroblastoma: a Seeding Clone. Clin Cancer Res. 2017 Aug 1;23(15): SIOP Europe Neuroblastoma Study. Br J Cancer. 2018 May; 4224-4232. doi: 10.1158/1078-0432.CCR-16-2082. 118(11):1502-1512. doi: 10.1038/s41416-018-0098-6. https://www.nature.com/articles/s41416-018-0098-6

Science Reports [ 68 ] Forschungsbericht St. Anna Kinderkrebsforschung | Science Report St. Anna Children’s Cancer Research Institute

Investigating bone marrow dissemination in metastatic neuroblastoma

The bone marrow is the most frequent site of metastasis in neuroblastoma - more than 95% of stage M neuroblastoma patients present with disseminated tumor cells (DTCs) in the bone marrow at diagnosis (Ambros et al., 2003, Figure 1). Bone marrow is a frequent homing organ for DTCs in many cancer types. It can serve as a metastatic niche for DTCs, promoting tumor cell survival and finally enabling disease recurrence after treatment. Thus, the presence of DTCs is considered as an important prognostic marker for poor outcome in patients with breast cancer, Ewing sarcoma and neuroblastoma.

Disease monitoring and early relapse detection Detection of DTCs by automatic immunofluores- cence imaging is highly sensitive (1 tumor cell in 1 Mio. bone marrow mononuclear cells) and state-of- the-art to evaluate treatment response and bone marrow minimal residual disease (MRD) in current neuroblastoma clinical trials (SIOPEN HR-NBL-1). Monitoring MRD is a reliable indicator for therapy response, and is now established as an integral part of treatment decision-making for children with acute lymphoblastic leukemia. Easy access to the tumor site (blood and bone marrow) in leukemia patients has driven this development more quickly, but it is just as

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well applicable for patients with solid tumors and disseminated/metastasized disease. The dynamics of bone marrow clearance can be used to precisely identify patients responding to treatment at an early time point. We monitor therapy response by detecting DTCs in bone marrow samples taken at several time points during treatment. The point at which DTCs are cleared from the bone marrow niche can function as a predictor for therapy response. Together with molecular profiling of the primary tumor and Figure 1. circulating biomarkers in liquid biopsies (Figure 1), In stage M (metastatic) our investigations are geared toward developing neuroblastoma tumor cells diagnostics to guide personalized decision making for from the primary site therapy selection and alterations in treatment. disseminate into the bone marrow in more than 90% of patients. While accessing the Tumor cell plasticity - adaptations upon primary tumor in order to dissemination and disease progression obtain biopsies is an invasive Although recent genomic and transcriptomic procedure, bone marrow studies have advanced our understanding of the aspiration is less invasive and frequently done to monitor therapy response and detect relapse early.

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transcripts that are encoded by this small genome. A numerical increase in mitochondria is a characteristic feature of various cancers, and it is well known that mitochondria can be actively taken up by tumor cells and impart considerable flexibility for cancer cells in harsh environments, such as the hypoxic bone marrow microenvironment. Surprisingly, despite accumulation of genomic aberrations in relapsing tumors and DTCs, the transcriptomic landscape of relapse DTCs largely resembled those of diagnostic DTCs with 113 disease, they have mainly focused on the primary differentially expressed genes. This is particularly tumor. However, DTCs are frequently refractory to remarkable, as these patients have experienced therapeutic interventions and/or cause disease mulit-modal treatment regimens including relapse in the bone marrow. In our recent studies, we high-dose combination cytotoxic and radiation have shown that bone marrow derived DTCs are therapy, suggesting that the protective bone marrow suitable for genomic and transcriptomic analyses niche has aided to retain DTC identity. Notably, (Abbasi et al., 2015, Rifatbegovic et al., 2015). relapse DTCs showed a positional enrichment of 31 Furthermore, it has been demonstrated that the down-regulated genes on chromosome 19, including relapse-seeding clone can often be identified already the tumor suppressor genes PUMA and CADM4, at the time point of diagnosis in DTCs rather than which were associated with an unfavorable the primary tumor (Abbasi et al., 2017). However, event-free survival (Figure 2). little is known about how tumor cells adapt to the Our study highlights the importance of studying bone marrow metastatic niche upon dissemination bone marrow metastases as they substantially differ and tumor progression. Therefore we aimed to from tumors in their expression programs, most characterize the transcriptomic landscape of DTCs likely as DTCs adapt to the microenvironment in the at diagnosis and relapse. metastatic niche, which might be influenced by vas- Diagnostic (n=22) and relapse (n=20) DTCs of cularization, hypoxia and other cellular and stage M neuroblastomas were enriched by extracellular components (Rifatbegovic et al., 2018). MACS-sorting using a tumor-specific marker, GD2, Based on novel single cell transcriptomics and and their mRNA was sequenced along with primary multiplex imaging technologies we currently attempt tumor biopsies sampled at diagnosis (n=16). We to obtain even deeper insights into DTC plasticity found that genes involved in metastasis initiation and the bone marrow microenvironment and how and angiogenesis were upregulated in the primary especially the latter is affected by therapeutic tumors in contrast to DTCs and bone marrow intervention. ■ mononuclear cells, which both reside in the highly vascularized bone marrow. Besides the downregulation of these genes, DTCs were particularly characterized by an increased amount of mitochondrial DNA and the corresponding

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Figure 2. Integration of genome-wide mRNA-se- quencing and DNA analysis reveals positional enrichment of genes encoded by chromosome 19. (a) Volcano plot of log2FC versus p-values comparing gene expression of diagnostic and relapse DTCs. A selection of genes which are upregulated in relapse DTCs and which are encoded by chromosome 19 is highlighted. (b) Ideogram showing the genomic location of 31 differentially expressed genes encoded by chromosome 19. Significantly enriched chromosomal regions (p < 0.01) are shown as red segments above the chromosome. Clonal deletions of particular genes are highlighted with solid circles in the heat map. Sub-clonal deletions are labeled with Publications: Rifatbegovic F, Frech C, Abbasi MR, non-solid circles. Names of tumor Taschner-Mandl S, Weiss T, Schmidt WM, Schmidt I, suppressor genes are highlighted (adapted Ladenstein R, Ambros IM, Ambros PF: Neuroblastoma cells from Rifatbegovic et al., 2018). undergo transcriptomic alterations upon dissemination into the bone marrow and subsequent tumor progression. International journal of cancer 2018, 142:297-307.

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Establishing diagnoses in children with rare hematological cytopenias via modern genomic approaches

Hematological cytopenias – a very heterogenous group of diseases Hematological cytopenias, deficiencies of one or several blood cell components, are the shared hallmark of an extremely heterogeneous group of either environmentally or genetically triggered diseases. The identification of the underlying genetic etiology of such diseases is particularly important in children in whom inborn disorders predominate. The spectrum of inborn disorders comprises for example red blood cell membrano- and enzymopathies, hemoglobinopathies, bone marrow failure syndromes, familial haemophago- cytic lymphohistiocytosis, mitochondriopathies, autoimmune diseases and primary immunodefici- encies. Of note, some of these disorders are associated with an increased risk of development of malignancies and except for hemoglobinopathies and hereditary spherocytosis (HS), all of these inborn diseases are orphan or even ultra-orphan diseases.

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Figure 1. Examples of targeted hematology disease groups/genes.

Correct disgnosis in patients with hematological establishing appropriate screening programs, or to cytopenias can be challenging counsel patients and parents on the pattern of Clinical features (e.g. jaundice, pallor, bruises, inheritance. Moreover, in rather ‘easy to diagnose’ skeletal and cardiac anomalies, splenomegaly, etc.) diseases like HS, elucidation of genotype/phenotype and standard haematological diagnostics like correlations may help to better predict the course of quantitative and morphological analyses of the disease. However, both diversity and phenotypic peripheral blood cells and their precursors in the overlap of distinct disease entities may make the bone marrow and assessment of parameters in the identification of underlying genetic etiologies by serum (e.g. bilirubin, haptoglobin, lactate-dehydro- classical Sanger sequencing challenging. genase, humoral immune analyses, etc.) coupled with specific diagnostic tests (e.g. eosine maleamid test, diethoxybutane test, telomere length Next generation sequencing measurement, elastase analysis in stool, based diagnostics haemoglobin analyses, etc.) can help to establish a In order to overcome shortcomings of above- diagnosis in more common diseases. However, mentioned diagnostics, Leo Kager and Kaan Boztug genetics plays an increasing role not only to along with colleagues developed and implemented a diagnose rare diseases, but also to better characterize systematic next generation sequencing (NGS)-based more common diseases. custom-designed targeted enrichment panel Establishing a precise genetic diagnosis is essential (HaloPlex™/Agilent Technologies) targeting 292 for several reasons including choosing the appropriate candidate genes (Figure 1) and screened 38 treatment, avoidance of secondary organ damage, consecutive children and young adults (17 males,

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Figure 2. Macrothrombycytes (left) and myosin inclusion bodies in granulocytes (right) in an infant who was diagnosed to have MYH9-related disease by demonstrating a known pathogenic mutation in MYH9, NM_002473.4:c.287C>T.

21 females with a median age of 7.5 years, range erythropoietic anemia type 1 (CDA1, n=2), 0.1 to 21.4 years) with a diagnosis of chronic RUNX1-related thrombocytopenia (n=1), haematological cytopenia for disease-associated GATA2-related neutropenia (n=1), myosin heavy mutations. chain 9 related macrothrombocytopenia Collectively, we identified a causative genetic (MYH9-RD, n=1) (Figure 2) and NHEJ1-related defect in 17/38 patients (44.7%) including novel combined immunodeficiency (n=1). mutations in 13 of them. In 8/17 (47%) patients the Besides extension of genotypes, we also observed results of molecular testing were considered as phenotype extensions. For example, we identified confirmatory and diagnostic in 9/17 (53%). Novel the first patient with myelodysplasia and NHEJ1 disease causing variants were identified in the deficiency. The patient presented at the age of 5 years ankyrin 1 gene (ANK1, n=3), the spectrin beta, in 2001 with refractory cytopenia of childhood erythrocytic gene (SPTB, N=2), in the piezo-type (RCC) with monosomy 7. Interestingly, mechanosensitive ion channel component 1 gene pancytopenia improved, and the monosomy 7 clone (PIEZO1, n=1), in the codanin 1 gene (CDAN1, was replaced by a deletion 20q clone. At the age of n=2), in the ribosomal protein genes S29 (RPS29, 21 years, the molecular genetic diagnosis was n=1) and L5 (RPL5, n=1), in the non-homologous established; i.e., a novel homozygous mutation in end joining factor 1 gene (NHEJ1, n=1), in the NHEJ1, NM_024782.2:c.236T>C, p.L79P was found. Fanconi anemia complementation group A gene Immunologic testing revealed profound CD19 (FANCA, n=1), and in the runt related transcription cytopenia and IgA deficiency. He has shortened factor 1 gene (RUNX1, n=1). telomeres, which is in-line with premature aging of HSCs. He is the second patient with NHEJ1 deficiency who survived without HSCT into Diagnosis of uncommon and unexpected diseases adulthood. This approach enabled us to include patients Moreover, we found compound heterozygous with diverse hematologic conditions and thereby to mutations in CDAN1 in a male patient discover uncommon and unexpected diseases, such (NM_138477.2: c.2015C>T, p.P672L (paternal, as hereditary xerocytosis (HX, n=2), congenital dys- novel); c.1189C>T, p.R397W (maternal, known)),

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Figure 3. Genotype-phenotype extension in a patient with congenital dyserypoietic anemia type 1 (CDA1). Smears of peripheral blood display severe anisocytosis (left above) and smears of bone marrow display marked dysery- thropoiesis with internuclear bridges between erythroblasts (left below). Magnetic resonance imaging shows thickening of the skull due to increased erythropoiesis, Arnold Chiari malformation type 1 (ACM1) and syringomyely (right above). Right below the skeletal defect who was referred at the age of 18 years with mild clinodactyly is depicted. macrocytic CHA. He received erythrocyte transfusions as infant and had undergone cardiac surgery because of partial anomalous pulmonary venous drainage (PAPVD) and atrial septal defect type II. Since the age of 16 years he suffered from evaluation of childhood cytopenias, their prevalent severe headache. Via MRI we diagnosed genetic origin, the plethora of potentially involved Arnold-Chiari malformation type 1 (ACM 1) with genes as well as heterogeneous types and syringomyelia as well as marked extramedullary distribution of mutations, together with the erythropoiesis of the skull (Figure 3). After surgical reassuring results presented herein strongly argue therapy of ACM1, the patient’s headache improved. for the implementation of such targeted sequencing He had typical findings in peripheral blood (i.e., an- screening programs already in the initial diagnostic isopoikilocytosis with some basophilic stippled work-up of such diseases. ■ RBCs) and bone marrow smears (i.e., chromatin- bridges between erythroblasts nuclei) and clinodactyly (Figure 3). He has yet no iron overload Publications: Kager L, Jimenez Heredia R, Hirschmugl T, documented. After establishing the diagnosis CDA1, Dmytrus J, Krolo A, Müller H, Bock C, Zeitlhofer P, Dworz- he was offered interferon-α therapy. He is the first ak M, Mann G, Holter W, Haas O, Boztug K. Targeted patient with CDA1 who had been observed to have mutation screening of 292 candidate genes in 38 children ACM1 and PAPVD. with inborn haematological cytopenias efficiently identifies Considering the inherent difficulties one often novel disease-causing mutations. Br J Haematol. experiences with the conventional diagnostic 2018 Jul;182(2):251-258.

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No evidence that addition of subcutaneous IL-2 to immunotherapy with dinutuximab beta, given as an 8 h infusion, improved outcomes in patients with high-risk neuroblastoma, hence, dinutuximab beta alone should thus be considered the standard of care

Background achieved a disease response that fulfilled prespecified Immunotherapy with the chimeric anti-GD2 criteria, had received high-dose therapy (busulfan monoclonal antibody dinutuximab, combined with and melphalan or carboplatin, etoposide, and alternating granulocyte-macrophage colony- melphalan) and had received radiotherapy to the stimulating factor and intravenous interleukin-2 primary tumour site. In this component of the trial, (IL-2), improves survival in patients with high-risk patients were randomly assigned (1:1) to receive neuroblastoma. We aimed to assess event-free dinutuximab beta (20 mg/m² per day as an 8 h survival after treatment with ch14.18/CHO infusion for 5 consecutive days) or dinutuximab beta (dinutuximab beta) and subcutaneous IL-2, plus subcutaneous IL-2 (6 × 10⁶ IU/m² per day on compared with dinutuximab beta alone in children days 1–5 and days 8–12 of each cycle) with the and young people with high-risk neuroblastoma. minimisation method to balance randomisation for national groups and type of high-dose therapy. All Methods participants received oral isotretinoin (160 mg/m² We did an international, open-label, phase 3, per day for 2 weeks) before the first immunotherapy randomised, controlled trial in patients with cycle and after each immunotherapy cycle, for six high-risk neuroblastoma at 104 institutions in 12 cycles. The primary endpoint was 3-year event-free countries. Eligible patients were aged 1–20 years and survival, analysed by intention to treat. This trial was had MYCN-amplified neuroblastoma with stages 2, registered with ClinicalTrials.gov, number 3, or 4S, or stage 4 neuroblastoma of any MYCN NCT01704716, and EudraCT, number 2006-001489- status, according to the International Neuroblastoma 17, and recruitment to this randomisation is closed. Staging System. Patients were eligible if they had been enrolled at diagnosis in the HR-NBL1/SIOPEN Findings trial, had completed the multidrug induction Between Oct 22, 2009, and Aug 12, 2013, 422 regimen (cisplatin, carboplatin, cyclophosphamide, patients were eligible to participate in the vincristine, and etoposide, with or without immunotherapy randomisation, of whom 406 (96%) topotecan, vincristine, and doxorubicin), had were randomly assigned to a treatment group (n=200

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Publications: Ladenstein R, Pötschger U, Valteau-Couanet D, Luksch R, Castel V, Yaniv I, Laureys G, Brock P, Michon JM, Owens C, Trahair T, Chan Number GCF, Ruud E, Schroeder H, Beck at risk (number Popovic M, Schreier G, Loibner H, censored) Ambros P, Holmes K, Castellani MR, CH14.18/CHO CH14.18/CHO+IL-2 Gaze MN, Garaventa A, Pearson ADJ, Lode HN. Interleukin 2 with anti-GD2 antibody ch14.18/CHO (dinutuximab beta) in patients with high-risk to dinutuximab beta and n=206 to dinutuximab beta neuroblastoma (HR-NBL1/SIOPEN): a with subcutaneous IL-2). Median follow-up was 4·7 multicentre, randomised, phase 3 trial. years (IQR 3·9–5·3). Because of toxicity, 117 (62%) of Lancet Oncol. 2018 Nov 12. 188 patients assigned to dinutuximab beta and pii: S1470-2045(18)30578-3. subcutaneous IL-2 received their allocated treatment, by contrast with 160 (87%) of 183 patients who received dinutuximab beta alone (p<0·0001). 3-year of 191), immunotherapy-related pain (19 [16%] of event-free survival was 56% (95% CI 49–63) with 122 vs 32 [26%] of 124), and impaired general dinutuximab beta (83 patients had an event) and 60% condition (30 [16%] of 185 vs 78 [41%] of 192). (53–66) with dinutuximab beta and subcutaneous IL-2 (80 patients had an event; p=0·76). Four patients Interpretation died of toxicity (n=2 in each group); one patient in There is no evidence that addition of each group while receiving immunotherapy (n=1 subcutaneous IL-2 to immunotherapy with congestive heart failure and pulmonary hypertension dinutuximab beta, given as an 8 h infusion, due to capillary leak syndrome; n=1 infection-related improved outcomes in patients with high-risk acute respiratory distress syndrome), and one patient neuroblastoma who had responded to standard in each group after five cycles of immunotherapy induction and consolidation treatment. (n=1 fungal infection and multi-organ failure; n=1 Subcutaneous IL-2 with dinutuximab beta was pulmonary fibrosis). The most common grade 3–4 associated with greater toxicity than dinutuximab adverse events were hypersensitivity reactions (19 beta alone. Dinutuximab beta and isotretinoin [10%] of 185 patients in the dinutuximab beta group without subcutaneous IL-2 should thus be vs 39 [20%] of 191 patients in the dinutuximab plus considered the standard of care until results of subcutaneous IL-2 group), capillary leak (five [4%] of ongoing randomised trials using a modified 119 vs 19 [15%] of 125), fever (25 [14%] of 185 vs 76 schedule of dinutuximab beta and subcutaneous [40%] of 190), infection (47 [25%] of 185 vs 64 [33%] IL-2 are available. ■

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Path to overcoming the threat of adenovirus infections in pediatric stem cell transplant recipients

Clinical relevance of adenovirus infections Human adenovirus (HAdV) infections are associated with life-threatening complications in immunocompromised individuals including particularly children undergoing human allogeneic stem cell transplantation (HSCT).1-2 In this setting, viral reactivation is reportedly more prevalent than de-novo infection. Primary infections in immuno- competent individuals occur most commonly in early childhood, often without any clinical symptoms, but HAdV can subsequently persist in tonsillar and adenoidal lymphocytes as well as in the gastrointestinal (GI) tract.3 The role of the GI-tract as a sanctuary for HAdV persistence has been discussed for decades, because shedding of the virus into stool was found in immunocompetent individuals even without any evidence of disease. In HSCT recipients, increasing HAdV loads in serial stool specimens have been documented during the post-transplant period prior to invasive infection, supporting the notion of intestinal virus persistence

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and reactivation.1-2 Our recent study in immuno- competent pediatric patients revealed HAdV persistence in mucosal lymphoid cells in the GI-tract, with particularly high prevalence in the ileum, whereas in HSCT recipients with HAdV reactivation post-transplant, an enormous virus density has been documented in intestinal epithelial cells.3 These observations revealed that intestinal lymphocytes apparently serve as a major site of HAdV persistence, but epithelial cells in the GI-tract HAdV DNA copy numbers exceeding defined are exploited for massive virus production.3 The threshold levels in serial stool samples herald highly productive virus replication in epithelial cells invasive infection days to weeks before the virus explains the rapidly expanding virus numbers in becomes detectable in PB. The monitoring of HAdV stool specimens of HSCT recipients prior to the DNA copy numbers in stool 5-8 could therefore serve onset of viremia and disseminated disease.1-2 as a basis for timely initiation of pre-emptive According to the current ECIL (European therapy, in attempts to prevent invasive infection. Conference of Infections in Leukemia) guidelines, Disseminated HAdV-mediated disease is associated peripheral blood (PB) samples are recommended as with dismal outcome, partly because delayed onset the primary material for HAdV screening in HSCT of therapy by current treatment options often fails to recipients.4 There is, however, increasing evidence control adenovirus-related complications.2 Since reported by different centers that rapidly increasing HAdV persistence in the GI-tract of immunocompe- tent children and post-transplant reactivation of the virus in pediatric patients were shown to occur at similar frequencies, we hypothesized that detection of HAdV shedding into stool before HSCT may provide an early marker for the risk of viremia and HAdV-related disease post-transplant. To address this notion, a study involving a large cohort of pediatric HSCT recipients was performed.

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Figure 1. Median values of HAdV DNA copies/g stool from patients shedding HAdV before HSCT (light grey bars) and from patients with first detection of HAdV positivity after transplantation (dark grey bars) are displayed. The virus copies were analyzed by RQ-PCR from stool specimens collected until day +56 post-transplant. The critical threshold for the risk of invasive infection at 1E + 06 HAdV DNA copies/g stool is indicated by the bold horizontal line, and the pertinent numbers of patient samples for each individual time point are indicated above the bars.

The impact of intestinal adenovirus shedding prior to transplantation Molecular HAdV monitoring of serial stool samples using RQ-PCR was performed in 304 children undergoing allogeneic HSCT. Analysis of stool and peripheral blood specimens was performed pre-transplant and at short intervals until day 100 post-HSCT. The virus was detected in the stool of 129 patients (42%), and 42 (14%) tested positive already before HSCT. The patients displaying HAdV shedding pre-transplant showed a significantly earlier increase of intestinal HAdV levels above the critical threshold associated with high risk of invasive infection (p<0.01). In this subset of patients, the occurrence of invasive infection characterized by viremia was significantly

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higher than in patients without HAdV shedding before HSCT (33% vs 7%; p<0.0001). The data demonstrate that intestinal HAdV shedding before HSCT confers a greatly increased risk for invasive infection and disseminated disease post-transplant, and highlights the need for timely HAdV monitoring and pre-emptive therapeutic considerations in HSCT recipients. The observation of low median virus copy numbers in stool before transplantation (Figure 1) might reflect a persistent state of intestinal virus infection associated with low-level viral leakage into the stool, as indicated in our recent study. 3 Intestinal HAdV shedding before transplantation documented in the present study, which in most instances was evident prior to conditioning, further supports the notion that presence of the virus in the in the GI-tract appears to be in the state of persistence rather than latency. In patients with inborn or acquired severe immuno- deficiency, intestinal shedding of the virus not be generally feasible in this clinical setting. pre-transplant might reflect an early onset of However, molecular detection of HAdV shedding reactivation. In individuals with most other into the stool pre-transplant is an easily applicable underlying diseases providing an indication for non-invasive approach facilitating the identification HSCT, virus reactivation is apparently triggered by of a subset of patients with persistence of the virus immunosuppression during the post-transplant in the GI-tract carrying an elevated risk of systemic period. Based on the present data, intestinal HAdV infection and disseminated disease. This persistence associated with virus shedding into the observation corroborates earlier findings in smaller stool prior to transplantation represents a risk factor cohorts for ensuing viremia which can be readily exploited indicating that pediatric patients without detectable in routine diagnostic monitoring in pertinent patient HAdV in stool apparently have a very low risk of cohorts. developing invasive infection in the clinical setting of HCST.3 Conversely, in line with earlier data, the risk of viremia is significantly increased in patients Diagnostic and therapeutic consequences testing positive for the virus in stool (Figure 2). It is conceivable that patients with persistent Children with documented intestinal HAdV HAdV infection in the GI-tract represent the most shedding prior to HSCT displayed particularly important group at risk of reactivating the virus rapid viral expansion in serial stool samples post-transplant, but the identification of viral exceeding 10E6 copies/g, the critical threshold for persistence would require the analysis of intestinal the probability of invasive infection, already within biopsy material obtained by endoscopy, which may 21 days post-transplant (Figure 1).

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Figure 2. Correlation between the time of first HAdV detection in stool and invasive infection post HSCT. The time intervals along the x-axis refer to the onset of first HAdV positivity in stool. The first HAdV detection in stool specimens (light grey columns) is displayed in relation to the corresponding frequency of viremia post-transplant (dark grey columns). The asterisks indicate the respective time spans between first detection of HAdV in stool and viremia, considering HAdV positivity in stool up to two weeks before HSCT: *median 35 days (range 8–92 days); **median 15 days (range 0–77 days). HSCT: hematopoietic stem cell transplantation.

These observations suggest that pre-transplant detection of HAdV in stool correlates with an elevated risk of early invasive infection, thus highlighting the importance of timely viral screening in stool specimens. Moreover, the presence of critically high HAdV DNA copy numbers in stool very early after HSCT might also herald impending onset of invasive infection prior to incipient immune reconstitution, thus possibly predisposing for particularly severe clinical manifestations. Since early initiation of treatment was shown to be critical for successful management of HAdV infections in this clinical setting, the time point of first HAdV occurrence in stool specimens could be of relevance for appropriate therapeutic considerations. In this regard, pre-transplant HAdV

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detection in stool may provide the impetus for postponing elective transplantations and offering early pre-emptive treatment in high-risk settings. This strategy might be considered particularly in patients with more than anecdotal HAdV positivity in serial stool specimens pre-transplant, with rather Publications: high virus copy numbers already at this stage. 1. Lion T, Kosulin K, Landlinger C, et al. Monitoring of Although this approach would result in the adenovirus load in stool by real-time PCR permits early overtreatment of a considerable proportion of detection of impending invasive infection in patients patients who might clear the virus even without after allogeneic stem cell transplantation. Leukemia 2010; therapeutic intervention, this option would appear 24(4): 706-14. reasonable provided that relatively non-toxic 2. Lion T. Adenovirus Infections in Immunocompetent treatment options were available. It is conceivable and Immunocompromised Patients. Clinical microbiology that early initiation of therapy aiming at the reviews 2014; 27(3): 441-62. prevention of invasive virus infection and 3. Kosulin K, Geiger E, Vecsei A, et al. Persistence and disseminated disease might contribute to improving reactivation of human adenoviruses in the gastrointestinal outcome. However, the risk of invasive infection tract. Clinical microbiology and infection 2016; must be judiciously balanced against the potential 22(4): 381 e1-8. side effects of current treatment approaches. The 4. Matthes-Martin S, Feuchtinger T, Shaw PJ, et al. availability of safer therapy options, such as the new European guidelines for diagnosis and treatment of adeno- compound Brincidofovir, might facilitate the virus infection in leukemia and stem cell transplantation: decision for early antiviral treatment once the summary of ECIL-4 (2011). Transplant infectious disease efficacy of pre-emptive treatment is firmly 2012; 14(6): 555-63. established. Moreover, the timely employment and 5. Ebner (Kosulin) K, Suda M, Watzinger F, Lion T. success of adoptive transfer of HAdV-specific Molecular detection of the entire spectrum of human T-cells using donor-derived or third-party cells adenoviruses by a two-reaction RQ-PCR assay. could be further improved by early recognition of Journal of clinical microbiology 2005; 43(7): 3049-53. high-risk patients in order to permit optimal 6. Kosulin K, Berkowitsch B, Lion T. Modified planning and timing of T-cell generation or pan-adenovirus real-time PCR assay based on genome acquisition. analysis of seventy HAdV types. Journal of clinical The present study in a large cohort of pediatric virology 2016; 80: 60-1. patients provides evidence for the higher risk of 7. Kosulin K, Dworzak S, Lawitschka A, Matthes-Leodolter viremia in individuals shedding HAdV into the stool S, Lion T. Comparison of different approaches to prior to HCST, and greatly reinforces our earlier quantitative adenovirus detection in stool specimens of findings.3 The current insights therefore provide a hematopoietic stem cell transplant recipients. Journal of rational basis for further clinical studies on early clinical virology. 2016;85:31-36 pre-emptive treatment approaches to determine the 8. Kosulin K, Pichler H, Lawitschka A, Geyeregger R, potential impact on successful management of Lion T. Diagnostic Parameters of Adenoviremia in Pediatric HAdV infections in highly immunocompromised Stem Cell Transplant Recipients. Frontiers in Microbiology. patients. ■ 2019 22;10:414

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Clinically important resistant mutations in Philadelphia chromosome- positive leukemias

Ponatinib is the most potent currently available tyrosine kinase inhibitor The third-generation tyrosine kinase inhibitor (TKI) ponatinib exerts a strong anti-neoplastic effect in all stages of chronic myeloid leukemia (CML) as well as in Philadelphia(Ph)-positive acute lymphoblastic leukemia (ALL). It is capable of suppressing the kinase activity of BCR-ABL1 carrying any single mutation in the tyrosine kinase domain (TKD) including the gate-keeper mutation T315I. However, therapy with ponatinib may be associated with serious side effects, and reports on apparently dose-dependent severe adverse events provided the basis for reduction of the recommended daily dose from 45 mg to 30 or even 15 mg. These modifications in the dosing regimen imply that the achievable levels of effective average

plasma concentrations (efCave) of ponatinib decrease from 28 nM to 23 and 10 nM, respectively. Based on available in vitro data, a 10 nM effective plasma concentration of ponatinib is expectedly adequate for CML cells carrying wild-type BCR-ABL1 or almost any individual mutation in the kinase domain, with the exception of E255V (Table 1). Nevertheless, resistance to ponatinib can evolve in

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Table 1. In vitro responses of native and mutant BCR-ABL1 to ponatinib. The indicated BCR-ABL1 constructs were expressed at equivalent levels in Ba/F3 cells, and responses to ponatinib were determined using survival assays. The indicated nanomolar IC50 values for ponatinib and the corresponding variation (±ΔIC50) are based on three biological and a minimum of four technical replicates. Mutations highly sensitive to ponatinib are highlighted in green, whereas increasing resistance to this TKI is indicated in yellow>orange>red. *Compound mutations previously characterized in vitro.

sub-clones carrying BCR-ABL1 variants with two or

more mutations on the same allele, if the IC50 values for this TKI exceed the maximum achievable effective plasma levels. These so-called compound mutations (CMs) can be associated with increased oncogenic potential in comparison to individual mutations and represent a powerful mechanism of resistance to all currently available TKIs. The occurrence of CMs in CML has been linked particularly to sequential treatment with different TKIs, and is far more frequently observed in

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established a transposon-based approach to rapid and efficient transfection of mutant BCR-ABL1 constructs into cells, and implemented a flow cyto- metry-assisted selection method facilitating targeted enrichment of cells carrying single gene construct insertions in the genome. This protocol was demonstrated to provide BCR-ABL1 expression levels similar to those observed in patient specimens and to permit unbiased testing of TKI sensitivity in Ph-positive ALL. The identification of their vitro.

responsiveness to ponatinib is of paramount The IC50 values of ponatinib were determined by importance for the subsequent clinical management. employing a widely used in vitro proliferation assay. Since the acquisition of mutations within the Most CMs involving sites with no previous evidence BCR-ABL1 TKD is thought to be a random process, for implication in resistance to ponatinib displayed

we sought to determine the spectrum of highly TKI- IC50 values below 10 nM. This efCave is readily resistant CMs, with a particular focus on ponatinib. achievable with the 15 mg daily dose of ponatinib, thus suggesting high sensitivity to treatment with this TKI. CMs revealing elevated resistance to ponatinib Efficacy of ponatinib against leukemic subclones in vitro almost invariably included T315I or F317L carrying BCR-ABL1 compound mutations mutations. In fact, the only CM constellation within The structure of the ABL1 KD has been the panel tested bearing neither T315I nor F317L and employed to map and assess the presumptive impact displaying reduced sensitivity to ponatinib was

of 27 different CMs involving important functional Y253H/F359V, with an IC50 value of 23.7 ± 1.7 nM sites of the BCR-ABL1 TKD and including (Table 1). This in vitro assessment is supported by a constellations expected to display high resistance to clinical observation made within the PACE trial, a ponatinib (Table 1). The majority of CMs analyzed phase 2 clinical study with ponatinib in heavily have subsequently been observed in patients with pretreated patients with resistant or intolerant Ph-positive leukemia. To assess the anticipated Ph-positive leukemia: a patient with CML had responses to ponatinib based on in vitro testing, we presented with a V299L/F359V prior to therapy with

have introduced all BCR-ABL1 CMs into Ba/F3 cells. ponatinib, a constellation displaying an IC50 for this This cellular background was selected because the TKI in the range of 10 nM (Table 1). However, the majority of published heat maps indicating the patient showed signs of progressive disease under an TKI-responses of individual BCR-ABL1 mutations average daily ponatinib dose of 26 mg, corresponding

had been established in this model. Our recently to an efCave of ~20 nM. Mutational analysis at this published observations indicate that the in vitro data time revealed the Y253H/F359V which displays an

on TKI resistance of some mutations presented in IC50 value for ponatinib beyond the efCave apparently earlier reports may be overestimated.1 This is likely achieved in this patient. It is conceivable therefore attributable to the testing of cells inadvertently that the actual dose of ponatinib may not have been carrying multiple insertions of mutant BCR-ABL1 sufficient to suppress the kinase activity of cassettes in the genome. We have therefore BCR-ABL1Y253H/F359V, thus resulting in disease

Science Reports Forschungsbericht St. Anna Kinderkrebsforschung | Science Report St. Anna Children’s Cancer Research Institute [ 87 ]

Figure 1. Competitive co-culture of BaF3 cells expressing BCR-ABL1Y253H/F359V and BCRABL1V299L/F359V. BaF3 cells expressing BCR-ABL1Y253H/F359V (black columns) and BCR-ABL1V299L/F359V (white columns) were mixed at a 1:9 ratio and incubated in the presence of 20 or 30 nM ponatinib. Clonal evolution was monitored by fluorescence of the co-expressed fluorescent proteins ZsGreen for BCR-ABL1Y253H/F359V and TdTomato for BCR-ABL1V299L/F359V. The results were confirmed by Sanger sequencing of the BCRABL1 TKD using gDNA and cDNA isolated and prepared at the indicated time points. The proportion of dead cells was calculated using Annexin/PI staining (striped columns). The indicated error bars are based on two biological and three technical replicates.

progression. In vitro competitive co-culture experiments have demonstrated that Ba/F3 cells expressing BCR-ABL1Y253H/F359V have a proliferative and survival advantage over Ba/F3 carrying BCR-ABL1V299L/F359V in the presence of 20 nM ponatinib, while efficient suppression of both CM clones was observed at 30 nM of ponatinib (Figure 1). Some of the predicted CMs containing F317L

displayed an IC50 for ponatinib in the range of 30-40 nM, which would expectedly indicate clinical resistance to ponatinib. In the PACE trial, three

Science Reports [ 88 ] Forschungsbericht St. Anna Kinderkrebsforschung | Science Report St. Anna Children’s Cancer Research Institute

functional properties, which also showed IC50 values

for ponatinib clearly above the efCave of 28 nM. The additional sites affected included the south lobe mutation E459K and the A-loop mutations L384M and L387M, which were predicted by the model employed and, for the E459K mutation, suggested previously to destabilize the inactive conformation of the ABL1 TKD. This functional property may play a critical role because ponatinib is a type II TKI CML patients with the F317L/E459K were reported binding primarily to the inactive conformation of before the start of ponatinib therapy. This the BCR-ABL1 TKD, thus requiring considerably

constellation showed an IC50 of 28.0 ± 2.0 nM in our higher concentrations of the drug to inhibit the analysis, which is near the limit of the clinically kinase activity. The constellation T315I/L384M was

achievable efCave (Table 1). In one of the patients, suggested by the BCR-ABL1 protein model to confer who had received an average daily dose of 32 mg resistance to ponatinib, and the prediction could be

(corresponding to an efCave of ~24 nM), it took 583 confirmed in vitro, but this combination has not days to reach major molecular response (MMR), been reported in patients with Ph-positive leukemia whereas the two other patients who had received 42 to date. By contrast, the compound mutations

and 45 mg daily (efCave ~26 and 28 nM) achieved T315I/L387M and T315I/E459K were already MMR in 168 and 87 days, respectively. These observed in the clinical setting, but their examples highlight the potential relevance of susceptibility to ponatinib had not been selecting the appropriate dose of ponatinib by characterized by in vitro analysis2.

considering the IC50 of mutations identified prior to the initiation of treatment or during therapy. In agreement with earlier reports, CMs involving Diagnostic and therapeutic implications

the T315I mutation displayed the highest IC50 Therapy with ponatinib has been associated with values for ponatinib, clearly exceeding the clinically considerable cardiovascular toxicity and other achievable plasma concentration of the drug. These potentially serious side effects which appear to be mutational constellations included combinations of dose-related. However, as highlighted by the data T315I with the P-loop mutations G250E, Y253H, presented, current strategies that aim at decreasing and E255V/K as well as T315I/M351T and T315I/ the dose of ponatinib should carefully consider the F359V, which have been described earlier to arise presence and type of mutations in the BCR-ABL1 during treatment with ponatinib. Our analysis of TKD to enable effective treatment. It would be these CMs also indicated high levels of resistance to highly desirable, therefore, to implement testing of

ponatinib, but the IC50 values determined were the effective plasma drug concentrations and lower than reported previously, conceivably for the monitoring the kinetics of mutant subclones technical reasons outlined above. In addition to covering also compound mutations3 in the routine constellations already shown to confer resistance to diagnostic surveillance to provide a basis for ponatinib, we have identified CMs involving T315I optimized clinical management of patients treated and residues at sites displaying very distinct with ponatinib. ■

Science Reports Forschungsbericht St. Anna Kinderkrebsforschung | Science Report St. Anna Children’s Cancer Research Institute [ 89 ]

Publications: 1. Byrgazov K, Lucini CB, Berkowitsch B, Koenig M, Haas OA, Hoermann G, Valent P, Lion T. Transposon-mediated generation of BCR-ABL1-expressing transgenic cell lines for unbiased sensitivity testing of tyrosine kinase inhibitors. Oncotarget. 2016, 22;7(47):78083-78094. 2. Byrgazov K, Lucini CB, Valent P, Hantschel O, Lion T. BCR-ABL1 compound mutants display differential and dose-dependent responses to ponatinib. Haematologica. 2018;103(1):e10-e12 3. Kastner R, Zopf A, Preuner S, Pröll J, Niklas N, Foskett P, Valent P, Lion T, Gabriel C. Rapid identification of compound mutations in patients with Philadelphia- positive leukaemias by long-range next generation sequencing. Eur J Cancer. 2014;50(4):793-800.

Science Reports [ 90 ] Forschungsbericht St. Anna Kinderkrebsforschung | Science Report St. Anna Children’s Cancer Research Institute

Allogeneic Stem Cell Transplantation for children with Acute Lymphoblastic Leukemia: Outcome after mismatched-donor transplantation is influenced by Disease Risk

Stem Cell Transplantation for Childhood ALL is restricted to high-risk-patients. Approximately 80% of pediatric patients presenting with acute lymphoblastic leukemia (ALL) diagnosed over age 1 year are currently treated with conventional polychemotherapy protocols However, approximately 10% of patients present with poor prognostic features at initial diagnosis, and 20% of ALL pediatric patients eventually relapse after first complete remission (CR1). Additional therapy is required for patients who achieve complete remission (CR) One option is allogeneic stem cell transplantation (HSCT). The overall results of HSCT have consistently improved over time

Science Reports Forschungsbericht St. Anna Kinderkrebsforschung | Science Report St. Anna Children’s Cancer Research Institute [ 91 ]

through reduction in nonrelapse mortality (NRM). The use of unrelated volunteer donors, unrelated cord blood (CB), or haploidentical donors makes HSCT feasible for every patient. Unfortunately, the relapse incidence has not changed and remains a substantial concern.

Nonsibling-Donors are increasingly used In many countries the number of allogeneic HSCTs from nonsibling donors has exceeded that of matched sibling donor (MSD) HSCT since the middle of 2005. In 2015 Peters et al. published the results obtained by the BFM group for pediatric patients with ALL in CR. In that publication the authors demonstrated the equivalence of the results obtained with MSDs and with matched unrelated donors (MUDs). The International BFM (I-BFM) consortium reproduced these results in the I-BFM ALL HSCT 2007 study. In both studies patients were allocated to different relapse risk groups. Patients with a very high relapse risk (VHRR; Table 1) were eligible for any donor type and available stem cell source. Here, we report the outcomes of 148 patients who were transplanted in CR1 or subsequent CR from an HLA-mismatched donor (MMD) whatever the per-protocol disease risk allocation. HLA-MMDs were defined as donors with more than 1 (>9/10) or more allelic or antigenic disparities up to a different haplotype (MMD), regardless of Harmonized Procedures allow sound relationship. Unrelated CB was also accepted as a outcome analysis stem cell source. HLA typing was defined using Transplant centers from 3 countries (Austria, low-resolution molecular techniques for HLA-A and Germany, and Switzerland) participated in the 2003 HLA-B and high-resolution typing for HLA-DR, and BFM ALL study from 2003 to 2011, and 10 less than 5/6 matches were classified as MMDs. additional countries (Czech Republic, Denmark, The patients were stratified according to BFM France, Israel, Italy, the Netherlands, Poland, eligibility criteria for transplantation. Standard Sweden, Slovakia, and Turkey) participated in the relapse risk patients were not eligible for any HSCT, I-BFM ALL SCT 2007 study from 2007 to 2011. high relapse risk (HRR) patients were eligible for Both studies were prospective, multicentre open either MSD or MUD HSCT, and VHRR patients also trials. had indication for MMD transplants. BM was the

Science Reports [ 92 ] Forschungsbericht St. Anna Kinderkrebsforschung | Science Report St. Anna Children’s Cancer Research Institute

recommended source according to the protocol, but granulocyte colony-stimulating factor–primed peripheral blood (PB) and CB stem cells were also acceptable sources, according to transplant or donor centre preference. Target doses > 3 × 108 nucleated cells/kg recipient body weight and >1.5 × 106 CD34+ cells/kg recipient body were recommended for both BM and PB stem cells (PBSCs). For CB the target doses were 3 × 107 nucleated cells/kg recipient body weight and >1 × 106 CD34+ cells/kg recipient Patients benefit from early stem cell body weight. A consistent myeloablative transplantation: better survival, less relapses conditioning regimen was performed depending on The 4-year rate of EFS was 52% ± 4% for the both recipient age and donor type and did not entire cohort of 148 patients. One patient developed depend on disease risk group. For patients in the a secondary myelodysplastic syndrome. No other present study (ie, those transplanted from MMDs secondary malignancies were reported up to the date and older than 2 years of age [except for 8 patients < of point. As for OS, univariate analysis revealed that 2 years old]), the conditioning regimen was based on HSCT for VHRR versus HRR disease resulted in hyperfractionated total body irradiation (TBI; total worse outcomes (42% ± 5% versus 80% ± 6%, P < dose 1200 cGy i.e. 200 cGy b.i.d. on days −10 to −8), .001). Similarly, transplantation in CR1 or CR2 fludarabine (40 mg/m2/day for 4 days from days –7 versus CR > 2 was a favorable prognostic factor to –4), and etoposide (40 mg/kg at day –3). Patients (66% ± 6% versus 50% ± 6% versus 27% ± 9%, P < younger than 2 years of age received body weight– .001). In a multivariate analysis adjusted for adjusted doses of either i.v. or oral busulfan from indication, disease status at HSCT, donor and days –11 to –8, followed by fludarabine (40 mg/m2/ recipient CMV status, GVHD prophylaxis, TBI in day from days –7 to –4) and cyclophosphamide (60 the conditioning regimen, and patient age, HSCT for mg/kg for 2 days, days –3 and −2). Graft-versus-host VHRR disease (P = .002; HR, 3.33; 95% CI, 1.55 to disease (GVHD) prophylaxis comprised 7.16) and HSCT for advanced disease (P < .001; HR, cyclosporine A, methotrexate, and antithymocyte 3.55; 95% CI, 1.76 to 7.16) were statistically globulin (Fresenius, Bad Homburg vor der Höhe, significant negative prognostic factors . Multivariate Germany; 20 mg/kg/dose, on days −4, −3, and −2). analysis adjusted for HSCT indication, disease status Methotrexate was substituted with steroids in CB at HSCT, GVHD prophylaxis, TBI in conditioning recipients. Some patients received ex vivo T cell– regimen, and age of patient showed that none of depleted grafts either by CD34+ selection or CD3/ these factors was significant. CD19 depletion according to protocols and Similarly, the negative impact of the combination physician/center decision. Acute and chronic GVHD of CMV-negative donors with CMV-positive (aGVHD and cGVHD, respectively) were graded as recipients reached only borderline significance (P = previously described. Patients, who were alive and in .12; HR, 1.96; 95% CI, .84 to 4.58). In an additional remission 100 days after HSCT were considered at multivariate analysis aGVHD of any grade was risk for cGVHD. The discontinuation of immuno- associated with higher NRM (P = .002; HR, 3.35; suppression was considered the absence of GVHD. 95% CI, 1.19 to 9.43).

Science Reports Forschungsbericht St. Anna Kinderkrebsforschung | Science Report St. Anna Children’s Cancer Research Institute [ 93 ]

(A) Overall Survival (OS) according to indication. (B) OS according to remission status at HSCT. (C) Event Free Survival (EFS) according to indication. (D) EFS according to remission status at HSCT.

(A) Cummulative Incidence of Relapse (CIR) according to indication. (B) CIR according to remission status at HSCT. (C) Non-Relapse Mortality (NRM) NRM according to indication. (D) NRM according to remission status at HSCT.

Science Reports [ 94 ] Forschungsbericht St. Anna Kinderkrebsforschung | Science Report St. Anna Children’s Cancer Research Institute

Hematopoetic Stem Cell Transplantation from HLA mismatched donors is still a challenge. The ability to use partially matched unrelated CB completed the repertoire of potential stem cell sources. However, HSCT from alternative donors— whether related or unrelated—remained associated with high treatment failure rates, reflecting both NRM and relapse. Thus, alternative HSCT is often limited to high-risk patients and to some experienced centers able to perform ex vivo graft manipulations, such as T cell depletion through either positive CD34+ cell selection or CD3+ ± CD19+ cell depletion [14,16-19]. However, these disappointing results were mainly described from retrospective and single-centre studies and should be cautiously interpreted. In 2003 the Austrian-German-Swiss BFM protocol initiated a prospective study to evaluate the feasibility of the systematic use of either 9 or 10/10 unrelated donors for patients < 18 years old with an indication of allogeneic HSCT for ALL in CR1 or subsequent CR. The results demonstrated the equivalence between MSDs and MUDs, regardless of the disease relapse risk [8]. The 4-year OS rates were 79% ± 4% and 73% ± 3% for patients transplanted from MSDs and MUDs, respectively (not significant). Both 4-year EFS and CIR were also similar in both groups. NRM was statistically better in the MSD group. This initial study was followed by an international study of 10 countries, and the global results were similar (Balduzzi et al., under revision). Here, we report the results of these 2 prospective studies on 148 HRR and VHRR patients transplanted from MMDs for ALL in CR1 or subsequent CR. Both 4-year OS and EFS rates (56% ± 4% and 52% ± 4%) were inferior to the results reported within the same studies for patients with better matched donors. However, the results remained remarkable and satisfying for HRR patients transplanted from MMDs,

Science Reports Forschungsbericht St. Anna Kinderkrebsforschung | Science Report St. Anna Children’s Cancer Research Institute [ 95 ]

with 4-year rates of OS and EFS of 82% ± 6% and 80% ± 6%, respectively. In the present study we demonstrated the feasibility of allogeneic HSCT from alternative donors in both HRR and VHRR pediatric patients with ALL in CR after myeloablative conditioning. In HHR patients, MMDs seem to offer the same chance of success as using better HLA-matched donors. However, further progress is needed to decrease overall treatment failure (ie, both relapse rate and treatment-related mortality) in VHRR patients. ■

Publications: 1. Dalle JH, Balduzzi A, Bader P, Lankes- 3. Peters, C., Schrappe, M., von Stackelberg, A., Schrau- ter A, Yaniv I, Wachowiak J, Pieczonka A, Bierings M, Yes- der, A., Bader, P., Ebell, W., Lang, P., Sykora, K.W., ilipek A, Sedlaçek P, Ifversen M, Sufliarska S, Toporski J, Schrum, J., Kremens, B., Ehlert, K., Albert, M.H., Glogova E, Poetschger U, Peters C. Allogeneic Stem Cell Meisel, R., Matthes-Martin, S., Gungor, T., Holter, W., Transplantation from HLA-Mismatched Donors for Pedia- Strahm, B., Gruhn, B., Schulz, A., Woessmann, W., tric Patients with Acute Lymphoblastic Leukemia Treated Poetschger, U., Zimmermann, M. & Klingebiel, According to the 2003 BFM and 2007 International BFM T. Stem-cell transplantation in children with acute Studies: Impact of Disease Risk on Outcomes. Biol Blood lymphoblastic leukemia: A prospective international Marrow Transplant. 2018 Sep;24(9):1848-1855. multicenter trial comparing sibling donors with 2. Bader, P., Kreyenberg, H., von Stackelberg, A., matched unrelated donors-The ALL-SCT-BFM-2003 trial. Eckert, C., Salzmann-Manrique, E., Meisel, R., Poe- J Clin Oncol 2015, 33, 1265-1274. tschger, U., Stachel, D., Schrappe, M., Alten, J., Schrau- der, A., Schulz, A., Lang, P., Muller, I., Albert, M.H., Willasch, A.M., Klingebiel, T.E. & Peters, C. Monitoring of minimal residual disease after allogeneic stem-cell transplantation in relapsed childhood acute lympho- blastic leukemia allows for the identification of impen- ding relapse: results of the ALL-BFM-SCT 2003 trial. J Clin Oncol 2015, 33, 1275-1284.

Science Reports

Career [ 98 ] Forschungsbericht St. Anna Kinderkrebsforschung | Science Report St. Anna Children’s Cancer Research Institute

Working at CCRI

A shared mission makes a difference in research! The Children’s Cancer Research Institute (CCRI) is an internationally renowned biomedical research institute, dedicated to the advancement of diagno¬sis, prognosis and treatment of childhood cancer. Science at the CCRI addresses a broad range of topics, covered by 12 research groups whose members work closely together and in cooperation with clinicians from St. Anna Children’s Hospital. We are at the forefront of international pediatric cancer research and scientists at CCRI are striving to make the most innovative and effective therapies available to young patients.

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.

The CCRI offers an intellectually stimulating environment with lots of opportunities for scientific exchange and discussions in regular seminars, retreats, and national and international conference attendances. The quality of our work is periodically reviewed by a panel of top international experts guaranteeing consistently high standards of CCRI research. PhD students are accompanied by PhD-committees to guide them through their thesis and postdocs have the option to exercise their ability to teach by mentoring and training younger colleagues.

Career Forschungsbericht St. Anna Kinderkrebsforschung | Science Report St. Anna Children’s Cancer Research Institute [ 99 ]

If you want to apply for a position

please proceed according to the details given on our website. www.ccri.at

Career [ 100 ] Forschungsbericht St. Anna Kinderkrebsforschung | Science Report St. Anna Children’s Cancer Research Institute

Postdoctoral Fellows and Staff Scientists ▷ Reza M. Abbasi Scientific ▷ Ingeborg Ambros ▷ Dave Aryee ▷ Jozef Ban Staff ▷ Marie Bernkopf ▷ Polyxeni Bozatzi 2017/2018 ▷ Eva Bozsaky ▷ Konstantin Byrgazov ▷ Annamária Cseh ▷ Stefan Czurda ▷ Stefan Fiedler ▷ Klaus Fortschegger Group Leader/PI* Affiliated Clinicians ▷ Zvenyslava Husak Clinical Research ▷ Peter Ambros ▷ Andishe Attarbaschi ▷ Stefanie Kirchberger ▷ Helga Björk Arnadóttir ▷ Kaan Boztug ▷ Heidrun Boztug ▷ Karin Kosulin ▷ Saelde Baumgartner ▷ Martin Distel ▷ Kaan Boztug ▷ Zsuzsanna Lehner ▷ Tijana Frank ▷ Michael Dworzak ▷ Sabine Breuer ▷ Manfred Lehner ▷ Dagmar Friede ▷ Gerhard Fritsch ▷ Christofer Diakos ▷ Chantal Lucini ▷ Nora Geissler ▷ René Geyeregger (PI) ▷ Michael Dworzak ▷ Sören Mai ▷ Evgenia Glogova ▷ Oskar Haas ▷ Gernot Engstler ▷ Margarita Maurer-Granofszky ▷ Temeida Graf ▷ Florian Halbritter (PI) ▷ Stefan Fiedler ▷ Gerda Modarres ▷ Corinne Grafl ▷ Wolfgang Holter ▷ Caroline Hutter ▷ Karin Nebral ▷ Dasa Janousek ▷ Caroline Hutter (PI) ▷ Leo Kager ▷ Filomena de Almeida Nogueira ▷ Susanne Karlhuber ▷ Heinrich Kovar ▷ Hubert Kogler ▷ Susanna Pascoal ▷ Barbara Kristufek ▷ Ruth Ladenstein ▷ Anita Lawitschka ▷ Wolfgang Paster ▷ Andrea Madunic ▷ Thomas Lion ▷ Georg Mann ▷ Branka Radic-Sarikas ▷ Nora Mühlegger ▷ Renate Panzer-Grümayer ▷ Susanne Matthes-Leodolter ▷ Marlene Remely ▷ Marek Nykiel ▷ Sonja Schneider-Schwarz ▷ Milen Minkov ▷ Fikret Rifatbegovic ▷ Ulrike Pötschger (SOM) * * ▷ Christina Peters ▷ Dagmar Schinnerl ▷ Ingrid Pribill ▷ Sabine Strehl ▷ Herbert Pichler ▷ Raphaela Schwentner ▷ Marion Sebek ▷ Sabine Taschner-Mandl ▷ Andreas Vécsei ▷ Murat Tugrul ▷ Eva Sorz (PI) ▷ Volker Witt ▷ Tamara Weiss ▷ Elfriede Thiem ▷ Eleni Tomazou (PI) ▷ Natalie Zubarovskaya ▷ Petra Zeitlhofer ▷ Jana Zimermann

* PI: Principal Investigator * * SOM: Senior Operations Manager Career Forschungsbericht St. Anna Kinderkrebsforschung | Science Report St. Anna Children’s Cancer Research Institute [ 101 ]

Technicians in ▷ Caterina Sturtzel Research & Diagnostics ▷ Adrian Stütz ▷ Bettina Berkowitsch ▷ Dijana Trbojevic ▷ Maria Berneder ▷ Eva Winkler ▷ Clemens Brunner ▷ Sven Wohlmacher ▷ Bettina Brunner-Herglotz ▷ Andrea Ziegler ▷ Ulrike Engel ▷ Elke Zipperer ▷ Susanna Fischer ▷ Michaela Fortschegger ▷ Nelli Frank ▷ Brigitte Grimm ▷ Angela Halfmann ▷ Nadine Hartl ▷ Sabrina Haslinger ▷ Christine Hoffmann-Freimüller ▷ Stefanie Hosiner ▷ Andrea Inthal PhD Students ▷ Jovana Jovanovic Bioinformatics ▷ Lisa Bierbaumer ▷ Gunhild Jug ▷ Maximilian Kauer ▷ Charlotte Brey Diploma/Master Students ▷ Michael Kainz ▷ Gerda Modarres ▷ Barbara Dillinger ▷ Jakob Berner ▷ Margit König ▷ Anna Roswitha Poetsch ▷ Teresa Gerber ▷ Verena Dastl ▷ Isabella Krickl ▷ Niko Popitsch ▷ Sarah Grissenberger ▷ Markus Dobersberger ▷ Astrid Mecklenbräuker ▷ Clara Hechenberger ▷ Filip Marjan Gallob ▷ Karin Mühlbacher Research Support Office ▷ Anna-Maria Husa ▷ Merit Alwine Hildebrandt ▷ Maya-Marisol Plank ▷ Nuno-Miguel ▷ Marion Janschitz ▷ Paula Hofbauer ▷ Nadine Nirtl Andrade Gomes ▷ Tahereh Javaheri ▷ Daria Lazic ▷ Bettina Nocker ▷ Caterina Barresi ▷ Anna Maria Katschnig ▷ Peter Peneder ▷ Michaela Pregesbauer ▷ Melanie Brunhofer ▷ Stefan Köhrer ▷ Lisa Saloberger- ▷ Sandra Preuner-Stix ▷ Zoltán Dobai ▷ Florian Kromp Sindhöringer ▷ Dieter Printz ▷ Orsolya Nagy ▷ Rahil Noorizadeh ▷ Michael Schöber ▷ Marion Riebler ▷ Karoline Noworyta ▷ Leonel Pereira ▷ Christina Schüller ▷ Daniela Scharner ▷ Martin Schalling ▷ Katarzyna Pietrzykowska ▷ Shirin Sharghi ▷ Thomas Schnöller ▷ Benjamin Salzer ▷ Sandra Sonderegger ▷ Angela Schumich Independent Project Staff ▷ Elisabeth Steindl ▷ Marcus Strobl ▷ Manuela Stadler ▷ Markus Diem ▷ Marcus Tötzl ▷ Jakob Winkler ▷ Julia Stemberger ▷ Michael Reiter

Career

Finanzbericht [ 104 ] Forschungsbericht St. Anna Kinderkrebsforschung | Sience Report St. Anna Children’s Cancer Research Institute

Richtlinien zur Spendenverwendung

ie St. Anna Kinderkrebsforschung wird zum überwiegenden Teil durch private Spenden finanziert. Für den Betrieb des Forschungsinstituts werden jährlich mehr als sieben Millionen Euro benötigt, der Verein verfügt jedoch Spendengütesiegel und steuerliche Absetzbarkeit Düber keine Basisfinanzierung durch die öffentliche Seit dem Jahr 2002 trägt die St. Anna Kinder- Hand. Zusätzliche Mittel werden im Rahmen von krebsforschung als eine der ersten Organisationen kompetitiv ausgeschriebenen Projektförderungen Österreichs das Spendengütesiegel der Kammer von anerkannten nationalen und internationalen der Wirtschaftstreuhänder. Für die jährliche Stellen akquiriert. Neuverleihung führt ein Wirtschaftsprüfer Wir fühlen uns unseren Spenderinnen und zusätzlich eine Prüfung der transparenten und ord- Spendern gegenüber zu einer sparsamen und nungsgemäßen Verwendung der Mittel gemäß den effizienten Verwendung der uns anvertrauten strengen Richtlinien des Spendengütesiegels durch. Gelder verpflichtet. Aus diesem Grund verwenden Auf Grundlage eines von der Finanzlandesdirektion wir weniger als 10 % für die Verwaltung und das für Wien erlassenen Bescheides zählt die St. Anna Fundraising, das bedeutet: Mehr als 90 % der Kinderkrebsforschung zum begünstigten Empfän- Spenden fließen direkt in die Forschung. gerkreis, sodass Spenden sowohl von der Lohnsteuer Der Jahresabschluss wird gemäß den als Sonderausgabe als auch von der Einkommen- Bestimmungen des Vereinsgesetzes für große steuer als Betriebsausgabe steuerlich absetzbar sind. Vereine erstellt, wobei die gleichen Richtlinien wie für Kapitalgesellschaften gelten. Die Finanzgebarung und der Jahresabschluss des Vereins werden jährlich von einem beeideten Wirtschaftsprüfer kontrolliert und mit einem uneingeschränkten Bestätigungs- vermerk versehen. Damit wird der sach- und zweckgemäße Umgang mit den erhaltenen Spenden sichergestellt und bestätigt.

Finanzbericht Forschungsbericht St. Anna Kinderkrebsforschung | Sience Report St. Anna Children’s Cancer Research Institute [ 105 ]

Qualitätssicherung der wissenschaftlichen Arbeit Das Forschungsinstitut verfügt über ein Scientific Advisory Board – ein Gremium aus externen Experten – mit der Aufgabe der laufenden Evaluierung der wissenschaftlichen Arbeiten und der Beratung der Institutsleitung. Darüber hinaus werden regelmäßig neue wissenschaftliche Projekte bei renommierten forschungsfördernden nationalen und internationalen Stellen eingereicht und Forschungsergebnisse in international anerkannten wissenschaftlichen Journalen publiziert. In regelmäßigen Abständen findet zusätzlich eine objektive Beurteilung der wissenschaftlichen Leistung durch ausgewiesene externe Fachleute auf dem Gebiet statt. ■

Finanzbericht [ 106 ] Forschungsbericht St. Anna Kinderkrebsforschung | Sience Report St. Anna Children’s Cancer Research Institute

Mittelherkunft

Beträge in Euro 2017 2018 I. Spenden a) ungewidmete Spenden 0,00 0,00 b) gewidmete Spenden 11.483.207,03 10.719.938,68 II. Mitgliedsbeiträge 0,00 480,00 III. Betriebliche Einnahmen a) betriebliche Einnahmen aus öffentlichen Mitteln 1.244.860,60 1.004.722,41 b) sonstige betriebliche Einnahmen 2.397.857,54 3.598.832,13 I V. Subventionen und Zuschüsse der öffentlichen Hand 0,00 0,00 V. Sonstige Einnahmen a) Vermögensverwaltung 0,00 0,00 b) sonstige andere Einnahmen, sofern nicht in Punkt I bis IV festgehalten 423.360,01 0,00 VI. Auflösung von Passivposten für noch nicht widmungsgemäß 0,00 0,00 verwendete Spenden bzw. Subventionen VII. Auflösung von Rücklagen 0,00 0,00 VIII. Jahresverlust 0,00 0,00 TOTAL 15.549.285,18 15.323.973,22

Finanzbericht Forschungsbericht St. Anna Kinderkrebsforschung | Sience Report St. Anna Children’s Cancer Research Institute [ 107 ]

Mittelverwendung

Beträge in Euro 2017 2018 I. Leistungen für die statutarisch festgelegten Zwecke 11.034.511,89 12.365.591,88 II. Spendenwerbung 762.861,41 888.389,69 III. Verwaltungsaufwand 445.379,46 485.978,56 I V. Sonstiger Aufwand, sofern nicht unter Punkt I bis III festgehalten 54.514,06 83.758,40 V. Zuführung zu Passivposten für noch nicht widmungsgemäß verwendete 3.252.018,36 1.500.254,69 Spenden bzw. Subventionen VI. Zuführung von Rücklagen 0,00 0,00 VII. Jahresüberschuss 0,00 0,00 TOTAL 15.549.285,18 15.323.973,22

Finanzbericht

Anhang [ 110 ] Forschungsbericht St. Anna Kinderkrebsforschung | Science Report St. Anna Children’s Cancer Research Institute

International fremdgeförderte Projekte 2017/2018 International Grants 2017/2018

PRedictive In-silico Multiscale Analytics (PRIMAGE) Paediatric Cancer European Reference Network ■ CCRI responsible principal investigator: (ERN PaedCan) Ruth Ladenstein ■ CCRI Coordinator: Ruth Ladenstein ■ European Commission Grant – ■ Grant from the European Consumers, Health, Call H2020-SC1-DTH-2018-2020 Agriculture and Food Executive Agency ■ Duration: 01/12/2018 to 30/11/2022 (CHAFEA) ■ Duration: 01/03/2017 to 28/02/2022 Integrated European Network on cGvHD ■ CCRI responsible principal investigator: ITCC Pediatric Preclinical POC Platform (ITCCP4) Anita Lawitschka ■ CCRI partner: Heinrich Kovar ■ European Commission Grant – COST Action ■ Coordinator: Stefan Pfister (Deutsches ■ Duration: 31/08/2018 to 30/08/2022 Krebsforschungszentrum DKFZ, Germany) ■ European Commission Grant – ERN-PAEDCAN-CEF-Telecom H2020 Innovative Medicines Initiative ■ CCRI Coordinator: Ruth Ladenstein ■ Duration: 01/01/2017 to 31/12/2021 ■ European Commission Grant – Call CEF-TC-2017-2 Overcoming Neuroblastoma Tumour ■ Duration: 01/05/2018 to 31/08/2019 HETerogeneity, Resistance and RecurrAnCe (ONTHETRRAC) ERN-PAEDCAN Partner: Paediatric Rare Tumours ■ CCRI responsible principal investigator: Peter Network - European Registry (PARTNER) Ambros ■ CCRI responsible principal investigator: ■ Grant from the Austrian Science Fund (FWF), Ruth Ladenstein ERA-Net Transcan ■ European Commission Grant – ■ Duration: 01/01/2016 to 30/09/2018 Call CEF-TC-2017-2 ■ Duration: 01/01/2018 to 31/12/2020 Optimized diagnostics for improved treatment stratification in invasive fungal diseases (FUNGITECT) ■ CCRI/Labdia Coordinator: Thomas Lion ■ European Commission Grant – FP7 Cooperation Project ■ Duration: 01/02/2014 to 31/01/2019

Anhang Forschungsbericht St. Anna Kinderkrebsforschung | Science Report St. Anna Children’s Cancer Research Institute [ 111 ]

Automation of flow cytometric analysis for TRANSCALL (Translational research in childhood quality-assured follow-up assessment to guide acute lymphoblastic leukemia) curative therapy for acute lymphoblastic leukaemia ■ CCRI responsible principal investigator: in children (AutoFLOW) Renate Panzer-Grümayer ■ CCRI/Labdia partner: Michael Dworzak ■ Grant from the Austrian Science Fund (FWF), ■ Coordinator: Martin Kampel ERA-Net Transcan (Technical University of Vienna, Austria) ■ Duration: 01/07/2013 to 30/06/2017 ■ European Commission Grant – FP7 Industry-Academia Partnerships and PROVABES (prospective validation of biomarkers Pathways (Marie Curie Actions) in Ewing sarcoma for personalized treatment) ■ Duration: 01/02/2014 to 31/01/2018 ■ CCRI responsible principal investigator: Heinrich Kovar EURO EWING Consortium – International clinical ■ Grant from the Austrian Science Fund (FWF), trials to improve survival from Ewing sarcoma (EEC) ERA-Net Transcan ■ CCRI partner: Heinrich Kovar ■ Duration: 01/04/2013 to 31/03/2017 ■ Coordinator: Jeremy Whelan (University College London, UK) International study for treatment of childhood ■ European Commission Grant – relapsed ALL 2010 (IntReALL) FP7 Cooperation Project ■ CCRI partners: Georg Mann, Andishe Attarbaschi ■ Duration: 01/10/2013 to 30/09/2019 and Ruth Ladenstein ■ Coordinator: Jeremy Whelan (University College Molecular mechanisms of human fungal pathogen London, UK) host interaction (ImResFun) ■ European Commission Grant – ■ CCRI partner: Thomas Lion FP7 Cooperation Project ■ Coordinator: Karl Kuchler ■ Duration: 01/10/2011 to 30/09/2017 (Medical University of Vienna, Austria) ■ European Commission Grant – FP7 Initial PanCare childhood and adolescent cancer survivor Training Network (Marie Curie Actions) care and follow-up studies (PanCareSurFup) ■ Duration: 01/10/2013 to 30/09/2017 ■ CCRI/St Anna Spital partners: Eva Frey ■ Coordinator: Lars Hjorth (Lund University, Expert paediatric oncology reference network for Sweden) diagnostics and treatment (ExPO-r-Net) ■ European Commission Grant – ■ CCRI Coordinator: Ruth Ladenstein FP7 Cooperation Project ■ Grant from the European Consumers, Health, ■ Duration: 01/02/2011 to 31/01/2017 Agriculture and Food Executive Agency (CHAFEA) ■ Duration: 01/03/2014 to 31/08/2017

Anhang [ 112 ] Forschungsbericht St. Anna Kinderkrebsforschung | Science Report St. Anna Children’s Cancer Research Institute

National fremdgeförderte Projekte 2017/2018 National Grants 2017/2018

ZF GGAA landscape Platform supporting an integrated analysis of image ■ CCRI responsible principal investigator: and multiOMICs data based on liquid biopsies for Niko Popitsch tumor diagnostics (VISIOMICS) ■ Grant from the Austrian Research Promotion ■ Labdia Coordinator: Sabine Taschner-Mandl, Agency (FFG), Call Talente FEMTech Praktika Peter Ambros Studentinnen 2018 ■ Grant from the Austrian Research Promotion ■ Duration: 01/08/2018 to 30/09/2018 Agency (FFG), COIN Programme ■ Duration: 01/11/2017 to 31/10/2019 Phylogenetic analysis of primary and disseminated Establishing a zebrafish disease model drug tumor cells to reconstruct the metastatic route of screening platform, (Danio4Can) relapsed neuroblastomas ■ CCRI responsible principal investigator: ■ CCRI responsible principal investigator: Martin Distel Reza Abbasi ■ Grant from the Austrian Research Promotion ■ Grant from the Ingrid Shaker Nessmann Agency (FFG), R&D Infrastrukturförderung, Krebsforschungsvereinigung (ISNK) – 1st Call Forschungsförderung 2018 ■ Duration: 01/05/2017 to 31/05/2019 ■ Duration: 01/02/2018 to 31/01/2019 Role of stress granules in Ewing sarcoma Analysis of temporal and spatial Ewing sarcoma susceptibility tumor evolution during chemotherapy and ■ CCRI responsible principal investigator: validation of its clinical implication Heinrich Kovar ■ CCRI coordinator: Eleni Tomazou ■ Grant from the Austrian Science Fund (FWF), ■ Grant from the Austrian National Bank (OeNB), Stand Alone Project Jubiläumsfonds ■ Duration: 01/06/2017 to 31/05/2020 ■ Duration: 01/09/2018 to 31/08/2021 Ewing sarcoma – an enhancer disease? ■ CCRI responsible principal investigator: Eleni Tomazou ■ Grant from the Austrian Science Fund (FWF), Elise Richter Programme ■ Duration: 01/01/2016 to 31/12/2019

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Myeloproliferative neoplasms Liquid biopsy in neuroblastoma: chance for ■ CCRI partner: Thomas Lion diagnosis, prognosis and disease monitoring ■ Coordinator: Peter Valent (Medical University ■ CCRI responsible principal investigator: of Vienna, Austria) Peter Ambros ■ Grant from the Austrian Science Fund (FWF), ■ Grant from the Austrian National Bank (OeNB), Special Research Programme (SFB) Jubiläumsfonds ■ Duration: 01/03/2013 to 28/02/2017 ■ Duration: 01/01/2016 to 30/09/2018

Verfahren zur hochautomatisierten Bewertung Permanent consequences in childhood Langerhans und Klassifikation von Zellen in Gewebeschnitten cell histiocytosis anhand räumlicher Markerprofile (TisQuant) ■ CCRI responsible principal investigator: ■ LABDIA responsible principal investigator: Milen Minkov Peter Ambros ■ Grant from the Austrian National Bank (OeNB), ■ Grant from the Austrian Research Promotion Jubiläumsfonds Agency (FFG), ERA-SME ■ Duration: 01/09/2015 to 31/05/2019 ■ Duration: 01/06/2014 to 31/05/2017 Verbesserte Patientenkommunikation in der Directed in vitro differentiation of induced Onkologie mittels INTERACCT App (OCCURSUS) pluripotent stem cells towards the B lymphoid ■ CCRI responsible principal investigator: lineage (B-different) Anita Lawitschka ■ CCRI coordinator: Klaus Fortschegger ■ Grant from the „Österreichische Gesellschaft für ■ Grant from the Austrian Research Hämatologie und Medizinische Onkologie“ Promotion Agency (FFG), Call Bridge ■ Duration: 01/05/2016 to 31/07/2017 (Brückenschlagprogramm) ■ Duration: 01/06/2014 to 22/12/2017 Virus-specific immunotherapy (VISIT) ■ CCRI/Labdia: coordinator René Geyeregger (Labdia), partner: Matthes-Leodolter (CCRI) ■ Grant from the Wirtschaftsagentur Wien, Call Life Sciences 2014 ■ Duration: 01/04/2015 to 31/03/2018

Automated MRD-assessment in AML (flowCLUSTER) ■ Labdia coordinator: Michael Dworzak ■ Grant from the Wirtschaftsagentur Wien, Call Life Sciences 2014 ■ Duration: 01/03/2015 to 28/02/2018

Anhang [ 114 ] Forschungsbericht St. Anna Kinderkrebsforschung | Science Report St. Anna Children’s Cancer Research Institute

Danksagung Acknowledgments

■ 3. Gesundheitsprogramm der Europäischen Kommission | 3rd Health Programme of the European Commission ■ Abbott Österreich GmbH | Abbott Austria GmbH ■ Alle der St. Anna Kinderkrebsforschung naheste- henden Institutionen, Verbände und Vereine | All institutions, associations and societies related to St. Anna Children‘s Cancer Research Institute ■ Deutsches Krebsregister Universität Mainz | ■ Alle MitarbeiterInnen des St. Anna Kinderspitals | German Cancer Registry, University of Mainz All employees of St. Anna Children‘s Hospital ■ EBMT Europäische Gruppe für Blut- und ■ Bezirksorganisation Alsergrund | Knochenmarktransplantation | District organisation Alsergrund EBMT European Society for Blood and ■ Bundesministerium für Arbeit, Soziales, Marrow Transplantation Gesundheit und Konsumentenschutz | ■ Ethikkommission der Medizinischen Federal Ministry of Labour, Social Affairs, Universität Wien | Health and Consumer Protection Ethics Commission of the Medical ■ Bundesministerium für Bildung, University of Vienna Wissenschaft und Forschung (BMBWF) | ■ Fonds der Stadt Wien für innovative Federal Ministry of Education, interdisziplinäre Krebsforschung | Science and Research (BMBWF) Fund of the City of Vienna for innovative ■ Bundesministerium für Gesundheit und Frauen | interdisciplinary cancer research Federal Ministry for Health and Women ■ Fonds zur Förderung der wissenschaftlichen ■ Bundesministerium für Inneres | Forschung (FWF) | Federal Ministry of the Interior Austrian Science Fund (FWF) ■ Forschungsrahmenprogramme der Europäischen Kommission | Research and Innovation Programmes of the European Commission

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■ Gesellschaft für Pädiatrische Onkologie und Hämatologie (GPOH) | Society for Paediatric Oncology and Haematology ■ Gigax Privatstiftung FL | Gigax Private Trust FL ■ Herzfeldersche Familienstiftung | Herzfeldersch e Family Foundation ■ Ingrid Shaker Nessmann Krebsforschungsvereinigung | Ingrid Shaker Nessmann Cancer Research Association ■ Kapsch AG | Kapsch AG ■ Liddy Shriver Sarkom Initiative | The Liddy Shriver Sarcoma Initiative ■ Marinomed Biotech AG | Marinomed Biotech AG ■ Medac Gesellschaft für klinische Spezialpräparate mbH, Deutschland | Medac Ltd for Specialized Clinical Products ■ Medizinische Universität Wien | Jugendheilkunde (ÖGKJ) | Medical University of Vienna Austrian Society for Paediatric Medicine (ÖGKJ) ■ Medizinisch-Wissenschaftlicher Fonds des ■ Österreichisches Stammzell-Register | Bürgermeisters der Stadt Wien | Austrian Stem Cell Register Medical-Scientific Fund of the ■ Private FörderInnen, MentorInnen, Mayor of the capital city of Vienna Vereinsmitglieder und SpenderInnen der St. Anna ■ Novartis Pharma Österreich GmbH | Kinderkrebsforschung | Novartis Pharma Austria GmbH Private sponsors, mentors, association members ■ Österreichische Akademie der and donors for the St. Anna Children´s Cancer Wissenschaften (ÖAW) | Research Institute Austrian Academy of Sciences (ÖAW) ■ Stadt Wien | City of Vienna ■ Österreichische Forschungsförderungsgesellschaft ■ Stadtrat für Soziales, Gesundheit und Sport | (FFG) | Senior City Councillor for Social Affairs, Health Austrian Research Promotion Agency (FFG) and Sport ■ Österreichische Gesellschaft für Hämatologie & ■ Verein für Dermatologie, Wien | Medizinische Onkologie (ÖGHO) | Association for Dermatology Vienna Austrian Society of Haematology and ■ Wiener Wissenschafts-, Forschungs- und Oncology (ASHO) Technologiefonds (WWTF) | ■ Österreichische Nationalbank (OeNB) | Vienna Science and Technology Fund (WWTF) Austrian National Bank (OeNB) ■ Wirtschaftsagentur Wien | ■ Österreichischen Gesellschaft für Kinder- und Vienna Business Agency

Anhang [ 116 ] Forschungsbericht St. Anna Kinderkrebsforschung | Science Report St. Anna Children’s Cancer Research Institute

Bachelor-, Master-, Diploma- and PhD-Theses

Verena Dastl Is the minimal residual disease (MRD) in HPC apheresis products in patients with neuroblastoma of importance for the prognosis. A retrospective data analysis. > Supervised by Volker Witt, MD and Assoc.-Prof. Peter Ambros, PhD Diploma Thesis

Helena Dodig Analysis of the anti-proliferative and differentiation-inducing effects of Schwann cells on neuroblastoma cells. > Supervised by Sabine Taschner-Mandl, PhD and Assoc.-Prof. Peter Ambros, PhD Master Thesis

Kristin Fischer Establishing an optogenetic system for spatially and temporally controlled transgene expression in zebrafish. Supervised by Univ.-Prof. Heinrich Kovar, PhD and Martin Distel, PhD Master Thesis

Anhang Forschungsbericht St. Anna Kinderkrebsforschung | Science Report St. Anna Children’s Cancer Research Institute [ 117 ]

Alwine Hildebrandt Lisa Saloberger Mechanisms of ETV6/RUNX1- Validation of pre-analytical parameters to introduce Mediated Gene Regulation in Leukemia cell-free tumor DNA as reliable minimal-invasive > Supervised by Univ.-Prof. Renate Panzer- diagnostic tool. Grümayer, MD > Supervised by Assoc.-Prof. Peter Ambros, PhD Master Thesis Master Thesis

Anna Maria Katschnig Klara Soukup The influence of EWS-FLI1 on the Rho/MRTF/ MK2 in immune regulation by dendritic cells. actin circuit in Ewing sarcoma. > Supervised by Alexander Dohnal, PhD > Supervised by Univ.-Prof. Heinrich Kovar, PhD PhD Thesis PhD Thesis

Maria Regina Strobl Sophie Lackner Establishment of induced pluripotent stem cell l DUX4 in human disease (BAC1). Detection of DUX4 ines carrying a CD34 transcriptional reporter using expression in paediatric B-cell precursor acute CRISPR/Cas9. lymphoblastic leukemia (BAC2). > Supervised by Klaus Fortschegger, PhD > Supervised by Sabine Strehl, PhD and Klaus Master Thesis Fortschegger, PhD Bachelor Thesis Tamara Weiss Investigation of human Schwann cells in nerve Christian Posch regeneration and neuroblastoma growth inhibition. Signaling of NRAS mutant melanoma and resulting > Supervised by Assoc.-Prof. Peter Ambros, PhD therapeutic consequences. and Sabine Taschner-Mandl, PhD > Supervised by Univ.-Prof. Renate Panzer- PhD Thesis Grümayer, MD PhD Thesis Jakob Winkler FACS2VIS – Data scraping, database implementation Fikret Rifatbegovic and visualization for analyzability of flow cytometry The transcriptomic landscape of bone marrow data related to pediatric stem cell transplantation derived disseminated neuroblastoma cells. > Dipl.-Sporting. Mario Heller, PhD and > Supervised by Assoc.-Prof. Peter Ambros, PhD Priv.-Doz. René Geyeregger, PhD PhD Thesis Master Thesis

Anhang [ 118 ] Forschungsbericht St. Anna Kinderkrebsforschung | Science Report St. Anna Children’s Cancer Research Institute

Publikationen/Publications 2017

Abbasi, M.R., F. Rifatbegovic, C. Brunner, G. Mann, A. Bigenzahn, S., B. Juergens, B. Mahr, J. Pratschke, A. Ziegler, U. Potschger, R. Crazzolara, M. Ussowicz, M. Koenigsrainer, T. Becker, D. Fuchs, G. Brandacher, A. Benesch, G. Ebetsberger-Dachs, G.C.F. Chan, N. Jones, Kainz, F. Muehlbacher, and T. Wekerle. R. Ladenstein, I.M. Ambros, and P.F. Ambros. No Augmentation of Indoleamine 2,3-Dioxygenase Impact of Disseminated Neuroblastoma Cells on (Ido) Activity through Belatacept Treatment in Liver the Identification of the Relapse-Seeding Clone. Transplant Recipients. Clin Exp Immunol, 2017. Clin Cancer Res, 2017. 23(15): p. 4224-4232. Bileck, A., R.L. Mayer, D. Kreutz, T. Weiss, S. Taschner- Aichhorn, S., A. Linhardt, A. Halfmann, M. Nadlinger, S. Mandl, S.M. Meier, A. Slany, and C. Gerner. Kirchberger, M. Stadler, B. Dillinger, M. Distel, A. Evaluation of inflammation-related signaling events Dohnal, I. Teasdale, and W. Schofberger. covering phosphorylation and nuclear translocation A pH-sensitive Macromolecular Prodrug as TLR7/8 of proteins based on mass spectrometry data. Targeting Immune Response Modifier. Chemistry, J Proteomics, 2017. 152: p. 161-171. 2017. 23(70): p. 17721-17726. Byrgazov, K., C.B. Lucini, P. Valent, O. Hantschel, and Amoroso, L., G. Erminio, G. Makin, A.D.J. Pearson, P. T. Lion. Brock, D. Valteau-Couanet, V. Castel, M. Pasquet, G. BCR-ABL1 compound mutants display differential Laureys, C. Thomas, R. Luksch, R. Ladenstein, R. Haupt, and dose-dependent responses to ponatinib. A. Garaventa, and S. Group. Haematologica, 2018 Epub 2017 Oct 5. 103(1): Topotecan-Vincristine-Doxorubicin in Stage 4 High- p. e10-e12. Risk Neuroblastoma Patients Failing to Achieve a Complete Metastatic Response to Rapid COJEC: A Colomer-Lahiguera, S., M. Pisecker, M. Konig, K. Nebral, SIOPEN Study. Cancer Res Treat, 2018 Epub 2017 W.F. Pickl, M.O. Kauer, O.A. Haas, R. Ullmann, A. Mar 21. 50(1): p. 148-155. Attarbaschi, M.N. Dworzak, and S. Strehl. MEF2C-dysregulated pediatric T-cell acute Balendran, S., S. Liebmann-Reindl, A.S. Berghoff, T. lymphoblastic leukemia is associated with Reischer, N. Popitsch, C.B. Geier, L. Kenner, P. Birner, B. CDKN1B deletions and a poor response to Streubel, and M. Preusser. glucocorticoid therapy. Leuk Lymphoma, Next-Generation Sequencing-based genomic 2017. 58(12): p. 2895-2904. profiling of brain metastases of primary ovarian cancer identifies high number of BRCA-mutations. J Neurooncol, 2017. 133(3): p. 469-476.

Anhang Forschungsbericht St. Anna Kinderkrebsforschung | Science Report St. Anna Children’s Cancer Research Institute [ 119 ]

Creutzig, U., M.N. Dworzak, K. Bochennek, J. Faber, C. Dworzak, M.N., B. Buldini, G. Gaipa, R. Ratei, O. Flotho, N. Graf, U. Kontny, C. Rossig, I. Schmid, A. von Hrusak, D. Luria, E. Rosenthal, J.P. Bourquin, M. Sartor, Stackelberg, J.E. Mueller, C. von Neuhoff, D. Reinhardt, A. Schumich, L. Karawajew, E. Mejstrikova, O. Maglia, and N. von Neuhoff. G. Mann, W.D. Ludwig, A. Biondi, M. Schrappe, G. First experience of the AML-Berlin-Frankfurt- Basso, and B.F.M.F.n. International. Munster group in pediatric patients with standard- AIEOP-BFM consensus guidelines 2016 for flow risk acute promyelocytic leukemia treated with cytometric immunophenotyping of Pediatric acute arsenic trioxide and all-trans retinoid acid. lymphoblastic leukemia. Cytometry B Clin Cytom, Pediatr Blood Cancer, 2017. 64(8). 2018 Epub 2017 Feb 21. 94(1): p. 82-93.

Creutzig, U., M.N. Dworzak, M. Zimmermann, D. Farrokhi, S., M. Shabani, Z. Aryan, S. Zoghi, A. Krolo, Reinhardt, L. Sramkova, J.P. Bourquin, H. Hasle, J. K. Boztug, and N. Rezaei. Abrahamsson, G. Kaspers, M.M. van den Heuvel, A.M.J. MHC class II deficiency: Report of a novel Reedijk, B. De Moerloose, F. Locatelli, and R. Masetti. mutation and special review. Characteristics and outcome in patients with Allergol Immunopathol (Madr), 2017. central nervous system involvement treated in European pediatric acute myeloid leukemia study Fernandez, A., L. Gruner-Nielsen, M. Andreana, M. groups. Pediatr Blood Cancer, 2017. 64(12). Stadler, S. Kirchberger, C. Sturtzel, M. Distel, L. Zhu, W. Kautek, R. Leitgeb, A. Baltuska, K. Jespersen, and A. Dillinger, B., S. Ahmadi-Erber, K. Soukup, A. Halfmann, Verhoef. S. Schrom, B. Vanhove, P. Steinberger, R. Geyeregger, S. Optimizing pulse compressibility in completely Ladisch, and A.M. Dohnal. all-fibered Ytterbium chirped pulse amplifiers for in CD28 Blockade Ex Vivo Induces Alloantigen- vivo two photon laser scanning microscopy. Specific Immune Tolerance but Preserves Biomed Opt Express, 2017. 8(8): p. 3526-3537. T-Cell Pathogen Reactivity. Front Immunol, 2017. 8(1152): p. 1152. Gardiner, J.D., L.M. Abegglen, X. Huang, B.E. Carter, E.A. Schackmann, M. Stucki, C.N. Paxton, R. Lor Dixon, Z.A., L. Nicholson, M. Zeppetzauer, E. Matheson, Randall, J.F. Amatruda, A.R. Putnam, H. Kovar, S.L. P. Sinclair, C.J. Harrison, and J.A. Irving. Lessnick, and J.D. Schiffman. CREBBP knockdown enhances RAS/RAF/MEK/ERK C/EBPbeta-1 promotes transformation and signaling in Ras pathway mutated acute chemoresistance in Ewing sarcoma cells. lymphoblastic leukemia but does not modulate Oncotarget, 2017. 8(16): p. 26013-26026. chemotherapeutic response. Haematologica, 2017. 102(4): p. 736-745. Gleixner, K.V., M. Schneeweiss, G. Eisenwort, D. Berger, H. Herrmann, K. Blatt, G. Greiner, K. Byrgazov, G. Hoermann, M. Konopleva, I. Waliul, A.A. Cumaraswamy, P.T. Gunning, H. Maeda, R. Moriggl, M. Deininger, T. Lion, M. Andreeff, and P. Valent. Combined targeting of STAT3 and STAT5: a novel approach to overcome drug resistance in chronic myeloid leukemia. Haematologica, 2017. 02(9): p. 1519-1529. Anhang [ 120 ] Forschungsbericht St. Anna Kinderkrebsforschung | Science Report St. Anna Children’s Cancer Research Institute

Gough, S.M., L. Goldberg, M. Pineda, R.L. Walker, Y.J. Katschnig, A.M., M.O. Kauer, R. Schwentner, E.M. Zhu, S. Bilke, Y.J. Chung, J. Dufraine, S. Kundu, E. Tomazou, C.N. Mutz, M. Linder, M. Sibilia, J. Alonso, Jacoby, T.J. Fry, S. Fischer, R. Panzer-Grumayer, P.S. D.N.T. Aryee, and H. Kovar. Meltzer, and P.D. Aplan. EWS-FLI1 perturbs MRTFB/YAP-1/TEAD target Progenitor B-1 B-cell acute lymphoblastic gene regulation inhibiting cytoskeletal leukemia is associated with collaborative mutations autoregulatory feedback in Ewing sarcoma. in 3 critical pathways. Blood Adv, 2017. Oncogene, 2017. 36(43): p. 5995-6005. 1(20): p. 1749-1759. Kirchberger, S., C. Sturtzel, S. Pascoal, and M. Distel. Haindl, R., S. Preisser, M. Andreana, W. Rohringer, C. Quo natas, Danio?-Recent Progress in Modeling Sturtzel, M. Distel, Z. Chen, E. Rank, B. Fischer, W. Cancer in Zebrafish. Front Oncol, 2017. 7: p. 186. Drexler, and M. Liu. Dual modality reflection mode optical coherence Kolenova, A., R. Schwentner, G. Jug, I. Simonitsch- and photoacoustic microscopy using an akinetic Klupp, C. Kornauth, L. Plank, J. Horakova, I. Bodova, T. sensor. Opt Lett, 2017. 42(21): p. 4319-4322. Sykora, L. Geczova, W. Holter, M. Minkov, and C. Hutter. Targeted inhibition of the MAPK pathway: emerging He, T., D. Surdez, J.K. Rantala, S. Haapa-Paananen, J. salvage option for progressive life-threatening Ban, M. Kauer, E. Tomazou, V. Fey, J. Alonso, H. Kovar, multisystem LCH. Blood Adv, 2017. 1(6): p. 352-356. O. Delattre, and K. Iljin. High-throughput RNAi screen in Ewing sarcoma Kostel Bal, S., S. Haskologlu, N.K. Serwas, C. Islamoglu, cells identifies leucine rich repeats and WD repeat C. Aytekin, T. Kendirli, Z. Kuloglu, G. Yavuz, B. Dalgic, domain containing 1 (LRWD1) as a regulator of Z. Siklar, A. Kansu, A. Ensari, K. Boztug, F. Dogu, and A. EWS-FLI1 driven cell viability. Ikinciogullari. Gene, 2017. 596: p. 137-146. Multiple Presentations of LRBA Deficiency: a Single-Center Experience. Hoeger, B., N.K. Serwas, and K. Boztug. J Clin Immunol, 2017. 37(8): p. 790-800. Human NF-kappaB1 Haploinsufficiency and Epstein-Barr Virus-Induced Disease-Molecular Krenn, M., E. Salzer, I. Simonitsch-Klupp, J. Rath, M. Mechanisms and Consequences. Wagner, T.B. Haack, T.M. Strom, A. Schanzer, M.W. Front Immunol, 2018, eCollection 2017. 8: p. 1978. Kilimann, R.L.J. Schmidt, K.G. Schmetterer, A. Zimprich, K. Boztug, A. Hahn, and F. Zimprich. Husak, Z. and M.N. Dworzak. Mutations outside the N-terminal part of RBCK1 Chronic stress induces CD99, suppresses may cause polyglucosan body myopathy with autophagy, and affects spontaneous adipogenesis in immunological dysfunction: expanding the human bone marrow stromal cells. genotype-phenotype spectrum. J Neurol, Stem Cell Res Ther, 2017. 8(1): p. 83. 2018 Epub 2017 Dec 19. 265(2): p. 394-401. Kainrath, S., M. Stadler, E. Reichhart, M. Distel, and H. Janovjak. Green-Light-Induced Inactivation of Receptor Signaling Using Cobalamin-Binding Domains. Angew Chem Int Ed Engl, 2017. 56(16): p. 4608-4611.

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Ladenstein, R., B. Lambert, U. Potschger, M.R. Castellani, Lipka, D.B., T. Witte, R. Toth, J. Yang, M. Wiesenfarth, P. V. Lewington, Z. Bar-Sever, A. Oudoux, A. Sliwinska, K. Nollke, A. Fischer, D. Brocks, Z. Gu, J. Park, B. Strahm, Taborska, L. Biassoni, G.A. Yanik, A. Naranjo, M.T. M. Wlodarski, A. Yoshimi, R. Claus, M. Lubbert, H. Parisi, B.L. Shulkin, H. Nadel, M.J. Gelfand, K.K. Busch, M. Boerries, M. Hartmann, M. Schonung, U. Matthay, J.R. Park, S.G. Kreissman, D. Valteau-Couanet, Kilik, J. Langstein, J.A. Wierzbinska, C. Pabst, S. Garg, A. and A. Boubaker. Catala, B. De Moerloose, M. Dworzak, H. Hasle, F. Validation of the mIBG skeletal SIOPEN scoring Locatelli, R. Masetti, M. Schmugge, O. Smith, J. Stary, M. method in two independent high-risk Ussowicz, M.M. van den Heuvel-Eibrink, Y. Assenov, M. neuroblastoma populations: the SIOPEN/HR-NBL1 Schlesner, C. Niemeyer, C. Flotho, and C. Plass. and COG-A3973 trials. Eur J Nucl Med Mol RAS-pathway mutation patterns define epigenetic Imaging, 2018 Epub 2017 Sep 23. 45(2): p. 292-305. subclasses in juvenile myelomonocytic leukemia. Nat Commun, 2017. 8(1): p. 2126. Ladenstein, R., U. Potschger, A.D.J. Pearson, P. Brock, R. Luksch, V. Castel, I. Yaniv, V. Papadakis, G. Laureys, J. Locatelli, F., M.G. Valsecchi, A. Moricke, M. Malis, W. Balwierz, E. Ruud, P. Kogner, H. Schroeder, Zimmermann, B. Gruhn, A. Biondi, A.E. Kulozik, D. A.F. de Lacerda, M. Beck-Popovic, P. Bician, M. Garami, Silvestri, N. Bodmer, M.C. Putti, S. Burdach, C. T. Trahair, A. Canete, P.F. Ambros, K. Holmes, M. Gaze, Micalizzi, A. Teigler-Schlegel, J. Ritter, A. Pession, G. G. Schreier, A. Garaventa, G. Vassal, J. Michon, D. Val- Cario, S. Bielack, G. Basso, T. Klingebiel, L. Vinti, C. teau-Couanet, and S.E.N. Group. Rizzari, A. Attarbaschi, N. Santoro, R. Parasole, G. Mann, Busulfan and melphalan versus carboplatin, L. Karawajew, O.A. Haas, V. Conter, and M. Schrappe. etoposide, and melphalan as high-dose Protocol II vs protocol III given twice during chemotherapy for high-risk neuroblastoma reinduction therapy in children with medium-risk (HR-NBL1/SIOPEN): an international, randomised, ALL. Blood, 2017. 130(19): p. 2146-2149. multi-arm, open-label, phase 3 trial. Lancet Oncol, 2017. 18(4): p. 500-514. Meier, S.M., D. Kreutz, L. Winter, M.H.M. Klose, K. Cseh, T. Weiss, A. Bileck, B. Alte, J.C. Mader, S. Jana, A. Li, J., T. Casteels, T. Frogne, C. Ingvorsen, C. Honore, M. Chatterjee, A. Bhattacharyya, M. Hejl, M.A. Jakupec, P. Courtney, K.V.M. Huber, N. Schmitner, R.A. Kimmel, Heffeter, W. Berger, C.G. Hartinger, B.K. Keppler, G. R.A. Romanov, C. Sturtzel, C.H. Lardeau, J. Wiche, and C. Gerner. Klughammer, M. Farlik, S. Sdelci, A. Vieira, F. Avolio, F. An Organoruthenium Anticancer Agent Shows Briand, I. Baburin, P. Majek, F.M. Pauler, T. Penz, A. Unexpected Target Selectivity For Plectin. Angew Stukalov, M. Gridling, K. Parapatics, C. Barbieux, E. Chem Int Ed Engl, 2017. 56(28): p. 8267-8271. Berishvili, A. Spittler, J. Colinge, K.L. Bennett, S. Hering, T. Sulpice, C. Bock, M. Distel, T. Harkany, D. Meyer, G. Superti-Furga, P. Collombat, J. Hecksher-Sorensen, and S. Kubicek. Artemisinins Target GABAA Receptor Signaling and Impair alpha Cell Identity. Cell, 2017. 168(1-2): p. 86-100 e15.

Anhang [ 122 ] Forschungsbericht St. Anna Kinderkrebsforschung | Science Report St. Anna Children’s Cancer Research Institute

Meyer, C., T. Burmeister, D. Groger, G. Tsaur, L. Fechina, Muller, H., R. Jimenez-Heredia, A. Krolo, T. Hirschmugl, A. Renneville, R. Sutton, N.C. Venn, M. Emerenciano, J. Dmytrus, K. Boztug, and C. Bock. M.S. Pombo-de-Oliveira, C. Barbieri Blunck, B. Almeida VCF.Filter: interactive prioritization of disease- Lopes, J. Zuna, J. Trka, P. Ballerini, H. Lapillonne, M. De linked genetic variants from sequencing data. Braekeleer, G. Cazzaniga, L. Corral Abascal, V.H.J. van Nucleic Acids Res, 2017. 45(W1): p. W567-W572. der Velden, E. Delabesse, T.S. Park, S.H. Oh, M.L.M. Silva, T. Lund-Aho, V. Juvonen, A.S. Moore, O. Mutz, C.N., R. Schwentner, D.N.T. Aryee, E.D.J. Heidenreich, J. Vormoor, E. Zerkalenkova, Y. Bouchard, E.M. Mejia, G.M. Hatch, M.O. Kauer, A.M. Olshanskaya, C. Bueno, P. Menendez, A. Teigler-Schlegel, Katschnig, J. Ban, A. Garten, J. Alonso, V. Banerji, and H. U. Zur Stadt, J. Lentes, G. Gohring, A. Kustanovich, O. Kovar. Aleinikova, B.W. Schafer, S. Kubetzko, H.O. Madsen, B. EWS-FLI1 confers exquisite sensitivity to Gruhn, X. Duarte, P. Gameiro, E. Lippert, A. Bidet, J.M. NAMPT inhibition in Ewing sarcoma cells. Cayuela, E. Clappier, C.N. Alonso, C.M. Zwaan, M.M. Oncotarget, 2017. 8(15): p. 24679-24693. van den Heuvel-Eibrink, S. Izraeli, L. Trakhtenbrot, P. Archer, J. Hancock, A. Moricke, J. Alten, M. Schrappe, Nogueira, F., F. Istel, L. Pereira, M. Tscherner, M. Stanulla, S. Strehl, A. Attarbaschi, M. Dworzak, O.A. and K. Kuchler. Haas, R. Panzer-Grumayer, L. Sedek, T. Szczepanski, A. Immunological Identification of Fungal Species. Caye, L. Suarez, H. Cave, and R. Marschalek. Methods Mol Biol, 2017. 1508: p. 339-359. The MLL recombinome of acute leukemias in 2017. Leukemia, 2018 Epub 2017 Jul 13. 32(2): p. 273-284. Nogueira, M., F. Istel, S. Jenull, L. Walker, N. Gow, and T. Lion. Millot, F., C. Dupraz, J. Guilhot, M. Suttorp, F. Brizard, T. Quantitative Analysis of Candida Cell Wall Leblanc, A.M. Gunes, P. Sedlacek, E. De Bont, C.K. Li, K. Components by Flow Cytometry with Triple- Kalwak, B. Lausen, S. Culic, M. Dworzak, E. Kaiserova, Fluorescence Staining. Journal of Microbiology and B. De Moerloose, F. Roula, A. Biondi, A. Baruchel, and F. Modern Techniques, 2017. 2(1): p. 1. Guilhot. Additional cytogenetic abnormalities and variant Obenauer, J.C., F.G. Kavelaars, M.A. Sanders, A.C.H. de t(9;22) at the diagnosis of childhood chronic Vries, V. de Haas, H.B. Beverloo, B. De Moerloose, T. myeloid leukemia: The experience of the Lammens, M. Dworzak, R.M. Hoogenboezem, P.J.M. International Registry for Chronic Myeloid Valk, I.P. Touw, and M.M. van den Heuvel-Eibrink. Leukemia in Children and Adolescents. Recurrently affected genes in juvenile Cancer, 2017. 123(18): p. 3609-3616. myelomonocytic leukaemia. Br J Haematol, 2017.

Millot, F., J. Guilhot, M. Suttorp, A.M. Gunes, P. Sedlacek, E. De Bont, C.K. Li, K. Kalwak, B. Lausen, S. Culic, M. Dworzak, E. Kaiserova, B. De Moerloose, F. Roula, A. Biondi, and A. Baruchel. Prognostic discrimination based on the EUTOS long- term survival score within the International Registry for Chronic Myeloid Leukemia in children and adole scents. Haematologica, 2017. 102(10): p. 1704-1708.

Anhang Forschungsbericht St. Anna Kinderkrebsforschung | Science Report St. Anna Children’s Cancer Research Institute [ 123 ]

Ozen, A., W.A. Comrie, R.C. Ardy, C. Dominguez Radic-Sarikas, B., K.P. Tsafou, K.B. Emdal, T. Conde, B. Dalgic, O.F. Beser, A.R. Morawski, E. Karakoc- Papamarkou, K.V. Huber, C. Mutz, J.A. Toretsky, K.L. Aydiner, E. Tutar, S. Baris, F. Ozcay, N.K. Serwas, Y. Bennett, J.V. Olsen, S. Brunak, H. Kovar, and G. Superti- Zhang, H.F. Matthews, S. Pittaluga, L.R. Folio, A. Furga. Unlusoy Aksu, J.J. McElwee, A. Krolo, A. Kiykim, Z. Combinatorial Drug Screening Identifies Baris, M. Gulsan, I. Ogulur, S.B. Snapper, R.H.J. Houwen, Ewing Sarcoma-specific Sensitivities. H.L. Leavis, D. Ertem, R. Kain, S. Sari, T. Erkan, H.C. Su, Mol Cancer Ther, 2017. 16(1): p. 88-101. K. Boztug, and M.J. Lenardo. CD55 Deficiency, Early-Onset Protein-Losing Ranft, A., C. Seidel, C. Hoffmann, M. Paulussen, A.C. Enteropathy, and Thrombosis. Warby, H. van den Berg, R. Ladenstein, C. Rossig, U. N Engl J Med, 2017. 377(1): p. 52-61. Dirksen, D. Rosenbaum, and H. Juergens. Quality of Survivorship in a Rare Disease: Panzer-Grumayer, R., S. Kohrer, and O.A. Haas. Clinicofunctional Outcome and Physical Activity in an The enigmatic role(s) of P2RY8-CRLF2. Observational Cohort Study of 618 Long-Term Oncotarget, 2017. 8(57): p. 96466-96467. Survivors of Ewing Sarcoma. J Clin Oncol, 2017. 35(15): p. 1704-1712. Park, J.R., R. Bagatell, S.L. Cohn, A.D. Pearson, J.G. Villablanca, F. Berthold, S. Burchill, A. Boubaker, K. Rasche, M., C. von Neuhoff, M. Dworzak, J.P. Bourquin, McHugh, J.G. Nuchtern, W.B. London, N.L. Seibel, O.W. J. Bradtke, G. Gohring, G. Escherich, G. Fleischhack, N. Lindwasser, J.M. Maris, P. Brock, G. Schleiermacher, R. Graf, B. Gruhn, O.A. Haas, T. Klingebiel, B. Kremens, T. Ladenstein, K.K. Matthay, and D. Valteau-Couanet. Lehrnbecher, A. von Stackelberg, J. Tchinda, Z. Revisions to the International Neuroblastoma Zemanova, C. Thiede, N. von Neuhoff, M. Zimmermann, Response Criteria: A Consensus Statement From the U. Creutzig, and D. Reinhardt. National Cancer Institute Clinical Trials Planning Genotype-outcome correlations in pediatric AML: Meeting. J Clin Oncol, 2017. 35(22): p. 2580-2587. the impact of a monosomal karyotype in trial AML- Pfajfer, L., M.G. Seidel, R. Houmadi, J. Rey-Barroso, T. BFM 2004. Leukemia, 2017. 31(12): p. 2807-2814. Hirschmugl, E. Salzer, I.M. Anton, C. Urban, W. Rifatbegovic, F., C. Frech, M.R. Abbasi, S. Taschner- Schwinger, K. Boztug, and L. Dupre. Mandl, T. Weiss, W.M. Schmidt, I. Schmidt, R. WIP deficiency severely affects human lymphocyte Ladenstein, I.M. Ambros, and P.F. Ambros. architecture during migration and synapse assembly. Neuroblastoma cells undergo transcriptomic Blood, 2017. 130(17): p. 1949-1953. alterations upon dissemination into the bone marrow and subsequent tumor progression. Pichler, H., K. Horner, G. Engstler, U. Poetschger, E. Int J Cancer, 2018 Epub 2017 Oct 4. 142(2): p. 297-307. Glogova, S. Karlhuber, M. Martin, W. Eibler, V. Witt, W. Holter, and S. Matthes-Martin. Schmah, J., B. Fedders, R. Panzer-Grumayer, S. Fischer, Cost-Effectiveness of Defibrotide in the Prophylaxis M. Zimmermann, E. Dagdan, S. Bens, D. Schewe, A. of Veno-Occlusive Disease after Pediatric Allogeneic Moericke, J. Alten, K. Bleckmann, R. Siebert, M. Stem Cell Transplantation. Biol Blood Marrow Schrappe, M. Stanulla, and G. Cario. Transplant, 2017. 23(7): p. 1128-1133. Molecular characterization of acute lymphoblastic leukemia with high CRLF2 gene expression in childhood. Pediatr Blood Cancer, 2017. 64(10).

Anhang [ 124 ] Forschungsbericht St. Anna Kinderkrebsforschung | Science Report St. Anna Children’s Cancer Research Institute

Schrappe, M., K. Bleckmann, M. Zimmermann, A. Sheffield, N.C., G. Pierron, J. Klughammer, P. Datlinger, Biondi, A. Moricke, F. Locatelli, G. Cario, C. Rizzari, A. A. Schonegger, M. Schuster, J. Hadler, D. Surdez, D. Attarbaschi, M.G. Valsecchi, C.R. Bartram, E. Barisone, Guillemot, E. Lapouble, P. Freneaux, J. Champigneulle, F. Niggli, C. Niemeyer, A.M. Testi, G. Mann, O. Ziino, B. R. Bouvier, D. Walder, I.M. Ambros, C. Hutter, E. Sorz, Schafer, R. Panzer-Grumayer, R. Beier, R. Parasole, G. A.T. Amaral, E. de Alava, K. Schallmoser, D. Strunk, B. Gohring, W.D. Ludwig, F. Casale, P.G. Schlegel, G. Basso, Rinner, B. Liegl-Atzwanger, B. Huppertz, A. Leithner, G. and V. Conter. de Pinieux, P. Terrier, V. Laurence, J. Michon, R. Reduced-Intensity Delayed Intensification in Ladenstein, W. Holter, R. Windhager, U. Dirksen, P.F. Standard-Risk Pediatric Acute Lymphoblastic Leukemia Ambros, O. Delattre, H. Kovar, C. Bock, and E.M. Defined by Undetectable Minimal Residual Disease: Tomazou. Results of an International Randomized Trial DNA methylation heterogeneity defines a (AIEOP-BFM ALL 2000). J Clin Oncol, disease spectrum in Ewing sarcoma. 2018 Epub 2017 Nov 17. 36(3): p. 244-253. Nat Med, 2017. 23(3): p. 386-395.

Schwab, C., K. Nebral, L. Chilton, C. Leschi, E. Smeenk, L., M. Fischer, S. Jurado, M. Jaritz, A. Azaryan, Waanders, J.M. Boer, M. Zaliova, R. Sutton, Ofverholm, B. Werner, M. Roth, J. Zuber, M. Stanulla, M.L. den Boer, II, K. Ohki, Y. Yamashita, S. Groeneveld-Krentz, E. C.G. Mullighan, S. Strehl, and M. Busslinger. Fronkova, M. Bakkus, J. Tchinda, T.D.C. Barbosa, G. Molecular role of the PAX5-ETV6 oncoprotein in Fazio, W. Mlynarski, A. Pastorczak, G. Cazzaniga, M.S. promoting B-cell acute lymphoblastic leukemia. Pombo-de-Oliveira, J. Trka, R. Kirschner-Schwabe, T. EMBO J, 2017. 36(6): p. 718-735. Imamura, G. Barbany, M. Stanulla, A. Attarbaschi, R. Panzer-Grumayer, R.P. Kuiper, M.L. den Boer, H. Cave, Soukup, K., A. Halfmann, B. Dillinger, F. Poyer, K. A.V. Moorman, C.J. Harrison, and S. Strehl. Martin, B. Blauensteiner, M. Kauer, M. Kuttke, G. Intragenic amplification of PAX5: a novel subgroup Schabbauer, and A.M. Dohnal. in B-cell precursor acute lymphoblastic leukemia? Loss of MAPK-activated protein kinase 2 enables Blood Adv, 2017. 1(19): p. 1473-1477. potent dendritic cell-driven anti-tumour T cell response. Sci Rep, 2017. 7(1): p. 11746. Sedlyarova, N., P. Rescheneder, A. Magan, N. Popitsch, N. Rziha, I. Bilusic, V. Epshtein, B. Zimmermann, M. Speckmann, C., S.S. Sahoo, M. Rizzi, S. Hirabayashi, A. Lybecker, V. Sedlyarov, R. Schroeder, and E. Nudler. Karow, N.K. Serwas, M. Hoemberg, N. Damatova, D. Natural RNA Polymerase Aptamers Regulate Schindler, J.B. Vannier, S.J. Boulton, U. Pannicke, G. Transcription in E. coli. Gohring, K. Thomay, J.J. Verdu-Amoros, H. Hauch, W. Mol Cell, 2017. 67(1): p. 30-43 e6. Woessmann, G. Escherich, E. Laack, L. Rindle, M. Seidl, A. Rensing-Ehl, E. Lausch, C. Jandrasits, B. Strahm, K. Sesti, A., K. Rappersberger, and C. Posch. Schwarz, S.R. Ehl, C. Niemeyer, K. Boztug, and M.W. Bullous Sweet‘s syndrome with pulmonary Wlodarski. involvement. Hautarzt, 2017. 68(8): p. 649-652. Clinical and Molecular Heterogeneity of RTEL1 Deficiency. Front Immunol, 2017. 8: p. 449.

Anhang Forschungsbericht St. Anna Kinderkrebsforschung | Science Report St. Anna Children’s Cancer Research Institute [ 125 ]

Sturtzel, C. Endothelial Cells. Adv Exp Med Biol, 2017. 1003: p. 71-91.

Thannesberger, J., H.J. Hellinger, I. Klymiuk, M.T. Kastner, F.J.J. Rieder, M. Schneider, S. Fister, T. Lion, K. Kosulin, J. Laengle, M. Bergmann, T. Rattei, and C. Steininger. Viruses comprise an extensive pool of mobile genetic elements in eukaryote cell cultures and human clinical samples. FASEB J, 2017. 31(5): p. 1987-2000.

Uffmann, M., M. Rasche, M. Zimmermann, C. von Neuhoff, U. Creutzig, M. Dworzak, L. Scheffers, H. Hasle, C.M. Zwaan, D. Reinhardt, and J.H. Klusmann. Therapy reduction in patients with Down syndrome and myeloid leukemia: the international ML-DS 2006 trial. Blood, 2017. 129(25): p. 3314-3321.

van der Lelij, P., S. Lieb, J. Jude, G. Wutz, C.P. Santos, K. Falkenberg, A. Schlattl, J. Ban, R. Schwentner, T. Hoffmann, H. Kovar, F.X. Real, T. Waldman, M.A. Pearson, N. Kraut, J.M. Peters, J. Zuber, and M. Petronczki. Synthetic lethality between the cohesin subunits STAG1 and STAG2 in diverse cancer contexts. Elife, 2017. 6.

Worel, N., N. Frank, C. Frech, and G. Fritsch. Influence of plerixafor on the mobilization of CD34+ cell subpopulations and lymphocyte subtypes. Transfusion, 2017. 57(9): p. 2206-2215.

Yanik, G.A., M.T. Parisi, A. Naranjo, H. Nadel, M.J. Gelfand, J.R. Park, R.L. Ladenstein, U. Poetschger, A. Boubaker, D. Valteau-Couanet, B. Lambert, M.R. Castellani, Z. Bar-Sever, A. Oudoux, A. Kaminska, S.G. Kreissman, B.L. Shulkin, and K.K. Matthay. Validation of Postinduction Curie Scores in High-Risk Neuroblastoma: A Children‘s Oncology Group and SIOPEN Group Report on SIOPEN/HR-NBL1. J Nucl Med, 2018 Epub 2017 Sep 8. 59(3): p. 502-508. Anhang [ 126 ] Forschungsbericht St. Anna Kinderkrebsforschung | Science Report St. Anna Children’s Cancer Research Institute

Publikationen/Publications 2018

Avery, D.T., Kane, A., Nguyen, T., Lau, A., Nguyen, Brey, C.U., Proff, J., Teufert, N., Salzer, B., Brozy, J., A., Lenthall, H., Payne, K., Shi, W., Brigden, H., Munz, M., Pendzialek, J., Ensser, A., Holter, W., and French, E., Bier, J., Hermes, J.R., Zahra, D., Sewell, Lehner, M. (2018). W.A., Butt, D., Elliott, M., Boztug, K., Meyts, I., A gB/CD3 bispecific BiTE antibody construct for Choo, S., Hsu, P., Wong, M., Berglund, L.J., Gray, P., targeting Human Cytomegalovirus-infected cells. O‘Sullivan, M., Cole, T., Holland, S.M., Ma, C.S., Sci Rep 8, 17453. Burkhart, C., Corcoran, L.M., Phan, T.G., Brink, R., Uzel, G., Deenick, E.K., and Tangye, S.G. (2018). Byrne, J., Alessi, D., Allodji, R.S., Bagnasco, F., Bardi, Germline-activating mutations in PIK3CD E., Bautz, A., Bright, C.J., Brown, M., Diallo, I., compromise B cell development and function. Feijen, E., Fidler, M.M., Frey, E., Garwicz, S., J Exp Med 215, 2073-2095. Grabow, D., Gudmundsdottir, T., Hagberg, O., Harila-Saari, A., Hau, E.M., Haupt, R., Hawkins, Berbegall, A.P., Bogen, D., Potschger, U., Beiske, K., M.M., Jakab, Z., Jankovic, M., Kaatsch, P., Kaiser, M., Bown, N., Combaret, V., Defferrari, R., Jeison, M., Kremer, L.C.M., Kuehni, C.E., Kuonen, R., Mazzocco, K., Varesio, L., Vicha, A., Ash, S., Castel, Ladenstein, R., Lahteenmaki, P.M., Levitt, G., Linge, V., Coze, C., Ladenstein, R., Owens, C., Papadakis, H., D, L.L., Michel, G., Morsellino, V., Mulder, R.L., V., Ruud, E., Amann, G., Sementa, A.R., Navarro, S., Reulen, R.C., Ronckers, C.M., Sacerdote, C., Skinner, Ambros, P.F., Noguera, R., and Ambros, I.M. (2018). R., Steliarova-Foucher, E., van der Pal, H.J., de Heterogeneous MYCN amplification in Vathaire, F., Vu Bezin, G., Wesenberg, F., Wiebe, T., neuroblastoma: a SIOP Europe Neuroblastoma Study. Winter, D.L., Falck Winther, J., Witthoff, E., Br J Cancer 118, 1502-1512. Zadravec Zaletel, L., and Hjorth, L. (2018). The PanCareSurFup consortium: research and Bigenzahn, S., Juergens, B., Mahr, B., Pratschke, J., guidelines to improve lives for survivors of childhood Koenigsrainer, A., Becker, T., Fuchs, D., Brandacher, cancer. Eur J Cancer 103, 238-248. G., Kainz, A., Muehlbacher, F., and Wekerle, T. (2018). No augmentation of indoleamine 2,3-dioxygenase (IDO) activity through belatacept treatment in liver transplant recipients. Clin Exp Immunol 192, 233-241.

Anhang Forschungsbericht St. Anna Kinderkrebsforschung | Science Report St. Anna Children’s Cancer Research Institute [ 127 ]

Casali, P.G., Bielack, S., Abecassis, N., Aro, H.T., Csillag, B., Ilencikova, D., Meissl, M., Webersinke, Bauer, S., Biagini, R., Bonvalot, S., Boukovinas, I., G., Laccone, F., Narumi, S., Haas, O., and Duba, Bovee, J., Brennan, B., Brodowicz, T., Broto, J.M., H.C. (2018). Brugieres, L., Buonadonna, A., De Alava, E., Dei Somatic mosaic monosomy 7 and UPD7q in a child Tos, A.P., Del Muro, X.G., Dileo, P., Dhooge, C., with MIRAGE syndrome caused by a novel SAMD9 Eriksson, M., Fagioli, F., Fedenko, A., Ferraresi, V., mutation. Pediatr Blood Cancer, e27589. Ferrari, A., Ferrari, S., Frezza, A.M., Gaspar, N., Gasperoni, S., Gelderblom, H., Gil, T., Grignani, G., Dauber, E.M., Mayr, W.R., Hustinx, H., Schonbacher, Gronchi, A., Haas, R.L., Hassan, B., Hecker-Nolting, M., Budde, H., Legler, T.J., Konig, M., Haas, O.A., S., Hohenberger, P., Issels, R., Joensuu, H., Jones, Fritsch, G., and Kormoczi, G.F. (2018). R.L., Judson, I., Jutte, P., Kaal, S., Kager, L., Kasper, Somatic mosaicisms of chromosome 1 at two B., Kopeckova, K., Krakorova, D.A., Ladenstein, R., different stages of ontogenetic development detected Le Cesne, A., Lugowska, I., Merimsky, O., by Rh blood group discrepancies. Haematologica. Montemurro, M., Morland, B., Pantaleo, M.A., Piana, R., Picci, P., Piperno-Neumann, S., Pousa, Depuydt, P., Boeva, V., Hocking, T.D., Cannoodt, R., A.L., Reichardt, P., Robinson, M.H., Rutkowski, P., Ambros, I.M., Ambros, P.F., Asgharzadeh, S., Safwat, A.A., Schoffski, P., Sleijfer, S., Stacchiotti, S., Attiyeh, E.F., Combaret, V., Defferrari, R., Fischer, Strauss, S.J., Sundby Hall, K., Unk, M., Van M., Hero, B., Hogarty, M.D., Irwin, M.S., Koster, J., Coevorden, F., van der Graaf, W.T.A., Whelan, J., Kreissman, S., Ladenstein, R., Lapouble, E., Laureys, Wardelmann, E., Zaikova, O., Blay, J.Y., Esmo G., London, W.B., Mazzocco, K., Nakagawara, A., Guidelines Committee, P., and Ern, E. (2018). Noguera, R., Ohira, M., Park, J.R., Potschger, U., Bone sarcomas: ESMO-PaedCan-EURACAN Clinical Theissen, J., Tonini, G.P., Valteau-Couanet, D., Practice Guidelines for diagnosis, treatment and Varesio, L., Versteeg, R., Speleman, F., Maris, J.M., follow-up. Ann Oncol 29, iv79-iv95. Schleiermacher, G., and De Preter, K. (2018). Genomic Amplifications and Distal 6q Loss: Novel Corrias, M.V., Parodi, S., Tchirkov, A., Lammens, T., Markers for Poor Survival in High-risk Neuroblastoma Vicha, A., Pasqualini, C., Trager, C., Yanez, Y., Patients. J Natl Cancer Inst 110, 1084-1093. Dallorso, S., Varesio, L., Luksch, R., Laureys, G., Val- teau-Couanet, D., Canete, A., Poetschger, U., Dillinger, B., Ahmadi-Erber, S., Lau, M., Hoelzl, Ladenstein, R., and Burchill, S.A. (2018). M.A., Erhart, F., Juergens, B., Fuchs, D., Heitger, A., Event-free survival of infants and toddlers enrolled Ladisch, S., and Dohnal, A.M. (2018). in the HR-NBL-1/SIOPEN trial is associated with the IFN-gamma and tumor gangliosides: Implications level of neuroblastoma mRNAs at diagnosis. for the tumor microenvironment. Pediatr Blood Cancer 65, e27052. Cell Immunol 325, 33-40.

Creutzig, U., Dworzak, M., von Neuhoff, N., Rasche, Duong, J.K., Veal, G.J., Nath, C.E., Shaw, P.J., M., and Reinhardt, D. (2018). [ Errington, J., Ladenstein, R., and Boddy, A.V. (2019). Acute Promyelocytic Leukemia: New treatment Population pharmacokinetics of carboplatin, strategies with ATRA and ATO - AML-BFM- etoposide and melphalan in children: a re-evaluation of Recommendations. Klin Padiatr 230, 299-304. paediatric dosing formulas for carboplatin in patients with normal or mild impairment of renal function. Br J Clin Pharmacol 85, 136-146. Anhang [ 128 ] Forschungsbericht St. Anna Kinderkrebsforschung | Science Report St. Anna Children’s Cancer Research Institute

Edwards, E.S.J., Bier, J., Cole, T.S., Wong, M., Hsu, P., Fauster, A., Rebsamen, M., Willmann, K.L., Berglund, L.J., Boztug, K., Lau, A., Gostick, E., Price, Cesar-Razquin, A., Girardi, E., Bigenzahn, J.W., D.A., O‘Sullivan, M., Meyts, I., Choo, S., Gray, P., Schischlik, F., Scorzoni, S., Bruckner, M., Konecka, Holland, S.M., Deenick, E.K., Uzel, G., and Tangye, J., Hormann, K., Heinz, L.X., Boztug, K., and S.G. (2019). Superti-Furga, G. (2018). Activating PIK3CD mutations impair human cytotoxic Systematic genetic mapping of necroptosis identifies lymphocyte differentiation and function and EBV SLC39A7 as modulator of death receptor trafficking. immunity. J Allergy Clin Immunol 143, 276-291 e276. Cell Death Differ.

Eghbal-Zadeh, H., Fischer, L., Popitsch, N., Kromp, Filbin, M.G., Tirosh, I., Hovestadt, V., Shaw, M.L., F., Taschner-Mandl, S., Gerber, T., Bozsaky, E., Escalante, L.E., Mathewson, N.D., Neftel, C., Frank, Ambros, P.F., Ambros, I.M., Widmer, G., and Moser, N., Pelton, K., Hebert, C.M., Haberler, C., Yizhak, K., B.A. (2018). Gojo, J., Egervari, K., Mount, C., van Galen, P., Bonal, DeepSNP: An End-to-End Deep Neural Network D.M., Nguyen, Q.D., Beck, A., Sinai, C., Czech, T., with Attention-Based Localization for Breakpoint Dorfer, C., Goumnerova, L., Lavarino, C., Carcaboso, Detection in Single-Nucleotide Polymorphism Array A.M., Mora, J., Mylvaganam, R., Luo, C.C., Peyrl, A., Genomic Data. J Comput Biol. Popovic, M., Azizi, A., Batchelor, T.T., Frosch, M.P., Martinez-Lage, M., Kieran, M.W., Bandopadhayay, P., Erhart, F., Blauensteiner, B., Zirkovits, G., Printz, Beroukhim, R., Fritsch, G., Getz, G., Rozenblatt-Ro- D., Soukup, K., Klingenbrunner, S., Fischhuber, sen, O., Wucherpfennig, K.W., Louis, D.N., Monje, K., Reitermaier, R., Halfmann, A., Lotsch, D., Spiegl- M., Slavc, I., Ligon, K.L., Golub, T.R., Regev, A., Kreinecker, S., Berger, W., Visus, C., and Dohnal, Bernstein, B.E., and Suva, M.L. (2018). A. (2019). Developmental and oncogenic programs in Gliomasphere marker combinatorics: multidimensional H3K27M gliomas dissected by single-cell RNA-seq. flow cytometry detects CD44+/CD133+/ITGA6+/ Science 360, 331-335. CD36+ signature. J Cell Mol Med 23, 281-292. Friesenbichler, W., Krizmanich, W., Lakatos, K., Erhart, F., Buchroithner, J., Reitermaier, R., Attarbaschi, A., Dworzak, M., Amann, G., Fischhuber, K., Klingenbrunner, S., Sloma, I., Hibsh, Furtwangler, R., Graf, N., and Kager, L. (2018). D., Kozol, R., Efroni, S., Ricken, G., Wohrer, A., Outcome of two patients with bilateral Haberler, C., Hainfellner, J., Krumpl, G., Felzmann, nephroblastomatosis/Wilms tumour treated with an T., Dohnal, A.M., Marosi, C., and Visus, C. (2018). add-on 13-cis retinoic acid therapy - Case report. Immunological analysis of phase II glioblastoma Pediatr Hematol Oncol, 1-7. dendritic cell vaccine (Audencel) trial: immune system characteristics influence outcome and Friesenbichler, W., Schumich, A., Simonitsch-Klupp, Audencel up-regulates Th1-related immunovariables. I., Panzer-Grumayer, R., Haas, O., Mann, G., and Acta Neuropathol Commun 6, 135. Dworzak, M. (2018). Concurrent Acute Myelofibrosis and Acute Lymphoblastic Leukemia in Childhood: Case Report and Review of the Literature. J Pediatr Hematol Oncol 40, 235-237.

Anhang Forschungsbericht St. Anna Kinderkrebsforschung | Science Report St. Anna Children’s Cancer Research Institute [ 129 ]

Garcia-Ramirez, I., Bhatia, S., Rodriguez-Hernandez, Hrusak, O., de Haas, V., Stancikova, J., Vakrmanova, G., Gonzalez-Herrero, I., Walter, C., Gonzalez de B., Janotova, I., Mejstrikova, E., Capek, V., Trka, J., Tena-Davila, S., Parvin, S., Haas, O., Woessmann, Zaliova, M., Luks, A., Bleckmann, K., Moricke, A., W., Stanulla, M., Schrappe, M., Dugas, M., Natkunam, Irving, J., Konatkowska, B., Alexander, T.B., Inaba, Y., Orfao, A., Dominguez, V., Pintado, B., Blanco, O., H., Schmiegelow, K., Stokley, S., Zemanova, Z., Alonso-Lopez, D., De Las Rivas, J., Martin-Lorenzo, Moorman, A.V., Rossi, J.G., Felice, M.S., A., Jimenez, R., Garcia Criado, F.J., Garcia Cenador, Dalla-Pozza, L., Morales, J., Dworzak, M., Buldini, M.B., Lossos, I.S., Vicente-Duenas, C., Borkhardt, A., B., Basso, G., Campbell, M., Cabrera, M.E., Marinov, Hauer, J., and Sanchez-Garcia, I. (2018). N., Elitzur, S., Izraeli, S., Luria, D., Feuerstein, T., Lmo2 expression defines tumor cell identity Kolenova, A., Svec, P., Kreminska, O., Rabin, K.R., during T-cell leukemogenesis. EMBO J 37. Polychronopoulou, S., da Costa, E., Marquart, H.V., Kattamis, A., Ratei, R., Reinhardt, D., Choi, J.K., Grunewald, T.G.P., Cidre-Aranaz, F., Surdez, D., Schrappe, M., and Stary, J. (2018). Tomazou, E.M., de Alava, E., Kovar, H., Sorensen, International cooperative study identifies treatment P.H., Delattre, O., and Dirksen, U. (2018). strategy in childhood ambiguous lineage leukemia. Ewing sarcoma. Nat Rev Dis Primers 4, 5. Blood 132, 264-276.

Haupt, R., Essiaf, S., Dellacasa, C., Ronckers, C.M., Husa, A.M., Strobl, M.R., Strajeriu, A., Wieser, M., Caruso, S., Sugden, E., Zadravec Zaletel, L., Muraca, Strehl, S., and Fortschegger, K. (2018). M., Morsellino, V., Kienesberger, A., Blondeel, A., Generation of CD34 Fluorescent Reporter Saraceno, D., Ortali, M., Kremer, L.C.M., Skinner, Human Induced Pluripotent Stem Cells for Monitoring R., Roganovic, J., Bagnasco, F., Levitt, G.A., De Rosa, Hematopoietic Differentiation. M., Schrappe, M., Hjorth, L., Ladenstein, R., Stem Cells Dev 27, 1376-1384. PanCareSurFup, E.W.G., and ExPo-r-Net Working, G. (2018). Juan Ribelles, A., Berlanga, P., Schreier, G., The ‚Survivorship Passport‘ for childhood cancer Nitzlnader, M., Brunmair, B., Castel, V., Essiaf, S., survivors. Eur J Cancer 102, 69-81. Canete, A., and Ladenstein, R. (2018). Survey on paediatric tumour boards in Europe: Hiwarkar, P., Kosulin, K., Cesaro, S., Mikulska, M., current situation and results from the Styczynski, J., Wynn, R., and Lion, T. (2018). ExPo-r-Net project. Clin Transl Oncol 20, 1046-1052. Management of adenovirus infection in patients after haematopoietic stem cell transplantation: Kager, L., Jimenez Heredia, R., Hirschmugl, T., State-of-the-art and real-life current approach: Dmytrus, J., Krolo, A., Muller, H., Bock, C., A position statement on behalf of the Infectious Zeitlhofer, P., Dworzak, M., Mann, G., Holter, W., Diseases Working Party of the European Society of Haas, O., and Boztug, K. (2018). Blood and Marrow Transplantation. Targeted mutation screening of 292 candidate Rev Med Virol 28, e1980. genes in 38 children with inborn haematological cytopenias efficiently identifies novel disease-causing mutations. Br J Haematol 182, 251-258.

Anhang [ 130 ] Forschungsbericht St. Anna Kinderkrebsforschung | Science Report St. Anna Children’s Cancer Research Institute

Kosulin, K., Berkowitsch, B., Matthes, S., Pichler, H., Ladenstein, R., Potschger, U., Valteau-Couanet, D., Lawitschka, A., Potschger, U., Fritsch, G., and Lion, Luksch, R., Castel, V., Yaniv, I., Laureys, G., Brock, P., T. (2018). Michon, J.M., Owens, C., Trahair, T., Chan, G.C.F., Intestinal Adenovirus Shedding Before Allogeneic Ruud, E., Schroeder, H., Beck Popovic, M., Schreier, Stem Cell Transplantation Is a Risk Factor for Invasive G., Loibner, H., Ambros, P., Holmes, K., Castellani, Infection Post-transplant. EBioMedicine 28, 114-119. M.R., Gaze, M.N., Garaventa, A., Pearson, A.D.J., and Lode, H.N. (2018). Kosulin, K., Kernbichler, S., Pichler, H., Lawitschka, Interleukin 2 with anti-GD2 antibody ch14.18/CHO A., Geyeregger, R., Witt, V., and Lion, T. (2018). (dinutuximab beta) in patients with high-risk Post-transplant Replication of Torque Teno Virus neuroblastoma (HR-NBL1/SIOPEN): a multicentre, in Granulocytes. Front Microbiol 9, 2956. randomised, phase 3 trial. Lancet Oncol 19, 1617-1629.

Kosulin, K., Lam, E., Heim, A., Dobner, T., and Lawson, J.T., Tomazou, E.M., Bock, C., and Sheffield, Rodriguez, E. (2018). N.C. (2018). Retreatment with sofosbuvir/velpatasvir in MIRA: an R package for DNA methylation-based cirrhotic patients with genotype-4 who failed a inference of regulatory activity. previous interferon-free regimen: a case series. Bioinformatics 34, 2649-2650. Antivir Ther 23, 475-483. Maas, C., Luftinger, R., Krois, W., Matthes-Martin, Kovar, H. (2018). S., Bayer, G., Boztug, K., and Metzelder, M. (2018). Selective enhancer changes in osteosarcoma lung EBV-positive B-cell lymphoma manifestation of the metastasis. Nat Med 24, 126-127. liver in an infant with RAG1 severe combined immuno- deficiency disease. Pediatr Blood Cancer 65, e27258. Kuhlen, M., Bader, P., Sauer, M., Albert, M.H., Gruhn, B., Gungor, T., Kropshofer, G., Lang, P., Machiela, M.J., Grunewald, T.G.P., Surdez, D., Lawitschka, A., Metzler, M., Pentek, F., Rossig, C., Reynaud, S., Mirabeau, O., Karlins, E., Rubio, R.A., Schlegel, P.G., Schrappe, M., Schrum, J., Schulz, A., Zaidi, S., Grossetete-Lalami, S., Ballet, S., Lapouble, Schwinger, W., von Stackelberg, A., Strahm, B., E., Laurence, V., Michon, J., Pierron, G., Kovar, H., Suttorp, M., Luettichau, I.T., Wossmann, W., Gaspar, N., Kontny, U., Gonzalez-Neira, A., Picci, P., Borkhardt, A., Meisel, R., Poetschger, U., Glogova, Alonso, J., Patino-Garcia, A., Corradini, N., Berard, E., and Peters, C. (2018). P.M., Freedman, N.D., Rothman, N., Dagnall, C.L., Low incidence of symptomatic osteonecrosis after Burdett, L., Jones, K., Manning, M., Wyatt, K., Zhou, allogeneic HSCT in children with high-risk or W., Yeager, M., Cox, D.G., Hoover, R.N., Khan, J., relapsed ALL - results of the ALL-SCT 2003 trial. Armstrong, G.T., Leisenring, W.M., Bhatia, S., Br J Haematol 183, 104-109. Robison, L.L., Kulozik, A.E., Kriebel, J., Meitinger, T., Metzler, M., Hartmann, W., Strauch, K., Kirchner, T., Dirksen, U., Morton, L.M., Mirabello, L., Tucker, M.A., Tirode, F., Chanock, S.J., and Delattre, O. (2018). Genome-wide association study identifies multiple new loci associated with Ewing sarcoma susceptibility. Nat Commun 9, 3184. Anhang Forschungsbericht St. Anna Kinderkrebsforschung | Science Report St. Anna Children’s Cancer Research Institute [ 131 ]

Mayr, V., Sturtzel, C., Stadler, M., Grissenberger, S., Morgenstern, D.A., Potschger, U., Moreno, L., and Distel, M. (2018). Papadakis, V., Owens, C., Ash, S., Pasqualini, C., Fast Dynamic in vivo Monitoring of Erk Activity at Luksch, R., Garaventa, A., Canete, A., Elliot, M., Single Cell Resolution in DREKA Zebrafish. Wieczorek, A., Laureys, G., Kogner, P., Malis, J., Front Cell Dev Biol 6, 111. Ruud, E., Beck-Popovic, M., Schleiermacher, G., Val- teau-Couanet, D., and Ladenstein, R. (2018). Meyer, C., Burmeister, T., Groger, D., Tsaur, G., Risk stratification of high-risk metastatic Fechina, L., Renneville, A., Sutton, R., Venn, N.C., neuroblastoma: A report from the HR-NBL-1/SIOPEN Emerenciano, M., Pombo-de-Oliveira, M.S., Barbieri study. Pediatr Blood Cancer 65, e27363. Blunck, C., Almeida Lopes, B., Zuna, J., Trka, J., Ballerini, P., Lapillonne, H., De Braekeleer, M., Mughal, T.I., Lion, T., Abdel-Wahab, O., Mesa, R., Cazzaniga, G., Corral Abascal, L., van der Velden, Scherber, R.M., Perrotti, D., Mauro, M., Verstovsek, S., V.H.J., Delabesse, E., Park, T.S., Oh, S.H., Silva, Saglio, G., Van Etten, R.A., and Kralovics, R. (2018). M.L.M., Lund-Aho, T., Juvonen, V., Moore, A.S., Precision immunotherapy, mutational landscape, Heidenreich, O., Vormoor, J., Zerkalenkova, E., and emerging tools to optimize clinical outcomes in Olshanskaya, Y., Bueno, C., Menendez, P., Teigler- patients with classical myeloproliferative neoplasms. Schlegel, A., Zur Stadt, U., Lentes, J., Gohring, G., Hematol Oncol 36, 740-748. Kustanovich, A., Aleinikova, O., Schafer, B.W., Kubetzko, S., Madsen, H.O., Gruhn, B., Duarte, X., Niktoreh, N., Lerius, B., Zimmermann, M., Gruhn, Gameiro, P., Lippert, E., Bidet, A., Cayuela, J.M., B., Escherich, G., Bourquin, J.P., Dworzak, M., Clappier, E., Alonso, C.N., Zwaan, C.M., van den Sramkova, L., Rossig, C., Creutzig, U., Reinhardt, D., Heuvel-Eibrink, M.M., Izraeli, S., Trakhtenbrot, L., and Rasche, M. (2019). Archer, P., Hancock, J., Moricke, A., Alten, J., Gemtuzumab ozogamicin in children with relapsed Schrappe, M., Stanulla, M., Strehl, S., Attarbaschi, or refractory acute myeloid leukemia: a report by A., Dworzak, M., Haas, O.A., Panzer-Grumayer, R., Berlin-Frankfurt-Munster study group. Sedek, L., Szczepanski, T., Caye, A., Suarez, L., Cave, Haematologica 104, 120-127. H., and Marschalek, R. (2018). The MLL recombinome of acute leukemias in 2017. Noort, S., Zimmermann, M., Reinhardt, D., Leukemia 32, 273-284. Cuccuini, W., Pigazzi, M., Smith, J., Ries, R.E., Alonzo, T.A., Hirsch, B., Tomizawa, D., Locatelli, F., Minkov, M. (2018). Gruber, T.A., Raimondi, S., Sonneveld, E., Cheuk, An update on the treatment of pediatric-onset D.K., Dworzak, M., Stary, J., Abrahamsson, J., Langerhans cell histiocytosis through pharmaco- Arad-Cohen, N., Czogala, M., De Moerloose, B., therapy. Expert Opin Pharmacother 19, 233-242. Hasle, H., Meshinchi, S., van den Heuvel-Eibrink, M., and Zwaan, C.M. (2018). Prognostic impact of t(16;21)(p11;q22) and t(16;21) (q24;q22) in pediatric AML: a retrospective study by the I-BFM Study Group. Blood 132, 1584-1592.

Anhang [ 132 ] Forschungsbericht St. Anna Kinderkrebsforschung | Science Report St. Anna Children’s Cancer Research Institute

Olesnicky, E.C., Antonacci, S., Popitsch, N., Potschger, U., Heinzl, H., Valsecchi, M.G., and Lybecker, M.C., Titus, M.B., Valadez, R., Derkach, Mittlbock, M. (2018). P.G., Marean, A., Miller, K., Mathai, S.K., and Assessing the effect of a partly unobserved, Killian, D.J. (2018). exogenous, binary time-dependent covariate on Shep interacts with posttranscriptional regulators to survival probabilities using generalised pseudo-values. control dendrite morphogenesis in sensory neurons. BMC Med Res Methodol 18, 14. Dev Biol 444, 116-128. Proff, J., Brey, C.U., Ensser, A., Holter, W., and Pastorczak, A., Sedek, L., Braun, M., Madzio, J., Lehner, M. (2018). Sonsala, A., Twardoch, M., Fendler, W., Nebral, K., Turning the tables on cytomegalovirus: targeting Taha, J., Bielska, M., Gorniak, P., Romiszewska, M., viral Fc receptors by CARs containing mutated Matysiak, M., Derwich, K., Lejman, M., Kowalczyk, CH2-CH3 IgG spacer domains. J Transl Med 16, 26. J., Badowska, W., Niedzwiecki, M., Kazanowska, B., Muszynska-Roslan, K., Sobol-Milejska, G., Rasche, M., Zimmermann, M., Borschel, L., Karolczyk, G., Koltan, A., Ociepa, T., Szczepanski, T., Bourquin, J.P., Dworzak, M., Klingebiel, T., and Mlynarski, W. (2018). Lehrnbecher, T., Creutzig, U., Klusmann, J.H., and Surface expression of Cytokine Receptor-Like Factor Reinhardt, D. (2018). 2 increases risk of relapse in pediatric acute Successes and challenges in the treatment of lymphoblastic leukemia patients harboring IKZF1 pediatric acute myeloid leukemia: a retrospective deletions. Oncotarget 9, 25971-25982. analysis of the AML-BFM trials from 1987 to 2012. Leukemia 32, 2167-2177. Pazmandi, J., Kalinichenko, A., Ardy, R.C., and Boztug, K. (2019). Schuh, A., Dreau, H., Knight, S.J.L., Ridout, K., Early-onset inflammatory bowel disease as a model Mizani, T., Vavoulis, D., Colling, R., Antoniou, P., disease to identify key regulators of immune Kvikstad, E.M., Pentony, M.M., Hamblin, A., homeostasis mechanisms. Immunol Rev 287, 162-185. Protheroe, A., Parton, M., Shah, K.A., Orosz, Z., Athanasou, N., Hassan, B., Flanagan, A.M., Ahmed, Pfajfer, L., Mair, N.K., Jimenez-Heredia, R., Genel, A., Winter, S., Harris, A., Tomlinson, I., Popitsch, N., F., Gulez, N., Ardeniz, O., Hoeger, B., Bal, S.K., Church, D., and Taylor, J.C. (2018). Madritsch, C., Kalinichenko, A., Chandra Ardy, R., Clinically actionable mutation profiles in patients with Gerceker, B., Rey-Barroso, J., Ijspeert, H., Tangye, cancer identified by whole-genome sequencing. S.G., Simonitsch-Klupp, I., Huppa, J.B., van der Cold Spring Harb Mol Case Stud 4. Burg, M., Dupre, L., and Boztug, K. (2018). Mutations affecting the actin regulator WD repeat-containing protein 1 lead to aberrant lymphoid immunity. J Allergy Clin Immunol 142, 1589-1604 e1511.

Popitsch, N. (2018). VARAN-GIE: Curation of Genomic Interval Sets. Bioinformatics.

Anhang Forschungsbericht St. Anna Kinderkrebsforschung | Science Report St. Anna Children’s Cancer Research Institute [ 133 ]

Schumich, A., Maurer-Granofszky, M., Attarbaschi, Serwas, N.K., Huemer, J., Dieckmann, R., A., Potschger, U., Buldini, B., Gaipa, G., Karawajew, Mejstrikova, E., Garncarz, W., Litzman, J., Hoeger, L., Printz, D., Ratei, R., Conter, V., Schrappe, M., B., Zapletal, O., Janda, A., Bennett, K.L., Kain, R., Mann, G., Basso, G., Dworzak, M.N., and Group, Kerjaschky, D., and Boztug, K. (2018). A.I.-B.-A.-F.-S. (2018). CEBPE-Mutant Specific Granule Deficiency Flow-cytometric minimal residual disease monitoring Correlates With Aberrant Granule Organization and in blood predicts relapse risk in pediatric B-cell pre- Substantial Proteome Alterations in Neutrophils. cursor acute lymphoblastic leukemia in trial AIEOP- Front Immunol 9, 588. BFM-ALL 2000. Pediatr Blood Cancer, e27590. Schuster, A.J., Kager, L., Reichardt, P., Baumhoer, D., Shen, J., Najafi, S., Stable, S., Fabian, J., Koeneke, E., Csoka, M., Hecker-Nolting, S., Lang, S., Lorenzen, Kolbinger, F.R., Wrobel, J.K., Meder, B., Distel, M., S., Mayer-Steinacker, R., von Kalle, T., Kevric, M., Heimburg, T., Sippl, W., Jung, M., Peterziel, H., Werner, M., Windhager, R., Wirth, T., and Bielack, Kranz, D., Boutros, M., Westermann, F., Witt, O., S.S. (2018). and Oehme, I. (2018). High-Grade Osteosarcoma of the Foot: Presentation, A kinome-wide RNAi screen identifies ALK as a target Treatment, Prognostic Factors, and Outcome of to sensitize neuroblastoma cells for HDAC8-inhibitor 23 Cooperative Osteosarcoma Study Group COSS treatment. Cell Death Differ 25, 2053-2070. Patients. Sarcoma 2018, 1632978. Siebert, N., Troschke-Meurer, S., Marx, M., Zumpe, Schwentner, R., Jug, G., Kauer, M.O., Schnoller, T., M., Ehlert, K., Gray, J., Garaventa, A., Manzitti, C., Waidhofer-Sollner, P., Holter, W., and Hutter, C. Ash, S., Klingebiel, T., Beck, J., Castel, V., Valteau- (2019). Couanet, D., Loibner, H., Ladenstein, R., and Lode, JAG2 signaling induces differentiation of CD14(+) H.N. (2018). monocytes into Langerhans cell histiocytosis-like cells. Impact of HACA on Immunomodulation and J Leukoc Biol 105, 101-111. Treatment Toxicity Following ch14.18/CHO Long-Term Infusion with Interleukin-2: Results from a Schwentner, R., Kolenova, A., Jug, G., Schnoller, T., SIOPEN Phase 2 Trial. Cancers (Basel) 10. Ahlmann, M., Meister, B., Lehrnbecher, T., Minkov, M., and Hutter, C. (2018). Longitudinal assessment of peripheral blood BRAFV600E levels in patients with Langerhans cell histiocytosis. Pediatr Res.

Schwinger, W., Urban, C., Ulreich, R., Sperl, D., Karastaneva, A., Strenger, V., Lackner, H., Boztug, K., Albert, M.H., Benesch, M., and Seidel, M.G. (2018). The Phenotype and Treatment of WIP Deficiency: Literature Synopsis and Review of a Patient With Pre-transplant Serial Donor Lymphocyte Infusions to Eliminate CMV. Front Immunol 9, 2554.

Anhang [ 134 ] Forschungsbericht St. Anna Kinderkrebsforschung | Science Report St. Anna Children’s Cancer Research Institute

Stanulla, M., Dagdan, E., Zaliova, M., Moricke, A., van Eijkelenburg, N.K.A., Rasche, M., Ghazaly, E., Palmi, C., Cazzaniga, G., Eckert, C., Te Kronnie, G., Dworzak, M.N., Klingebiel, T., Rossig, C., Leverger, Bourquin, J.P., Bornhauser, B., Koehler, R., Bartram, G., Stary, J., De Bont, E., Chitu, D.A., Bertrand, Y., C.R., Ludwig, W.D., Bleckmann, K., Groeneveld-Kr- Brethon, B., Strahm, B., van der Sluis, I.M., Kaspers, entz, S., Schewe, D., Junk, S.V., Hinze, L., Klein, N., G.J.L., Reinhardt, D., and Zwaan, C.M. (2018). Kratz, C.P., Biondi, A., Borkhardt, A., Kulozik, A., Clofarabine, high-dose cytarabine and liposomal Muckenthaler, M.U., Basso, G., Valsecchi, M.G., daunorubicin in pediatric relapsed/refractory acute Izraeli, S., Petersen, B.S., Franke, A., Dorge, P., myeloid leukemia: a phase IB study. Steinemann, D., Haas, O.A., Panzer-Grumayer, R., Haematologica 103, 1484-1492. Cave, H., Houlston, R.S., Cario, G., Schrappe, M., Zimmermann, M., Consortium, T., and van Rijn, J.M., Ardy, R.C., Kuloglu, Z., Harter, B., International, B.F.M.S.G. (2018). van Haaften-Visser, D.Y., van der Doef, H.P.J., van IKZF1(plus) Defines a New Minimal Residual Hoesel, M., Kansu, A., van Vugt, A.H.M., Thian, M., Disease-Dependent Very-Poor Prognostic Profile in Kokke, F.T.M., Krolo, A., Basaran, M.K., Kaya, N.G., Pediatric B-Cell Precursor Acute Lymphoblastic Aksu, A.U., Dalgic, B., Ozcay, F., Baris, Z., Kain, R., Leukemia. J Clin Oncol 36, 1240-1249. Stigter, E.C.A., Lichtenbelt, K.D., Massink, M.P.G., Duran, K.J., Verheij, J., Lugtenberg, D., Nikkels, Troll, J.V., Hamilton, M.K., Abel, M.L., Ganz, J., P.G.J., Brouwer, H.G.F., Verkade, H.J., Scheenstra, R., Bates, J.M., Stephens, W.Z., Melancon, E., van der Spee, B., Nieuwenhuis, E.E.S., Coffer, P.J., Janecke, Vaart, M., Meijer, A.H., Distel, M., Eisen, J.S., and A.R., van Haaften, G., Houwen, R.H.J., Muller, T., Guillemin, K. (2018). Middendorp, S., and Boztug, K. (2018). Microbiota promote secretory cell determination in Intestinal Failure and Aberrant Lipid Metabolism in the intestinal epithelium by modulating host Notch Patients With DGAT1 Deficiency. signaling. Development 145. Gastroenterology 155, 130-143 e115. Velicky, P., Meinhardt, G., Plessl, K., Vondra, S., Tsafou, K., Katschnig, A.M., Radic-Sarikas, B., Mutz, Weiss, T., Haslinger, P., Lendl, T., Aumayr, K., C.N., Iljin, K., Schwentner, R., Kauer, M.O., Mairhofer, M., Zhu, X., Schutz, B., Hannibal, R.L., Muhlbacher, K., Aryee, D.N.T., Westergaard, D., Lindau, R., Weil, B., Ernerudh, J., Neesen, J., Egger, Haapa-Paananen, S., Fey, V., Superti-Furga, G., G., Mikula, M., Rohrl, C., Urban, A.E., Baker, J., Toretsky, J., Brunak, S., and Kovar, H. (2018). Knofler, M., and Pollheimer, J. (2018). Identifying the druggable interactome of Genome amplification and cellular senescence are EWS-FLI1 reveals MCL-1 dependent differential hallmarks of human placenta development. sensitivities of Ewing sarcoma cells to apoptosis PLoS Genet 14, e1007698. inducers. Oncotarget 9, 31018-31031. Weinhofer, I., Zierfuss, B., Hametner, S., Wagner, M., Popitsch, N., Machacek, C., Bartolini, B., Zlabinger, G., Ohradanova-Repic, A., Stockinger, H., Kohler, W., Hoftberger, R., Regelsberger, G., Forss-Petter, S., Lassmann, H., and Berger, J. (2018). Impaired plasticity of macrophages in X-linked adrenoleukodystrophy. Brain.

Anhang Forschungsbericht St. Anna Kinderkrebsforschung | Science Report St. Anna Children’s Cancer Research Institute [ 135 ]

Weiss, T., Taschner-Mandl, S., Ambros, P.F., and Ambros, I.M. (2018). Detailed Protocols for the Isolation, Culture, Enrichment and Immunostaining of Primary Human Schwann Cells. Methods Mol Biol 1739, 67-86.

Whelan, J., Le Deley, M.C., Dirksen, U., Le Teuff, G., Brennan, B., Gaspar, N., Hawkins, D.S., Amler, S., Bauer, S., Bielack, S., Blay, J.Y., Burdach, S., Castex, M.P., Dilloo, D., Eggert, A., Gelderblom, H., Gentet, J.C., Hartmann, W., Hassenpflug, W.A., Hjorth, L., Jimenez, M., Klingebiel, T., Kontny, U., Kruseova, J., Ladenstein, R., Laurence, V., Lervat, C., Marec-Berard, P., Marreaud, S., Michon, J., Morland, B., Paulussen, M., Ranft, A., Reichardt, P., van den Berg, H., Wheatley, K., Judson, I., Lewis, I., Craft, A., Juergens, H., Oberlin, O., Euro, E.W.I.N.G., and Investigators, E.-. (2018). High-Dose Chemotherapy and Blood Autologous Stem-Cell Rescue Compared With Standard Chemotherapy in Localized High-Risk Ewing Sarcoma: Results of Euro-E.W.I.N.G.99 and Ewing-2008. J Clin Oncol, JCO2018782516.

Zoghi, S., Ziaee, V., Hirschmugl, T., Jimenez-Here- dia, R., Krolo, A., Boztug, K., and Rezaei, N. (2018). Exome sequencing revealed C1Q homozygous mutation in Pediatric Systemic Lupus Erythematosus. Allergol Immunopathol (Madr) 46, 594-598.

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Impressum

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