- Children`s Cancer Research Institute

Transcription

St. Anna
Children’s Cancer Research Institute
25 Years. Science Report 2011–2012
St. Anna
Kinderkrebsforschung
25 Jahre. Forschungsbericht 2011–2012
St. Anna
Children’s Cancer Research Institute
25 Years. Science Report 2011–2012
St. Anna
Kinderkrebsforschung
25 Jahre. Forschungsbericht 2011–2012
Quotes by Helmut Gadner
Extracts from past Science Reports
25 Years / 25 Jahre
A Brief Review
Ein kurzer Rückblick
Extracts from past CCRI Science Reports
Auszüge aus vergangenen Jahresberichten
der St. Anna Kinderkrebsforschung
Helmut Gadner, Director of the Institute:
Quotes from an Interview in October 2012
Helmut Gadner, Institutsleiter:
Zitate aus einem Interview im Oktober 2012
Time Travel through
Iconic Images
Eine Zeitreise in Bildern
25 Years / 25 Jahre
Among all types of malignant childhood cancer we had to deal with,
leukaemia was certainly the biggest challenge.
Leukämie war unter den bösartigen Erkrankungen im
Kindes- und Jugendalter damals diejenige, wo es den größten
Handlungsbedarf gab.
After successful implementation of a novel treatment concept
for children with cancer, I knew that we would need to see further progress.
Being the Medical Director of the St. Anna Children’s Hospital,
in 1985, I realised that all our therapeutic possibilities seemed to have hit
the end of the road.
Nach erfolgreicher Einführung eines neuen Behandlungskonzeptes
für krebskranke Kinder habe ich als ärztlicher Direktor des St. Anna Kinderspitals
im Jahr 1985 gewusst, dass wir noch mehr tun müssen, um weiter zu kommen.
Die therapeutischen Möglichkeiten führten in eine Sackgasse.
Thanks to the continuous financial support of our donors and
various funds, it was possible to establish seven different research groups
in the five labs that were available.
Dank anhaltendem Spendenfluss und Unterstützung durch
diverse Fonds war es möglich, in den fünf zur Verfügung stehenden Labors
sieben Arbeitsgruppen zu beschäftigen.
(Science Report 1990)
After only one year of construction time, and financed exclusively
by private donations, the Children’s Cancer Research Institute went
operational in June 1988.
Das Forschungsinstitut für krebskranke Kinder wurde im Juni 1988
nach einer einjährigen Bauzeit, deren Kosten ausschließlich aus Spenden
der Öffentlichkeit getragen worden sind, seiner Bestimmung übergeben.
25 Years / 25 Jahre
In some areas, discoveries made at the CCRI have already
been translated into new treatment strategies.
In einzelnen Bereichen ist es gelungen, Erkenntnisse,
die im Forschungsinstitut gewonnen wurden, in die Etablierung neuer
Behandlungsstrategien umzusetzen.
The close cooperation of clinicians and researchers
proves to be extremely beneficial to the patients.
Die enge Zusammenarbeit von Klinikern und Forschern,
wenn es um Belange des Patienten geht, hat sich hervorragend bewährt.
(Science Report 1995/96)
The primary goal of the newly founded institute
was the improvement of diagnostics using novel technologies.
In addition, we wanted to establish basic research and conduct clinical trials.
Our expectations were exceeded in every respect.
Die wichtigste Aufgabe des neu gegründeten Forschungsinstituts
war die verbesserte Diagnostik mittels neuer Technologien.
Zudem wollten wir auch die Grundlagenforschung etablieren
und klinische Studien durchführen.
Diese Ziele wurden bereits in wenigen Jahren mehr als übertroffen.
In 1992 the CCRI’s company profile changed: applied research,
sophisticated diagnostics, and basic research are now equally important.
Im Jahr 1992 vertiefte sich das für das Forschungsinstitut
charakteristische Profil: angewandte Forschung, verfeinerte Diagnostik
und Grundlagenforschung stehen gleichberechtigt nebeneinander.
25 Years / 25 Jahre
...the research activities of the CCRI are based partly on the work of
diploma and PhD students from different disciplines…Thus we perform
an important role in the training of future researchers...
... so baut die wissenschaftliche Arbeit zu einem guten Teil auf der Mitarbeit
und dem Einsatz von Studenten … aus verschiedenen Disziplinen auf ...
Damit übernimmt das Forschungsinstitut auch eine große Aufgabe in der
Ausbildung dieser angehenden Wissenschafter ...
...the fast development in haemato-oncology, which was observed
in the last decade world-wide, led to a further diversification
of the questions in this field also in our Institute...the transfer of examinations,
which have in the meantime become routine examinations,
to an especially equipped laboratory...
... weltweit beobachtete rasante Entwicklung auf dem Gebiet
der Hämato-Onkologie hat auch in unserem Institut dazu geführt,
dass eine weitere Diversifizierung der Fragestellungen aufgetreten ist …
Auslagerung von inzwischen in Routine übergangenen Untersuchungen
in ein speziell dafür eingerichtetes Labor ...
Enduring commitment of each individual research group builds
the institute’s mission statement. The steadily increasing number of
external grants makes the visions of CCRI scientists become reality.
Das Engagement der einzelnen Forschungsgruppen ist heute das Leitbild
des Instituts. Die zunehmenden Erfolge in der Einwerbung
von Drittmitteln zeigen, dass man hier am CCRI sehr bemüht ist
eigene Visionen zu realisieren.
...it was possible to raise the contribution of external competitive grants
to the research budget to 38%, which in part was due to the successful
participation in national...and international...cluster projects...as a result,
it was possible to initiate in-house bioinformatic support
... konnte der Anteil extern finanzierter Projektmittel auf 38% gehoben werden
was zu einem guten Teil auf die Teilnahme an nationalen …
und internationalen …Kooperationsprojekten zurückzuführen ist ...
in der Folge die Bioinformatik als Unterstützung im eigenen Haus zu etablieren ...
(Science Report 2006/07/08)
Good networking and international collaborations are still essential
to make the best therapies available to young cancer patients.
Gute Vernetzung sowie nationale und internationale Zusammenarbeit
sind nach wie vor essentiell, um die Forschung weiter voran zu treiben und
jungen Krebspatienten die beste Therapie zu ermöglichen.
25 Years / 25 Jahre
Zukunftsweisend sind personalisierte Therapien sowie Genomanalysen,
die uns Einblicke in die Ursachen einer bestimmten Krebserkrankung ermöglichen.
In the future, genome analyses will enable us to further personalise therapies
and reveal the mechanisms of cancer development and progression.
Reflections on the past 25 years of children’s cancer research
point to a promising future: There are many open questions
that still need to be answered.
Die Rückschau auf 25 Jahre Kinderkrebsforschung lässt uns
hoffnungsvoll in die Zukunft blicken: Es sind noch so viele Fragen offen.
...In January 2009 we were able to relocate our research laboratories
into the newly-constructed institutional building...it also opened up new paths
as regards translational and applied research...more third-party funding
than in previous years...networks enabled our researchers to gain access
to very expensive genomic and post-genomic high-throughput technology...
... Im Jänner 2009 konnten die Forschungslabors in das neu errichtete
Institutsgebäude … übersiedeln ... dies eröffnet neue Wege in der
translatorischen und angewandten Forschung ... mehr Drittmittelgelder
als in den Jahren zuvor ... Zugang zu sehr teuren genomischen und
postgenomischen Hochdurchsatz-Technologien ...
My wish is that this institute can sustain its outstanding
reputation by keeping up with novel developments and expanding
its research efforts.
Ich wünsche mir, dass das Institut auch in Zukunft
mit dem starken Aufwärtstrend der letzte Jahre Schritt halten kann
und die Forschung hier so weiter besteht wie bisher.
25 Years / 25 Jahre
3
25 Years Children’s Cancer
Research Institute
A Brief Review
16Preface
Research Report
Leukaemias
20
Immunological Diagnostics
Michael N. Dworzak
24
Biology of Leukaemias
Renate Panzer-Grümayer
30
Genetics of Leukaemia
Sabine Strehl
36
42
Solid Tumours
Tumour Biology
Peter F. Ambros
Molecular Biology of Solid Tumours
Heinrich Kovar
Immunology
48
Tumour Immunology
Thomas Felzmann
54
Clinical Cell Biology and FACS Core Unit
Gerhard Fritsch
60Transplantation-Immunology
Andreas Heitger
66
Development of Cellular Therapeutics
Wolfgang Holter
72
Molecular Microbiology and
Labdia Labordiagnostik GmbH
Thomas Lion
80
Clinical Research
S2IRP, Studies & Statistics of
Integrated Research and Projects
Ruth Ladenstein
92
Services, Administration,
PR and Donations Department
Respective Departments
Appendix
95
Scientific Advisory Board
96Awards
97
Completed MSc Diplomas and PhD Theses
98
External Grants and Research Funding Bodies
102Publications
110Imprint
111Acknowledgements
3
25 Jahre St. Anna Kinderkrebsforschung
Ein kurzer Rückblick
17Vorwort
Forschungsbericht
Leukämien
20
Immunologische Diagnostik
Michael N. Dworzak
24
Biologie der Leukämien
30Leukämiegenetik
Sabine Strehl
Solide Tumoren
36Tumorbiologie
Peter F. Ambros
42
Molekularbiologie Solider Tumoren
Heinrich Kovar
Immunologie
48Tumor-Immunologie
Thomas Felzmann
54
Klinische Zellbiologie und FACS Core Unit
Gerhard Fritsch
60Transplantations-Immunologie
Andreas Heitger
66
Entwicklung zellulärer Therapeutika
Wolfgang Holter
72
Molekulare Microbiologie und
Labdia Labordiagnostik GmbH
Thomas Lion
80
Klinische Forschung
S2IRP, Studien und Statistik
Ruth Ladenstein
92
Service-Einrichtungen, Administration,
PR und Spendenabteilung
Entsprechende Abteilungen
Anhang
95
Wissenschaftlicher Beirat
96Preise
97
Abgeschlossene Diplomarbeiten
und Dissertationen
98
Extern geförderte Projekte und Fördergeber
102Publikationen
110Impressum
111Danksagung
While we are all very much looking forward to a new era
in the history of this institute, we are also looking back
with gratitude on those years in which Prof. Gadner
nursed the CCRI from a mere idea in his head to an
internationally renowned research institute that gives
children with cancer in Austria access to research
results, as well as state-of-the art diagnostics and
treatments. In these past years, biomedical research
has achieved significant progress and experienced
several technological revolutions. The CCRI has
always kept up with these important developments,
and has contributed significantly to the translation
of these achievements into paediatric cancer research.
This also holds true for the period 2011/12, documented
in this bi-annual report. The adaptation of modern
genomic and biological concepts of cancerogenesis,
immune surveillance, and targeted therapy to the
specifics of childhood cancer is achieved by a highly
motivated multidisciplinary team of CCRI researchers
from different countries, of which ambitious graduate
and PhD students form an indispensable part.
Guided by our experienced group leaders and
postdocs, several of these students successfully
completed their theses in the reporting period.
The strength of CCRI research is based on its close
proximity to the clinics at St. Anna Kinderspital.
To further strengthen the practical and intellectual
relationship between these two institutions, we have
installed an in-house program especially supporting
clinical-translational research driven by treating
physicians from the hospital. In addition, the area of
clinical research has further expanded, and the CCRI
has taken leadership of a European network (ENCCA)
to improve multi-centric clinical and translational
research through collaboration, coordination, and
communication with European health authorities.
ENCCA is only one example of several, in which CCRI
researchers successfully applied to the European
commission for funding of collaborative initiatives on
an international level. This success has been greatly
facilitated by our dedicated research support office
which has been of invaluable help in the successful
recruitment of a considerable number of competitive
grants supporting high-end research at the CCRI.
These grants and the continuous support of our donors
and mentors allow the CCRI to continue growing and
prospering. A new research group, working on the
development of cellular therapeutics, was established
with the arrival of the new director of the institute.
The increasing needs for bioinformatics analyses of high
throughput genomics data and for innovative in vivo
models is met by the recruitment of an additional data
analyst and a new junior research group, respectively,
in 2013. In order to fulfill our responsibility towards our
donors, we keep them informed on research outcomes
and their application to the benefit of sick children
and for this reason have also invested in a science
communication team. This report is the most recent
product of their important work.
Heinrich Kovar
Scientific Director
Es ist erstaunlich: 2013 feiert das Children’s Cancer
Research Institute (CCRI) bereits seinen 25. Geburtstag. Von Spenden und vielen kompetitiven Projektförderungen genährt, unterstützt von anhaltendem
Wohlwollen wuchs das Institut heran. Ende 2012 ist nun
der Gründungsvater und langjährige Leiter des CCRI,
Prof. Helmut Gadner, in den wohlverdienten Ruhestand
getreten, und ein neuer Direktor, Prof. Wolfgang Holter,
hat das Institut übernommen. So markiert 2012 das
Ende einer bemerkenswerten, fruchtbaren und auf
regenden Periode in der Entwicklung des CCRI.
Während wir uns schon sehr neugierig auf eine neue
Ära in der Geschichte des Institutes freuen, blicken
wir auch voll Dankbarkeit auf die Jahre zurück, in
denen Prof. Gadner das CCRI von einer Idee in seinem
Kopf zu einer international höchst anerkannten
Forschungseinrichtung geführt hat, die krebskranken
Kindern in Österreich Zugang zu den neuesten wissenschaftlichen Ergebnissen in Diagnostik und Therapie
ermöglicht. In diesen vergangenen Jahren machte die
biomedizinische Forschung signifikante Fortschritte
und erlebte mehrere technologische Revolutionen.
Das CCRI hat immer mit diesen Entwicklungen Schritt
gehalten und trug maßgeblich zur Umsetzung dieser
Errungenschaften für die Erforschung von und den
Einsatz bei Krebserkrankungen des Kindesalters bei.
Das gilt auch für die Periode 2011/12, die in diesem
Jahresbericht dokumentiert wird. Die Anpassung
moderner genomischer und biologischer Konzepte der
Krebsentstehung, Immunüberwachung und gezielter
Therapien an die speziellen Eigenheiten von Kinderkrebserkrankungen wird von einem hoch motivierten,
multidisziplinären Team von CCRI Wissenschaftlern
getragen, in dem ambitionierte Diplom-/Master und
DissertationsstudentInnen eine wesentliche Rolle
spielen. Angeleitet von erfahrenen GruppenleiterInnen
und PostdoktorandInnen konnten mehrere StudentInnen ihre Dissertation im Berichtszeitraum abschließen.
Die Stärke der Forschung am CCRI liegt in der großen
Nähe zum St. Anna Kinderspital. Um die enge
Zusammenarbeit von Klinikern und Forschern weiter
zu stärken, haben wir ein Programm zur Förderung
von klinisch-translationalen Projekten eingerichtet,
welche durch behandelnde Ärzte des Kinderspitals
vorangetrieben werden. Zudem wurde die klinische
Forschung weiter ausgedehnt, und das CCRI übernahm
die Führung eines europäischen Netzwerkes (ENCCA),
dessen Ziel die Verbesserung multi-zentrischer
klinischer und translationaler Studien durch
Zusammenarbeit, Koordination und Kommunikation
mit europäischen Gesundheitsbehörden ist. ENCCA
ist nur ein Beispiel von mehreren, in denen CCRI
Wissenschaftler erfolgreich Unterstützung für
kollaborative Initiativen auf internationaler Ebene
von der Europäischen Kommission einwerben konnten.
Dieser Erfolg wurde durch die Einrichtung eines
„Research Support Office“ erleichtert, welches von
unschätzbarer Hilfe in der Akquisition einer beein
druckenden Anzahl kompetitiver Drittmittelprojekte
war. Diese Projekte und die fortgesetzte Unterstützung
unserer SpenderInnen und MentorInnen ermöglichten
ein weiteres Wachstum des CCRI. Mit der Ankunft
des neuen Institutsdirektors wurde eine neue
Arbeitsgruppe, die sich mit der Entwicklung zellulärer
Therapeutika beschäftigt, etabliert. Der steigende
Bedarf an bioinformatischen Analysen von Hoch
durchsatz genomischen Daten sowie von innovativen
in vivo Modellen erfordert 2013 sogar die Einstellung
eines weiteren Datenanalysten und Anwerbung einer
jungen neuen Arbeitsgruppe. Um der Verantwortung
gegenüber unseren SpenderInnen gerecht zu werden,
informieren wir sie über den Einsatz unserer Mittel
für Forschung und deren Anwendung zum Wohl kranker
Kinder und haben dafür auch in ein Wissenschafts
kommunikationsteam investiert. Der vorliegende
Jahresbericht ist das bislang letzte Produkt seiner
wichtigen Arbeit.
Heinrich Kovar
Wissenschaftlicher Leiter
16–17
It is amazing: In 2013, the Children’s Cancer Research
Institute celebrates its 25th anniversary. Bottle-fed by
donations, supported by many benefactors, and raised
on competitive grants and continuing philanthropy, the
CCRI has come of age. At the end of 2012, the founding
father and head of the CCRI, Prof. Helmut Gadner,
retired from the directorship of the institute, and a new
director, Prof. Wolfgang Holter, took over. Thus, 2012
marked the end of a remarkably fruitful and exciting era
in the development of the CCRI.
Vorwort
St. Anna Kinderkrebsforschung/CCRI Scientific Report 2011–2012
Preface
Research
Report
Forschungs
bericht
Leukaemias
Immunological
Diagnostics
Leukämien
Immunologische
Diagnostik
Group leader
Michael N. Dworzak
[email protected]
Staff scientist
Zvenyslava Husak
Diploma/MSc student
Romana Breitler1
Technician
Angela Schumich
Susanne Suhendra2
1 since Dec. 2012
2 since Aug. 2011
Our group’s research focus resides in developing
and validating new diagnostic methods based
on flow cytometry – mainly in the field of childhood
leukaemia and lymphoma, which make up about
50% of all cancer cases in children and adolescents.
Our working directions embrace investigations
into disease-associated peculiarities of protein
expression which can be exploited clinically
for elaborate diagnostics [1], risk stratification
and treatment tailoring to individual needs [2]:
e.g. highly sensitive and specific detection of
minimal residual disease in acute lymphoblastic
as well as myeloid leukaemia1. In an additional but
related focus we also aim at supporting novel
therapeutic options against childhood leukaemias
with new diagnostic approaches, e.g. by deciphering
the activated signalling network of leukaemic cells
potentially relevant for patient-tailored signal
transduction inhibitor treatment2, or by unravelling
signal regulation towards augmenting anti-leukaemic
defence by the innate immune system [3].
Die Entwicklung und Validierung neuer diagnostischer
Methoden basierend auf Durchflußzytometrie ist
das Hauptziel unserer Forschungsgruppe. Dabei
konzentrieren wir uns vor allem auf die pädiatrischen
Leukämien sowie Lymphome, die gemeinsam etwa
50% aller Krebserkrankungen im Kindes- und Jugendalter ausmachen. Zusammenfassend sind die Themen
unserer Arbeit einerseits die krankheitsassoziierten
Besonderheiten in der Proteinexpression und die sich
daraus ergebenden Möglichkeiten für Feindiagnostik [1],
Prognoseevaluation, Risikostratifizierung und indivi
dualisierte Behandlungsplanung [2], basierend vor
allem auf hochsensitiver und spezifischer Detektion
minimaler Resterkrankung sowohl bei akuter lympho
blastischer wie auch bei myeloischer Leukämie1.
Andererseits beschäftigen wir uns auch mit leukämie
biologischen Fragestellungen2. Wir analysieren die
Zellsignalwegaktivierung in Hinblick auf den patienten
gerechten Einsatz neuer Medikamente (Signal
weginhibitoren) bzw. auf neue Möglichkeiten, die
antileukämische Immunabwehr zu stimulieren [3].
CD99, an antigen implicated in cell survival and
adhesion, is strongly expressed on Ewing tumours
and on acute leukaemias. We previously found
that modulation of this antigen induces cell death in
TEL/AML1-positive ALL [1]. We also observed that
CD99-modulation up-regulates the heat shock
protein hsp70 in leukaemic cells. Based on these
findings, we speculated that CD99-mediated hsp70
up-regulation would induce NK-cell cytotoxicity.
In our subsequent investigations supported by
the Austrian National Bank1 we demonstrated that
CD99-mediated hsp70 up-regulation induces
increased NK-cell cytotoxicity against ALL cells,
both in primary samples and cell lines, but only
weakly if at all against Ewing tumour cell lines [2].
This observation puts CD99-targeting forward as
a potential tool to augment defence mechanisms
of the immune system against ALL.
1 ENCCA grant 261474 (2011–2014)
2 WWTF grant Nr. LS07–037 (2008–2013)
1 ENCCA grant 261474 (2011–2014)
2 WWTF grant Nr. LS07–037 (2008–2013)
In collaboration with
Prof. Giusepe Basso, Laboratory of Pediatric Onco-Hematology,
University of Padova, Italy.
Prof. Andrea Biondi and Dr. Giuseppe Gaipa, Tettamanti
Research Center, Monza – University Milano-Bicocca, Italy.
Dr. Richard Ratei and Dr. Leonid Karawajew, Zellmarkerlabor,
RRK, HELIOS Klinikum Berlin, Charite MS, Berlin, Germany.
Prof. Dirk Reinhardt, Abteilung für Kinderheilkunde –
Onko-Haematologie, Medizinische Hochschule Hannover,
Hannover, Deutschland.
Prof. Veronika Sexl, Vet. Med. Uni. Wien, Wien, Österreich
In Zusammenarbeit mit
Prof. Giusepe Basso, Laboratory of Pediatric Onco-Hematology,
University of Padova, Italy.
Prof. Andrea Biondi and Dr. Giuseppe Gaipa, Tettamanti
Research Center, Monza – University Milano-Bicocca, Italy.
Dr. Richard Ratei and Dr. Leonid Karawajew, Zellmarkerlabor,
RRK, HELIOS Klinikum Berlin, Charite MS, Berlin, Germany.
Prof. Dirk Reinhardt, Abteilung für Kinderheilkunde –
Onko-Haematologie, Medizinische Hochschule Hannover,
Hannover, Deutschland.
Prof. Veronika Sexl, Vet. Med. Uni. Wien, Wien, Österreich
For further reading
[1] Fišer, Sieger et al. 2012
[2] Gaipa, Cazzaniga et al. 2012
[3] Husak and Dworzak 2012
Literaturangaben
[1] Fišer, Sieger et al. 2012
[2] Gaipa, Cazzaniga et al. 2012
1 ÖNB grant 13081; 2008–2010
For further reading
[1] Husak, Printz et al. 2010
s HSP70*
s HSP70**
cy HSP70**
22–23
CD99 and HSP70 crosstalk
in ALL cells increases NK
cytotoxicity
00
101
102
103
00
101
102
103
00
101
102
103
Fig. 1 – HSP up-regulation in primary ALL cells (red: CD99stimulation;
blue and grey: (mock) controls.
BCP-ALL
100
control
DN16
80
60
40
20
0
0:1
2.5:1
10:1
E:T ratio
Jurkat
P<0.05
100
80
60
40
control
DN16
20
0
0:1
1:1
2.5:1
10:1
E:T ratio
Fig. 2 – Apoptosis induction by CD99 stimulation (grey line) versus
control (black) with and without NK-cells in soaring ratios versus target
ALL cells [2].
Innovative immunologische
Diagnostik für Leukämie
im Kindesalter
% of apoptotic targeted cells
Innovative diagnostic
tools for paediatric
leukaemia
Leukaemias
Specific project
Cell counts
Forschungsschwerpunkt
% of apoptotic targeted cells
Research focus
Leukaemias
Biology of
Leukaemias
Leukämien
Biologie der
Leukämien
Group leader
[email protected]
Postdoctoral fellows
Reinhard Grausenburger
Kamilla Malinowska-Ozdowy1
Maria Morak
PhD students
Stephan Daniel Bastelberger2
Gerhard Fuka
Ulrike Kaindl
Christine Nassimbeni3
Technicians
Susanna Fischer
Andrea Inthal4
Astrid Mecklenbräuker5
Marion Zeginigg6
1 since Aug. 2012
2 since May 2011
3 since Febr. 2011
4 maternity leave
from Nov. 2011
5 since Aug. 2011
6 since May 2011
7 Vice Director
Clinicians,
St. Anna Children’s Hospital
Andische Attarbaschi
Georg Mann
LabDia Labordiagnostik GmbH
Oskar A. Haas7
Childhood acute lymphoblastic leukaemia (ALL), the
most frequent cancer in childhood and adolescence,
has reached an overall cure rate of approximately 80%
when treated according to contemporary risk-directed
protocols. Yet despite this remarkable progress, a
substantial proportion of cases still suffers a relapse,
making leukaemia the 4th most common cause of
death in this age group. Consequently, current research
focuses on poorly responding ALL subgroups and the
identification of new genetic markers. Additionally, given
the high cure rates of children with ALL, novel treatment
strategies that are less toxic than the currently applied
regimens are urgently needed. Therefore, the ultimate
goal of our research is to identify and characterise new
prognostic and predictive molecular lesions but also to
understand how such changes collude to produce and
sustain overt leukaemia, progress to relapse and cause
drug resistance. To reach this ambitious goal, we i)
try to understand why and how leukaemia develops and
progresses, and ii) use molecular methods to assess
the in vivo treatment response of children with ALL.
What mutations drive leukaemia?
The main focus of our basic and translational research
lies on two large, genomically homogenous ALL subgroups, comprising approximately 20% of all ALL cases
each. The first subgroup carries the ETV6/RUNX1, the
second a high hyperdiploid karyotype (HD). They share
similar clinical features, as both generally lack high risk
criteria and respond rapidly to initial chemotherapy.
They relapse, however, in up to 20% of cases from which
a substantial proportion is resistant to treatment [1].
Both genetic entities have been shown to develop during
fetal life, and need – in addition to the ETV6/RUNX1
gene fusion and the non-disjunction event that leads
to the gain of additional chromosomes – cooperating
aberrations for progression to overt leukaemia [3][4].
During the last two years, we specifically addressed the
function of ETV6/RUNX1 in the fully malignant cell, as
well as the timing and nature of presumably pivotal sec
ondary aberrations in both ALL subgroups [1][2][5][6].
Entschlüsselung der
Leukämieentstehung – Prognose verbessern
Why do some leukaemias re-occur?
To gain new insights into the relapse mechanisms, we
have employed high resolution SNP arrays and, more
recently, also next generation sequencing, for the
unbiased genome-wide identification of copy number
aberrations and somatic sequence mutations in
various leukaemia subgroups. These studies are complemented by targeted re-sequencing and functional
analysis of candidate genes. Thereby we detected
CREBBP deletion and sequence mutations in more
than half of all HD ALL cases at relapse. Moreover, these
mutations seem to prevail at initial presentation of
relapse cases – albeit to a lesser extent than at relapse.
Thus, CREBBP mutations might eventually be explored
as biomarkers for refined risk stratification and cus
tomized treatment [2]. Similar strategies were applied
to explore relapse mechanisms of ETV6/RUNX1-positive leukaemias and late relapsing T cell precursor ALL.
The majority of relapses were either derived from a
common ancestral clone or differed completely from
the initial leukaemia [1][7]. All these studies required
large patient cohorts and sample collections and were
only possible through international collaborations.
Individualisation of leukaemia treatment
Molecular detection of in vivo response to chemo
therapy is currently one of the best predictive
parameters and therefore an integral part of most
treatment protocols [8][9]. Methods and applications
are subjected to continuous improvement and
adaptation within the EURO-MRD Group.
For further reading
[1] Kuster, Grausenburger et al. 2011
[2] Inthal, Zeitlhofer et al. 2012
[3] Panzer-Grümayer, Fasching et al. 2002
[4] Ford, Bennett et al. 1998
[5] Fuka, Kauer et al. 2011
[6] Fuka, Kantner et al. 2012
[7] Szczepanski, van der Velden et al. 2011
[8] Schrappe, Valsecchi et al. 2011
[9] Conter, Bartram et al. 2010
Die akute lymphoblastische Leukämie (ALL) ist als
häufigste Krebsart des Kindes- und Jugendalters mit
derzeitigen risiko-angepassten Therapieprotokollen
in ungefähr 80% heilbar. Dieser Erfolg hat dazu geführt,
dass nun auch die Lebensqualität bei Kindern mit ALL
ein wichtiges Kriterium für die Beurteilung der Therapie
darstellt und daher weniger toxische therapeutische
Maßnahmen, als die bisher angewandten, notwendig
geworden sind. Aus diesem Grund haben wir uns zum
übergeordneten Forschungsziel gesetzt, einerseits
neue prognostisch relevante molekulare Läsionen zu
entdecken und zu charakterisieren, andererseits aber
auch generell zum besseren Verständnis beizutragen,
wie Mutationen zusammenwirken und zu einer klinisch
manifesten Leukämie führen. Darüber hinaus möchten
wir untersuchen, ob bestimmte genomische Ver
änderungen für den Bestand der Leukämieerkrankung
notwendig sind, welche Rolle sie in der Rezidiventwicklung spielen und wie sie zu einer Resistenz gegen
Chemotherapeutika beitragen.
Welche Mutationen sind für ALL wichtig?
Der Hauptfokus unserer Grundlagen- und transla
tionalen Forschung liegt auf zwei großen, genomisch
einheitlich definierten Untergruppen der ALL, die
jeweils ca. 20% aller Leukämien ausmachen. Die eine
Gruppe ist durch das Vorhandensein des ETV6/RUNX1
Fusionsgens, die zweite durch einen hyperdiploiden
Chromosomensatz charakterisiert; beide Leukämie
formen sind generell nicht mit Hochrisiko-Kriterien
assoziiert und sprechen sehr gut auf die Ersttherapie
an. Nichtsdestoweniger treten bei bis zu 20% der Kinder
Rezidive auf. Im Gegensatz zur früheren Annahme, dass
diese Rezidive abermals gut auf die Therapie ansprechen, ist bei einer beträchtlichen Anzahl der Patienten
nun eine Resistenz gegen die verabreichte Therapie
beobachtet worden [1][2]. Beide Leukämieformen
entstehen bereits während der fötalen Entwicklung
im Mutterleib und benötigen weitere Mutationen,
um eine klinisch manifeste Leukämie zu erzeugen [3][4].
Während der letzten zwei Jahre haben wir uns
insbesondere auf die Funktion von ETV6/RUNX1 in der
leukämischen Zelle, sowie die zeitliche Abfolge und Beschaffenheit von kritischen sekundären Veränderungen
in beiden Leukämiegruppen konzentriert [1][2][5][6].
Warum treten Leukämien erneut auf?
Um neue Erkenntnisse auf dem Gebiet der Rezidiventwicklung zu gewinnen, haben wir hochauflösende
SNP Arrays und genomweite Sequenzierungen für den
uneingeschränkten Nachweis von genetischen Veränderungen verwendet. Diese Studien wurden durch
funktionelle Analysen ausgewählter Gene ergänzt. Wir
konnten so einerseits bei hyperdiploiden Leukämien
und ETV6/RUNX1-positiven, andererseits bei T ZellLeukämien feststellen, dass die meisten Rezidive
von einer gemeinsamen Vorläuferzelle abstammen
oder sogar komplett unterschiedlich sind [1][2][7].
Da alle diese Studien große Patientenzahlen benötigen,
wurden sie im Rahmen von internationalen Kollabora
tionen, insbesondere der BFM Studiengruppen,
durchgeführt.
Erfassung des Therapieansprechens
Mittels hochauflösender molekularer Methoden
bestimmen wir das individuelle Therapieansprechen
aller Kinder mit ALL in Österreich. Diese Form der
Diagnostik stellt derzeit einen der besten prädiktiven
Parameter da und ist in die meisten Therapiestudien
integriert [8][9]. Die Methoden und Anwendungen
werden innerhalb der Europäischen MRD Gruppe
ständig verbessert und erweitert.
Literaturangaben
[2] Inthal, Zeitlhofer et al. 2012
[3] Panzer-Grümayer, Fasching et al. 2002
[4] Ford, Bennett et al. 1998
[7] Szczepanski, van der Velden et al. 2011
[8] Schrappe, Valsecchi et al. 2011
[9] Conter, Bartram et al. 2010
26–27
Unraveling leukaemia –
pathogenesis to improve
prognosis of affected children
Leukaemias
Research focus
We also found that PI3K/mTOR inhibitors sensitised
resistant cell lines to drugs. Our findings thus
demonstrate that – together with a severely impaired
repopulation capacity of leukaemia cell lines upon
suppression of ETV6/RUNX1 in NOD/SCID mice –
continuous expression of the ETV6/RUNX1 fusion
gene i) interferes with key regulatory functions that
shape the biology of this leukaemia subtype and ii)
is essential for disease maintenance. Importantly,
these results provide the first rationale and
justification for targeting the fusion gene and the
PI3K/AKT/mTOR pathway therapeutically [2].
This is of particular relevance given the enormous
clonal heterogeneity of secondary aberrations that
precludes their therapeutic targeting and leaves
ETV6/RUNX1 as the only common marker [1][2][3].
A
B
Fig. 1 – Meta-groups of functional annotations for up-regulated
genes upon E/R knock-down. Meta-groups were curated based on
gene clustering of annotation terms.
A: Top 100 annotation terms from knock-down-UP genes, their
P-values and their affiliation to meta-groups. Similarity of the metagroups was based on the number of shared genes. For distance
calculations between the meta-groups genes from all contributing
terms were taken together.
B: Change in expression of individual genes in meta-groups that
contain significant annotation terms. The color code at the bottom of
the figure indicates the extent of log2-fold changes in gene expression.
The P2RY8-CRLF2 fusion defines a particular relapseprone subset of ALL in non-Down Syndrome (DS)
children. It derives from an interstitial deletion in
the PAR1 region, results in the overexpression of the
cytokine receptor-like factor 2 (CRLF2) and practically never concurs with ETV6-RUNX1, MLL, TCF3 or
BCR-ABL fusions. Previous reports suggested that
P2RY8-CRLF2 represents a driver mutation and that
the abrogation of its signaling may by therapeutically
exploited. Prompted by the observation of subclonal
distribution of the fusion, we investigate whether
and to what extent different clone sizes influence
disease and relapse development. For this purpose,
we quantified the genomic P2RY8-CRLF2 fusion
product and correlated it with the corresponding
CRLF2 expression levels in patients enrolled in the
BFM-ALL 2000 protocol in Austria.
Of 268 cases without recurrent chromosomal
translocations and high-hyperdiploidy (representing
approximately 50% of all cases), 67 (25%) were
P2RY8-CRLF2-positive [4]. The respective clone sizes
were ≥20% in 27% and <20% in 73% of the cases. The
cumulative incidence of relapse of the entire fusionpositive group was clone size independent and significantly higher than that of the fusion-negative group
(35±8% vs. 13±3%, P=0.008); also, these cases were
primarily confined to the non-high-risk groups. Of 22
P2RY8-CRLF2-positive diagnosis/relapse pairs, only 4/8
had the fusion-positive dominant clone conserved at
relapse, whereas none of the original 14 fusion-positive
small clones reappeared as the dominant relapse
clone. We conclude that the majority of P2RY8-CRLF2positive clones are small at diagnosis and virtually never
generate a dominant relapse clone. Our findings therefore suggest that P2RY8-CRLF2-positive clones do not
have the necessary proliferative or selective advantage
to evolve into a disease-relevant relapse clone [4][5].
Kofler R. and Rainer J., Tyrolian Cancer Research Institute
and Biocenter – Division Molecular Pathophysiology,
Innsbruck Medical University, Austria
Bauer E., Kanter H.P. and Stoiber D., Ludwig Boltzmann
Institute for Cancer Research, Vienna, Austria
Hall A. and Russel L., Northern Institute for Cancer Research,
Newcastle University, Newcastle upon Tyne UK
Metzler M., Department of Paediatrics, University of
Erlangen-Nuernberg, Erlangen, Germany
Meyer L.H., Universitätsklinik für Kinder- und Jugendmedizin,
Ulm, Germany
Meyer C. and Marschalek R., Institute of Pharmacological
Biology/DCAL, Goethe-University, Frankfurt, Germany
Harbott J., Oncogenetic laboratory, Dept. Ped. Haematol/Oncol.,
Justus-Liebig University, Gießen, Germany
Associazione Italiana di Ematologia ed Oncologia
Pediatrica (AIEOP)-Berlin-Frankfurt-Münster (BFM)
Study Group, European Study Group for the Detection
of Minimal Residual Disease (Euro-MRD-Group).
For further reading
[4] Attarbaschi, Morak et al. 2012
[5] Morak, Attarbaschi et al. 2012
28–29
To evaluate the impact of ETV6/RUNX1 on the regulation of genes and pathways in leukaemia, we performed
shRNA-mediated knock-down of the endogeneous
fusion gene in two leukaemic cell lines and investi
gated the ensuing consequences on genome-wide
gene expression patterns and deducible regulatory
functions [1]. We thereby identified 777 genes whose
expression was substantially altered with approximately
equal numbers up- or down-regulated. The up-
regulated set was significantly enriched with genes
included in cell activation, immune response,
apoptosis, signal transduction and development, and
differentiation categories (Fig. 1). In the ETV6/RUNX1
knock-down set, only PI3K/AKT/mTOR signaling and
haematopoietic stem cell category were observed.
Comparable gene expression signatures obtained
from primary ETV6/RUNX1-positive ALL samples
also underlined the relevance of these pathways and
molecular functions. As a consequence, we analysed
the interaction between the fusion gene and
PI3K/AKT/mTOR pathway activation in more detail
and showed that ETV6/RUNX1 indeed activates this
pathway and that pharmacological inhibition
significantly increases the apoptosis rate in these
leukaemias.
Challenging P2RY8-CRLF2’s
role as main relapse-driving
force in leukaemia
ETV6/RUNX1 perturbs key
processes and is essential
for leukaemia
Leukaemias
Specific project
Specific project
Leukaemias
Genetics of
Leukaemia
Leukämien
Leukämiegenetik
Group leader
Sabine Strehl
[email protected]
Postdocs
Dagma Denk1
Klaus Fortschegger
Karin Nebral2
PhD students
Stefanie Anderl3
Sara Colomer Lahiguera4
Dagmar Denk5
Diploma/MSc student
Teresa Preglej6
Technician
Margit König
Clinicians,
Andishe Attarbaschi
Michael N. Dworzak
Georg Mann
1 since Aug. 2012
2 until Febr. 2012
3 since Apr. 2011
4 since Dec. 2011
5 until July 2012
6 since Sept. 2012
Although in the majority of leukaemia the primary
genetic alterations responsible for the pathogenesis
of the disease have already been unraveled [1][2], in
many instances the underlying molecular genetic
lesions are still unknown. Furthermore, the biological
consequences as well the clinical impact of numerous
specific genetic alterations remain to be determined.
Therefore, the research of the Genetics of Leukaemia
group is centred around two main aspects: On the one
hand, we focus on the identification of novel genetic
alterations that are involved in the pathogenesis and
progression of childhood acute leukaemia and the
evaluation of genetic lesions as potential predictive
biomarkers for the refinement of risk stratification and
risk-adapted therapy. On the other hand, our aim is to
gain further insights into the functional consequences
of specific genetic alterations, in particular those
potentially oncogenic fusion genes that have been
identified by our group [3][4].
Genetic alterations and prognosis
To accomplish these tasks, we conduct comprehensive population-based screening studies for genetic
alterations in leukaemia samples of patients enrolled
in the Austrian BFM clinical trials employing a range of
molecular genetic approaches. Genetic and clinical
datasets are then used to determine whether some
recurring genetic alteration may serve as biomarkers
for treatment failure and whether its occurrence may,
therefore, justify therapy intensification. The assessment of the prognostic relevance of specific genetic
aberrations is performed in close collaboration with the
clinicians of the St. Anna Children’s Hospital and within
the framework of the International BFM Study Group.
Genetische und
biologische Charakterisierung
akuter Leukämien
PAX5 aberrations in B-cell leukaemia
To gain further insights into the pathogenesis of B-cell
precursor acute lymphoblastic leukaemia (BCP-ALL),
we are focusing on the functional consequences of
PAX5 alterations. The rationale for the selection of this
particular gene is derived from the fact that PAX5 plays
a pivotal role in B-cell commitment and maintenance
and is affected by mutations – including deletions,
amplifications, point mutations, and gene rearrangements – in about 30% of paediatric BCP-ALL, suggesting a crucial role in the pathogenesis of the disease.
Based on our identification of several PAX5 fusion
genes, which encode potentially oncogenic aberrant
transcription factors [3–5], we now aim to elucidate
their role in leukaemogenesis.
References
[1] Pui, Carroll et al. 2011
[2] Pui, Mullighan et al. 2012
[3] Nebral, König et al. 2007
[4] Nebral, Denk et al. 2009
[5] Denk, Nebral et al. 2012
In der Mehrheit der akuten Leukämien im Kindes- und
Jugendalter wurden die für die Krankheitsentstehung
verantwortlichen primären genetischen Veränderungen
bereits aufgeklärt [1][2]. Dennoch sind in vielen Fällen
die genetischen Aberrationen, die zur Leukämie
entwicklung führen, nach wie vor unerforscht, und
sowohl deren biologische Auswirkungen als auch deren
Einfluss auf die Prognose der PatientInnen unbekannt.
Deshalb fokussiert die Arbeitsgruppe für Leukämiegenetik ihre Arbeit auf zwei Schwerpunktthemen: Einerseits auf die Identifizierung genetischer Veränderungen,
die in die Krankheitsentstehung und das Fortschreiten
einer akuten Leukämie involviert sind, sowie die
Bewertung der prognostischen Relevanz bestimmter
genetischer Veränderungen, die in Folge als sogenannte
Biomarker zur Risikobewertung und Therapieab
stimmung herangezogen werden könnten. Andererseits
ist es Ziel unserer Forschungsarbeit, die biologischen
Auswirkungen bestimmter genetischer Veränderungen
zu erforschen, insbesondere jener Fusionsgene, die
unsere Arbeitsgruppe identifiziert hat [3][4].
Die Bedeutung genetischer Veränderungen
Um diese Ziele zu erreichen, führen wir umfassende
genetische Analysen der leukämischen Zellen aller
PatientInnen durch, die in den österreichischen
Leukämiestudien registriert sind. Dafür setzen wir eine
Reihe molekularer Techniken ein. Um festzustellen,
ob eine spezifische genetische Veränderung mit einem
hohen Risiko eines Therapieversagens einhergeht
und als prognostisch aufschlussreicher genetischer
Biomarker eingesetzt werden kann, werden genetische
und klinische Daten miteinander korreliert. Die Bewertung der prognostischen Relevanz der genetischen
Marker, die zu einer verfeinerten Einteilung in Risikogruppen führen könnte, wird in enger Zusammenarbeit
mit den ÄrztInnen des St. Anna Kinderspitals und
der Internationalen BFM Studiengruppe durchgeführt.
Leukämie und PAX5
Um neue Erkenntnisse in Bezug auf die Krankheitsentstehung der B-Vorläuferzell akuten lymphatischen
Leukämie (B-ALL) zu gewinnen, erforschen wir schwer
punktmäßig die Auswirkungen von Veränderungen
des PAX5 Gens. Die Motivation, gerade dieses Gen zu
untersuchen, begründet sich darauf, dass PAX5 eine
zentrale Funktion in der Entwicklung normaler B-Zellen
und deren Erhaltung spielt und in ungefähr 30% aller
BALL von genetischen Veränderungen – Deletionen,
Amplifikationen, Mutationen und Genrearrangements,
die zu Genfusionen führen – betroffen ist. Nahe
liegenderweise könnte PAX5 deshalb auch eine Rolle
in der Leukämieentstehung spielen. Ausgehend von
unserer Identifikation einiger PAX5 Partnergene [3–5]
erforschen wir nun, welche Rolle diese potentiell
onkogenen Fusionsgene in der Pathogenese der akuten
Leukämie spielen.
Literaturangaben
[1] Pui, Carroll et al. 2011
[2] Pui, Mullighan et al. 2012
32–33
Genetic and biological
characterisation of paediatric
acute leukaemia
Leukaemias
Research focus
Properties of PAX5 fusion proteins
The consistent retention of the N-terminal PAX5 DNAbinding paired domain, which is fused to the C-terminal
domains of one of the highly diverse partner proteins,
indicates that PAX5 chimeric proteins may have
properties both common to and distinct from those
of wild-type PAX5. Indeed, independent of the genuine
subcellular localisation of the individual partner protein,
all PAX5 fusion proteins display a predominantly
nuclear localisation (Fig. 1), which appears to be mainly
dictated by the paired domain. Intriguingly, in contrast
to the cytoplasmic localisation of all other JAK2 fusion
proteins analysed to date, PAX5-JAK2 is also a nuclear
protein. As a second common key characteristic
of all PAX5 fusion proteins, the PAX5 paired domain
mediates their DNA interactions at specific PAX5 target
sequences, albeit with obviously variable affinities,
pointing at potential differences in their effects on
gene transcription. In contrast to these common
biochemical properties, our experiments showed that
only some PAX5 fusion proteins are capable of homooligomerisation while others are not. Since PAX5 wildtype protein is known to bind to DNA as monomer, the
interfaces for self-interaction are provided by distinct
moieties such as coiled-coil domains present in the
respective partner proteins. In terms of their impact on
PAX5 target gene expression, at least in reporter gene
assays, some of the PAX5 fusion proteins appeared to
have activation potential rather than mere antagonizing
or repressive capacity. However, in a murine cell line
model, the effect of PAX5 chimeric proteins on the
transcriptional activation of the PAX5 target Cd79a
could not be confirmed and thus, additional studies
are required to determine the impact of PAX5 chimeric
proteins on PAX5 target gene transcription.
Constitutive kinase activity of PAX5-JAK2
Intriguingly, amongst all PAX5 fusion proteins, PAX5JAK2 appears to have a unique function, because it has
constitutive kinase activity and activates the JAK-STAT
signaling pathway. More specifically, PAX5JAK2 itself
is phosphorylated and, in turn, phosphorylates and
activates downstream STATs. Furthermore, gene
expression profiling of PAX5-JAK2 positive patient
samples revealed high similarities with BCR-ABL1
and JAK2-mutated BCP-ALL, further substantiating
that in the primary leukaemia, the JAK-STAT pathway
is indeed activated. Importantly, at least in vitro, the
kinase activity of PAX5-JAK2 can be efficiently blocked
by JAK2 inhibitors, rendering it a potential target for
therapeutic intervention.
References
[1] Mullighan, Goorha et al. 2007
[4] Coyaud, Struski et al. 2010
In international collaborations, we continually
contribute to studies regarding the prognostic
relevance of specific genetic alterations in the context
of the I-BFM and in comparison to other treatment
protocols. These multicentre studies are a necessity
to increase the size of patient cohorts to gain more
statistical power, and to analyse rare disease entities.
of leukaemia, we have analysed 350 cases lacking the
major sentinel fusion genes by an IGH@ break-apart
FISH assay and have identified IGH@ translocations
in ~5% of patients. This study confirmed CRLF2 to be
the most frequent IGH@ partner gene, however, in
50% of cases the IGH@ translocation partners remain
unknown and their identification is still ongoing.
MLL and CRLF2 rearrangements
STAT5B-RARA-positive acute
promyelocytic leukaemia
The I-BFM study group has extended its meta-analysis
assessing the clinical impact of specific MLL rearrangements in paediatric acute myeloid leukaemia (AML)
and showed that not only the MLL translocation partner
itself [1] but also secondary cytogenetic aberrations
independently predict clinical outcome [2]. Moreover,
in a comparative study it has been determined that
the cumulative relapse incidence of P2RY8-CRLF2+
patients treated on the AIEOP-BFM or UK trials did
not differ statistically. However, the relapse rate of
IGH@-CRLF2+ patients was much higher in patients
enrolled in UK trials suggesting treatment-related
differences [3].
IGH@ translocations
In another international project with the aim to
ascertain the true incidence of IGH@ translocations
in BCP-ALL and to further characterise this subtype
Furthermore, we have determined that, in contrast
to PML-RARA+ acute promyelocytic leukaemia (APL),
which is responsive to treatment with all-trans
retinoic acid (ATRA) and arsenic trioxide (ATO),
the STAT5B-RARA fusion variant defines a highly
relapse-prone APL subgroup, which is insensitive
to both therapeutic agents. Hence, the accurate
identification of this rare subtype of APL is therefore
essential for therapeutic decision-making [4].
The Austrian (represented by Andishe Attarbaschi,
Michael N. Dworzak, Georg Mann, all St. Anna Children’s Hospital)
and the International Berlin-Frankfurt-Münster Study Group
(I-BFM-SG).
References
[1] Balgobind, Raimondi et al. 2009
[2] Coenen, Raimondi et al. 2011
[3] Attarbaschi, Morak et al. 2012
[4] Strehl, König, et al. 2012
Fig. 1 – The indicated PAX5 fusion proteins predominantly localise
to the nucleus as determined by immunofluorescence and confocal
microscopy; white arrow points out punctate cytoplasmic staining
and white bars indicate 20 μm.
34–35
In about 30% of B-cell precursor acute lymphoblastic
leukaemia (BCP-ALL), PAX5, a master regulator of
B-cell development, is affected by genetic alterations
including deletions, amplifications, point mutations,
and the formation of fusion genes [1]. While deletions
and point mutations frequently coincide with known
sentinel alterations such as the ETV6-RUNX1 and
BCR-ABL1 fusion genes, and are therefore considered
as secondary events, PAX5 rearrangements, which
result in the expression of potentially oncogenic
fusion genes, most likely represent primary genetic
events. The chimeric proteins encoded by the fusion
transcripts are hypothesised to act as trans-dominant
aberrant transcription factors, which antagonize
wild-type PAX5 function. Based on our own studies and
those of others, in which it has been determined that
the N-terminus of PAX5 is fused to a markedly heterogeneous group of C-terminal fusion partners including
transcription factors, structural proteins or the tyrosine
kinase JAK2 [1–5], we currently aim to investigate
the biochemical and functional properties of these
distinct chimeric proteins.
Treatment outcome of
childhood leukaemia with
specific genetic alterations
Functional consequences
of PAX5 fusion proteins
Leukaemias
Specific project
Specific project
Solid Tumours
Tumour Biology
Solide Tumoren
Tumorbiologie
Group leader
Peter F. Ambros
[email protected]
Staff scientist
Ingeborg M. Ambros
Sabine Taschner-Mandl
PhD students
M. Reza Abbasi1
Dominik Bogen2
Fikret Rifatbegovic3
Tamara Weiss4
Diploma/MSc student
Magdalena Schwarz5
Technicians
Bettina Brunner-Herglotz
Andrea Ziegler
Clinicians
Leo Kager
Ruth Ladenstein
1 since Mar. 2012
2 currently at NIH
3 since May 2012
4 since Oct. 2011
5 since Sep. 2012
Our research is primarily devoted to understanding
the genomic and cell biological aspects of neuro
blastomas and other paediatric solid tumours to
establish a sound basis for patient specific treatment
options and to provide information on therapeutically
useful compounds that induce tumour cell senescence
and tumour cell death.
Genomic data helps to direct therapy
Neuroblastomas are the most frequent solid tumours
of infancy and early childhood. Their biological
behaviour is unique and does not necessarily
correspond to tumour dissemination but is largely
dependent on tumour genomics. A favourable course,
which occurs in more than a third of patients, is based
on spontaneous regression or maturation of tumour
cells. In the majority of patients, however, neuro
blastomas behave aggressively [1]. Over the last two
decades a number of research teams, including ours,
have provided evidence that genomic features help
to differentiate between prognostically different
subgroups. Austria was in fact one of the first countries
to apply a genome based treatment stratification,
resulting in a dramatic increase of surviving patients [2].
This worldwide accepted concept is now being applied
and developed further in European clinical studies
aiming at reducing or intensifying cytotoxic treatment
in genetically defined patient groups [3]. Since these
current clinical studies range from a “wait and see
strategy” after complete/incomplete resection or
even no resection at all to the application of high dose
chemotherapy and stem cell transplantation, the
tumour-genome based therapy stratification has to be
as secure, reliable, sensitive and specific as possible.
Hence, we apply the most advanced moleculargenomic diagnostic tools currently available to us.
The detection of tumour genomic changes is performed by a high resolution array technique (besides
the application of interphase FISH). The simultaneous
quantification of more than 2.6 million probes on
one array represents the highest genomic coverage
Einblicke in die
Tumorbiologie erlaubt
individuelle Behandlung
presently available, allowing detection of copy number
changes that affect even single exons, exact breakpoint
localisations, gene amplifications, uniparental disomies,
low-level-mosaicisms and chromothripsis, a recently
described state of genomic catastrophe indicating
unfavourable prognosis [4]. This information, provided
to the clinician in charge, enables a patient-tailored
treatment strategy.
Another crucial diagnostic pillar is the proof of tumour
cells in the bone marrow, performed by a highly
sophisticated combination of genetic and immunologic
techniques and an automatic microscope [5].
International studies
Currently, two European neuroblastoma studies cover
the vast majority of patients throughout Europe by
implementing genomic features to direct therapy:
The Low/Intermediate Risk Neuroblastoma Study
(LINES) and the High Risk Neuroblastoma (HR-NBL)
Study. In both studies, our group has the role of chairman and co-chairman of the European Biology Group
to guarantee the best possible quality of the genomic
data [6]. The members of the Biology Group are from
12 European and 4 non-European countries (Australia,
Hong Kong, Israel und Japan). Furthermore, our group
is embedded in two international efforts to link genomic
and MRD (minimal residual disease) information with
clinical data to establish consensus criteria that will
then be used worldwide (International Neuroblastoma
Risk Group – INRG, and International Neuroblastoma
Response Criteria – INRC) [7].
For further reading
[1] Brodeur 2003; Maris, Hogarty et al. 2007
[2] Ladenstein, Ambros et al. 1996
[3] Cohn, Pearson et al. 2009
[4] Molenaar, Koster et al. 2012
[5] Ambros, Mehes et al. 2003
[6] Ambros, Benard et al. 2003; Ambros, Brunner et al. 2011
[7] Ambros, Ambros et al. 2009; Cohn, Pearson et al. 2009;
Monclair, Brodeur et al. 2009; London, Castel et al. 2011;
Moroz, Machin et al. 2011; Taggart, London et al. 2011;
Angelini, London et al. 2012; Schleiermacher, Mosseri et al. 2012
Es ist unser Ziel, Einblicke in Genomveränderungen
und die zellbiologischen Eigenschaften von Neuroblastomen und anderen soliden Tumoren bei Kindern und
Jugendlichen zu gewinnen, um eine wissenschaftliche
Grundlage für individuelle Therapien zu begründen.
Zudem identifizieren wir Wirkstoffe, die Tumorzellen
altern bzw. absterben lassen.
Eine Basis für individuelle Therapien
Neuroblastome sind die häufigsten Tumoren bei Säug
lingen und Kleinkindern mit einem biologisch einzig
artigen Verhalten, das oftmals von den Genomver
änderungen des Tumors abhängig ist. In mehr als einem
Drittel der Patienten verhalten sich die Neuroblastome
durch spontane Rückbildung (Absterben der Tumor
zellen) oder durch Ausreifung gutartig. Bei der Mehrzahl
der Patienten werden allerdings aggressive Neuro
blastome diagnostiziert [1]. Unsere Forschungsarbeiten
haben dazu beigetragen, Patienten, die sich hinsichtlich ihrer Prognose erheblich unterscheiden, anhand
tumorgenomischer Eigenschaften zu identifizieren.
Österreich war weltweit eines der ersten Länder, in
denen an Hand von Genomanalysen die Behandlungsstrategien festgesetzt wurden, was zu einem beachtlichen Anstieg der Überlebensraten führte [2]. Dieses
Therapiekonzept ist heute weltweit anerkannt und wird
in europäischen Studien stetig weiter entwickelt und
verbessert [3]. Aktuelle klinische Studien verfolgen
eine Strategie des Zuwartens ohne Chemotherapie
nach kompletter, teilweiser oder gar keiner Resektion
bis hin zu hochdosierter Chemotherapie und Stamm
zelltransplantation. Aufgrund dieser Behandlungsunterschiede müssen die Untersuchungsmethoden,
welche den Entscheidungsprozess mitbestimmen,
sicher, zuverlässig, sensitiv und hochspezifisch sein.
Wir verwenden höchstauflösende molekulargenetische
Array Methoden, die nur an diesem Zentrum in der
Neuroblastomdiagnostik eingesetzt werden. Derartige
Analysen geben Aufschluss über die Organisation der
Erbsubstanz und die Art und Anzahl von Abweichungen:
Veränderungen einzelner Exons, Aufklärung exakter
Bruchstellen, Nachweis von Genvervielfältigungen,
und Chromothripsis, einer Form von sog. katastrophaler
genomischer Umlagerung, die mit einer schlechten
Prognose einhergeht [4]. Auch die klassische Methode
der Interphase Fluoreszenz-in-situ-Hybridisierung
wird in der Diagnostik angewandt. Mithilfe dieser
Information können Therapien auf den Patienten
maßgeschneidert werden.
Ein weiterer Eckpfeiler der Diagnostik ist die B
eurteilung
des Therapieansprechens mittels Nachweis von
Tumorzellen im Knochenmark bei Einsatz von genetischen und immunologischen Techniken und einem
vollautomatischen Fluoreszenzmikroskop [5].
Internationale Studien
Aktuell werden in zwei europäischen NeuroblastomStudien die Therapien auf Basis von Genominforma
tionen angepasst: die Niedrig/Mittelhoch-Risiko
Neuroblastom-Studie (LINES) und die Hoch-Risiko
Neuroblastom-Studie (HR-NBL). Wir leiten die
europäische Biologie Gruppe, die die Genomanalysen
durchführt. Der Zusammenschluss internationaler
Labors und permanente Qualitätskontrollen garantieren höchste Datenqualität. Zudem sind wir in zwei
Initiativen aktiv, die genomische bzw. minimale Rest
erkrankungsdaten mit klinischen Daten verbinden, um
weltweit verbindliche, diagnostische Standards und
Kriterien für den Therapieerfolg festzulegen (Inter
national Neuroblastoma Risk Group – INRG, and Inter
national Neuroblastoma Response Criteria – INRC) [6].
Literaturangaben
[1] Brodeur 2003, Maris, Hogarty et al. 2007
[2] Ladenstein, Ambros et al. 1996
[3] Cohn, Pearson et al. 2009
[4] Molenaar, Koster et al. 2012
[6] Ambros, Ambros et al. 2009; Cohn, Pearson et al. 2009;
Monclair, Brodeur et al. 2009; London, Castel et al. 2011;
Moroz, Machin et al. 2011; Taggart, London et al. 2011;
Angelini, London et al. 2012; Schleiermacher, Mosseri et al. 2012
38–39
Understanding paediatric
tumours facilitates
patient-specific treatment
Solid Tumours
Research focus
Disseminated tumour cells
Induction of tumour cell senescence
Disseminated tumour cells (DTCs) can be responsible
for disease relapse in neuroblastoma patients. At
diagnosis, virtually all stage 4 (M – metastatic) patients
present with DTCs in the bone marrow (BM). After
initial therapy, however, two patient subgroups can
be identified: patients with a drastic reduction of
the tumour cell load below the detection limit of the
applied AIPF (automatic immune-fluorescence plus
FISH) technique (<1 tumour cell/3 million BM cells) [1][2]
and patients retaining tumour cells in the BM. In order
to further elucidate the findings from the latter group,
BM evaluation results of approximately three thousand
BM samples from about seven hundred patients (from
before, during and after chemotherapy or from the
time of relapse) were generated and partly linked with
clinical data. This correlation revealed significant better
(p<0.0005) event-free and overall survival of patients
that showed rapid elimination of DTCs from the BM as
compared to the “non-clearing” group. Thus, monitoring and quantifying the tumour cell load during and
after cytotoxic treatment is a reliable and sensitive way
to monitor treatment success. It will no doubt vastly
improve therapeutic strategies in the future.
Another part of our research focuses on the potential
therapeutic application of low dose cytotoxic drugs
capable of inducing cellular senescence in a subset
of genetically defined tumours. This permanent state
of proliferative arrest can be induced in tumours with
gene amplifications (MYCN amplification, in the case
of neuroblastoma) by low dose hydroxyurea [3] and
camptothecin, a topoisomerase I inhibitor, in vitro.
Topotecan, a camptothecin derivate, is currently used
only at high doses in neuroblastoma treatment
regimens and, therefore, the effects evoked by lowdose topotecan and the involved pathways required
further scrutiny. We found that senescence induction
only works in cell lines with amplification of the
MYCN oncogene and is linked to a drastic reduction of
the MYCN amplification, frequently leading to a nonamplified state. In this manner, the tumour cells lose a
crucial hallmark of aggressiveness and rapid growth.
In two ongoing projects, we characterise DTCs on two
levels: the genomic status and the global expression
profile by applying SNParrays and expression arrays/RNA
sequencing approaches. To facilitate these analyses,
DTC fractions have to be enriched up to more than
1000-fold. The data from these studies are expected
to identify genomic/expression markers that are able
to further discriminate favourable from unfavourable
tumours at diagnosis, to indicate a tumour’s sensitivity
to therapy and to provide information for a targeted
tumour therapy (e.g. ALK inhibitors, receptor tyrosine
kinase inhibitors, CDK inhibitors). Moreover, consecutive array data obtained from DTCs of individual
patients provide information on the genomic tumour
evolution which helps to identify mechanisms leading
to resistance against cytotoxic agents.
In addition, besides a drastically reduced proliferation
activity, marked changes in the expression profile
were detected in low-dose hydroxyurea and topo
tecan treated neuroblastoma cell lines. In co-cultures,
senescent cells reduced cell growth and GD2 levels
of non-senescent tumour cells. Moreover, senescent
neuroblastoma-cells induced a strong immune
reaction, probably mediated through cell-bound but
not secreted factors. This is in line with an increased
expression of MHCI and other immune-responserelated molecules. Senescent neuroblastoma-cells
also showed elevated levels of MICA and MICB which
are ligands of the activating receptor NKG2D, present
on natural killer cells and other immune-reactive cells.
In addition, CD44 a marker of favourable neuro
blastoma, and several genes involved in the process of
adhesion and negative regulation of cell proliferation as
well as secreted factors were found to be up-regulated
by senescent tumour cells. Our current work also
addresses detailed evaluation of the expression profile
with emphasis on the consequences for cell-cell
interactions and analysis of the secretome of senescent neuroblastoma cells. Furthermore, we collaborate
with the Institute of Pharmacology in Vienna to simulate
the process of tumour cell senescence in a xenograft
model. Preliminary data showed a reduced tumour
growth and occurrence of tumour cell senescence in
topotecan treated xenografts. On-going studies aim at
verifying these first observations and will pave the way
for long-term studies. However, further work is needed
to clarify whether neuroblastoma patients may benefit
from senescence-inducing, low-dose treatment.
Schwann cell factors
In contrast to aggressive neuroblastomas, spontane
ously maturing and mature neuroblastomas (ganglio
neuro-blastoma/ganglioneuroma) harbour a special
characteristic, the Schwann cell (SC) stroma.
Importantly, the presence and volume of Schwannian
stroma closely correlate with neuroblastoma growth
inhibition and a favourable prognosis (the exceptions
are histologically unequivocally identifiable
ganglioneuroblastoma subtypes, i.e. the nodular
type of ganglioneuroblastoma) [4]. In previous studies,
we showed that SCs in neuroblastomas are normal
cells that supposedly have been recruited by neuro
blastic cell signals [5]. Furthermore, we postulated a
“cross-talk” between SCs and neuroblastoma cells
causing SC attraction and proliferation, and, in turn,
tumour cell apoptosis and/or differentiation into
ganglionic cells. These assumptions were verified
by us and others by in vitro and in vivo experiments
where SCs have been shown to act also on aggressive
neuroblastoma cells [6][7]. The ongoing project is
dedicated to characterising the Schwann cell factors
involved in tumour growth inhibition by applying a
cytokine antibody array. Moreover, collaboration with
the Institute of Analytical Chemistry shall result in a
comprehensive view of the SC proteome by applying
mass spectrometry. Using this approach, we aim at
identifying the SC secretory protein profile responsible
for inducing growth inhibition and/or apoptosis in
aggressive neuroblastoma cells.
For further reading
[1] Mehes, Luegmayr et al. 2003
[3] Narath, Ambros et al. 2007
[4] Shimada, Ambros et al. 1999
[5] Ambros, Zellner et al. 1996
[6] Ambros, Attarbaschi et al. 2001
[7] Chlenski, Liu et al. 2002
Fig. 1 – A fluorescence microscopic image of Schwann cells,
which fulfill a broad range of tasks in the peripheral nervous system,
in cell culture. Since this cell type plays a crucial role in the
maturation process of neuroblastoma, Schwann cells were isolated,
cleaned up for in vitro experiments and stained with S100 (green),
cytokeratin (red) antibodies and nuclear staining (blue).
Disease monitoring
and novel therapeutic
strategies
40–41
Solid Tumours
Specific project
Solid Tumours
Molecular Biology
of Solid Tumours
Solide Tumoren
Molekularbiologie
Solider Tumore
Group leader
Heinrich Kovar
[email protected]
Staff scientist
Dave Aryee
Jozef Ban
Postdoct. fellows
Argyro Fourtouna1
David Herrero Martin1
Caroline Hutter2
Stefan Nieden3
Raphaela Schwentner3
Eleni Tomazou4
PhD students
Anna Katschnig5
Cornelia Mutz5
Raphaela Schwentner
Technicians
Gunhild Jug
Karin Mühlbacher
1 since Mar. 2011
2 since Oct. 2011
3 since Jan. 2013
4 since Mar. 2012
5 since Nov. 2012
Cancer is caused by mutations in key genes that
withdraw body cells from regular growth control and
immune surveillance. Typical cancers are characterised
by hundreds of genetic alterations, which differ from
patient to patient. In these instances it is frequently
difficult to distinguish disease driving from passenger
mutations. In contrast, Ewing sarcoma, a group of
aggressive bone tumours in children and adolescents,
is characterised by a specific recurrent gene fusion
which is primarily responsible for the disease.
Already since the nineties, our research group has
been investigating the molecular pathogenesis of
Ewing sarcoma. Based on the use of modern genomic
high-throughput technologies, we are characterising
the gene regulatory networks that are controlled by
the specific gene fusion in this disease. Our goal
is to develop basic concepts for novel biology-based
therapeutic strategies. To achieve this goal, the
Austrian Research Fund FWF1 and the collaborative
European systems biology project ASSET2 enable
us to generate and integrate large genomic data
sets and to model and validate molecular disease
mechanisms on a holistic network level.
Our group’s basic research is complemented by our
activities in clinical research networks3–5, which allow
us to test and validate laboratory results in patients.
Our aim is the prioritisation of novel biomarkers and
novel therapeutic concepts for clinical use [1].
In the reporting period 2011–2012, significant progress
has been achieved in the characterisation of the role
of micro RNAs and of a developmental signaling pathway (NOTCH) involved in normal tissue differentiation
for Ewing sarcoma pathogenesis [2][3]. Investigation
of gene regulatory mechanisms in Ewing sarcoma led
to the completion of three PhD theses6–8.
Molekulare Mechanismen der
Ewing-Sarkom-Pathogenese
Langerhans cell histiocytosis’ mechanisms
In addition to our focus on Ewing sarcoma, we have
been investigating pathogenic mechanisms in
Langerhans cell histiocytosis (LCH). This disease of
enigmatic origin is characterised by the accumulation
of Langerhans cell-like cells, and presents either
localised or widely disseminated. It is still unknown
if it has to be considered a reactive (inflammatory)
or a malignant disease. We recently showed that in
LCH, similar to many leukaemias, the NOTCH s ignaling
pathway is activated [4]. Future investigations will
explore the therapeutic potential of this finding.
1 P20665-B12; P223208-B09; P24708-B21
2 EU-FP7 259348 “Assessing and Striking the Sensitivities
of Embryonal Tumours”
3 EU-FP7 “European Network for Cancer Research in
Children and Adolescents”;
4 EuroEwing99;
5 ERA-NET TRANSCAN: Prospective Validation of Biomarkers
in Ewing Sarcoma for personalized translational medicine
“PROVABES” I1225-B19
6 Raphaela Schwentner: Mechanisms of target regulation
by the chimeric oncogene EWS-FLI1 in Ewing’s sarcoma
7 Stephan Niedan: Mechanisms of transcriptional repression
by EWS-FLI1 in Ewing sarcoma.
8 Lucia Riedmann: The effect of EWS and its oncogenic
derivative EWS-FLI1 on transcriptional and post-transcriptional
gene regulation in Ewing sarcoma.
For further reading
[1] Kovar, Alonso et al. 2012
[2] Ban, Jug et al. 2011
[3] Bennani-Baiti, Aryee et al. 2011
[4] Hutter, Kauer et al. 2012
Auslöser und Wirkung von Krebs sind Mutationen von
Schlüsselgenen, welche Körperzellen der normalen
Wachstumskontrolle und Immunüberwachung ent
ziehen. Typische Krebserkrankungen sind durch mehrere
hundert genetische Veränderungen gekennzeichnet,
welche sich von Patient zu Patient unterscheiden.
In diesen Fällen ist es vielfach schwierig, Schlüssel
veränderungen von Trittbrettfahrermutationen zu
unterscheiden. Ewing Sarkome, eine Gruppe äußerst
bösartiger Knochentumore bei Kindern und Jugendlichen, sind hingegen durch eine spezifische, immer
wiederkehrende Genfusion charakterisiert, die für
die Erkrankung hauptverantwortlich ist.
Seit den Neunzigerjahren befasst sich unsere Arbeitsgruppe mit der molekularen Pathogenese des Ewing
Sarkoms. Basierend auf dem Einsatz moderner
genomischer Hochdurchsatzmethoden charakteri
sieren wir genregulatorische Netzwerke, welche von
der für die Erkrankung typischen pathogenen Gen
fusion gesteuert werden. Unser Ziel dabei ist, Grund
lagen für neue therapeutische Ansätze zu entwickeln.
Zur Erreichung unserer Ziele unterstützen uns der
österreichische Forschungsförderungsfonds FWF1
und das Europäische Systembiologieprojekt ASSET2
und ermöglichen uns nicht nur, große Datensätze zu
gewinnen und zu integrieren, sondern auch molekulare
Krankheitsmechanismen auf einer ganzheitlichen
Netzwerkebene zu modellieren und zu überprüfen.
Die grundlagenwissenschaftliche Forschung unserer
Arbeitsgruppe wird durch unsere aktive Rolle in
Europäischen klinischen Forschungsnetzwerken3–5
ergänzt. Diese ermöglichen uns, die im Labor gewonnen
Erkenntnisse auf ihre Gültigkeit in Patienten zu über
prüfen. Ziel ist die Priorisierung von neuen Biomarkern
und neuen therapeutischen Strategien für den
Einsatz in der Klinik [1].
Im Berichtszeitraum 2011–2012 wurden vor allem
Fortschritte in der Charakterisierung der Rolle von
MikroRNAs [2] und eines in der normalen Embryonal
entwicklung und Differenzierung vieler Gewebe
wichtigen Signalweges (NOTCH) [3] für die Pathogenese
des Ewing Sarkoms erzielt. Untersuchungen zu den
Mechanismen der Genregulation in Ewing Sarkomen
führten zum Abschluss dreier Dissertationen6–8.
Langerhanszell-Histiozytose-Mechanismen
Neben dem Forschungsschwerpunkt Ewing Sarkom
beschäftigen wir uns mit den molekularen Mechanismen der Langerhanszell Histiozytose (LCH). Diese
Erkrankung unbekannter Herkunft zeichnet sich durch
die Anhäufung Langerhanszell-ähnlicher Zellen aus
und kann sowohl lokalisiert als auch disseminiert auf
treten. Bis heute ist unklar, ob es sich um eine reaktive
(entzündliche) oder eine bösartige Tumorerkrankung
handelt. Wir konnten zeigen, dass in dieser Erkrankung
der NOTCH Signaltransduktionsweg, ähnlich wie bei
vielen Leukämien, aktiviert vorliegt [4]. Zukünftige
Untersuchungen werden das therapeutische Potential
dieses Befundes erforschen.
1 P20665-B12; P223208-B09; P24708-B21
2 EU-FP7 259348 „Assessing and Striking the Sensitivities
of Embryonal Tumours”
3 EU-FP7 „European Network for Cancer Research in
Children and Adolescents”;
4 EuroEwing;
5 ERA-NET TRANSCAN: Prospective Validation of Biomarkers
in Ewing Sarcoma for personalized translational medicine
„PROVABES” I1225-B19
6 Raphaela Schwentner: Mechanismen der Zielgenregulation
durch das chimäre Onkogen EWS-FLI1 im Ewing Sarkom
7 Stephan Niedan: Mechanismen der transkriptionellen
Repression durch EWS-FLI1 im Ewing Sarkom.
8 Lucia Riedmann: Die Wirkung von EWS und seinem
onkogenen Derivat EWS-FLI1 auf die transkriptionelle und
post-transkriptionelle Genregulation im Ewing Sarkom
Literaturangaben
[1] Kovar, Alonso et al. 2012
[2] Ban, Jug et al. 2011
44–45
Molecular mechanisms of
Ewing sarcoma pathogenesis
Solid Tumours
Research focus
From genomics to novel
therapeutic concepts in
Ewing sarcoma
Re-activating the
p53 tumour suppressor
NOTCH in Langerhans
cell histiocytosis
The EWS-FLI1 chimeric protein is produced specifically
in Ewing sarcoma as a result of a diagnostic chromosomal gene rearrangement. It functions as an aberrant
sequence-specific ETS1 transcription factor with both
gene activating and gene repressive consequences.
Since this is specific to Ewing sarcoma and not existent
in any normal tissue, EWS-FLI1 would constitute an
ideal therapeutic target in this disease. However,
efficient targeting of nuclear transcription factors still
constitutes an unsolved pharmacological problem.
We therefore used an integrated genomic approach to
characterise mechanisms and downstream pathways
of EWS-FLI1 activity. Two major patterns of gene regulation were observed: i) Gene activation was found predominantly associated with EWS-FLI1 binding proximal
to the transcription start sites mainly of genes involved
in cell cycle and growth regulation, RNA metabolism
and translation. Here, we found functional cooperation
with the family of E2F1 transcription factors [1].
ii) Gene repression was most frequently observed
when EWS-FLI1 was binding to chromatin distal (>4kb)
to transcriptional initiation sites and affected pre
dominantly genes involved in mesenchymal differen
tiation, neural development and bone remodeling.
Activation of the p53 tumour suppressor pathway is
central to different kinds of cellular stress responses.
We have previously demonstrated modulation of
p53 basal levels by EWS-FLI1 in Ewing sarcoma [3].
We found that EWS-FLI1 suppresses an important
developmental signaling pathway, NOTCH2 [3][4].
Upon experimental silencing of EWS-FLI1, or ectopic
stimulation of NOTCH activity, p53 was found to
be reactivated leading to tumour cell growth arrest.
Stability and activity of p53 is generally regulated by
protein modifications. In the reporting period, we
identified impaired acetylation as the cause of p53 in-
activation in EWS-FLI1 expressing Ewing sarcoma cells.
A deacetylase, SIRT1, which links cellular metabolism to
p53 regulation, was identified as a NOTCH suppressed
target. In the presence of EWS-FLI1, NOTCH signaling
is repressed, and consequently SIRT1 expression is
induced preventing p53 from activating acetylation.
There is still relatively little known about the nature of
the cells that are found in LCH, except that they seem
to be immature dendritic cells (DC) that exhibit much
of the surface markers of Langerhans cells – hence
also the name Langerhans Cell Histiocytosis. In order
to elucidate the relationship of the LCH-DCs with
normally occurring DC subsets, we have performed
comparative gene expression analysis of LCH cells and
three major, functionally divergent human DC lineages,
i.e. epidermal LC, myeloid dendritic cells (mDC1), and
plasmacytoid dendritic cells (pDC) in close collabo
ration with Ernst Kriehuber from the Department of
Dermatology/Medical University of Vienna. We found
that LCH cells are distinct from LC, mDC, and pDC
and display a unique combination of hallmark lineage
antigens, indicating that these cells form a separate
DC entity. Interestingly, LCH cells seem to be
equidistantly related to mDC1 and LC, challenging
the idea that LCH cells are aberrant LCs.
We found that the proximal regions of EWS-FLI1
repressed genes are enriched in recognition motifs of
FOX1 transcription factors, suggesting an indirect mode
of transcriptional repression of a subset of EWS-FLI1
controlled genes. We identified the responsible protein
as the tumor suppressor FOXO1 and characterised a
multilayered mechanism of FOXO1 repression by
EWS-FLI1 through transcriptional and post-transcriptional mechanisms. Reactivation of FOXO1 in Ewing
sarcoma cells resulted in cell death induction. We
identified a small molecule capable of inducing active
FOXO1 in Ewing sarcoma cells associated with significantly reduced tumour growth in an in-vivo disease
model. These results provide a proof of principle for a
successful pharmacological intervention downstream
of EWS-FLI1 and potentially hold promise for innovative
therapeutic strategies in Ewing sarcoma [2].
Using a pharmacological SIRT1 inhibitor, we were able
to re-activate the tumour suppressor p53 and induce
cell death in Ewing sarcoma cells in vitro. These findings
suggest that SIRT1 inhibition may hold therapeutic
promise, which now needs to be confirmed in an in vivo
tumour model. About 10% of Ewing sarcomas carry p53
mutations that perturb its tumour suppressive function.
This subgroup has a particularly bad prognosis [5].
P53 mutations frequently change the normal structure
of the protein. We have been validating the potentially
therapeutic efficacy of a p53 refolding drug. Preliminary
in vitro experiments suggest that p53 refolding drugs
can activate at least some normal p53 stress response
functions and induce cell death. However, we also
identified a so far unknown high variability in responsiveness to this drug which cannot merely be assigned
to the specific type of p53 mutation but also to other,
still uncharacterised host factors.
Together, our studies identified several novel drug
targets downstream of EWS-FLI1 in Ewing sarcoma.
Future studies will focus on their therapeutic potential
in combination chemotherapy.
In addition, we have identified transcripts that are
uniquely expressed by LCH cells. Among these
transcripts is the NOTCH ligand JAG2, which is highly
expressed in LCH. LCH cells also express NOTCH1
and because LCH cells within the lesions often form
densely packed aggregates, it is conceivable that
efficient stimulation of the NOTCH signaling pathway
occurs in these cells in trans, leading to activation
of NOTCH1 downstream targets. We demonstrated
that JAG2 signaling can induce LCH-like features in
vitro: JAG2 signaling induces the differentiation of
monocytes into cells carrying the LCH hallmark markers
CD1a and CD207 and can induce matrix metallo
proteases which are highly expressed in LCH lesions
and might be responsible for local tissue destruction.
Since NOTCH inhibitors are currently evaluated in
clinical trials and would therefore also be available
for the treatment of LCH, the hypothesis that NOTCH
signaling is a player in LCH pathogenesis is interesting
also from a clinician’s perspective, since it could set
off new approaches to the treatment of this perplexing
disease [5].
Solid Tumours
Specific project
1 ETS, E2F and FOX transcription factors are distinct
families of gene regulatory proteins that bind to DNA at
specific recognition motifs.
2 NOTCH is a transmembrane receptor involved in cellular
communication playing a role during morphogenesis and
differentiation of many tissues.
Sven Bilke and Paul Meltzer, Genetics Branch,
National Cancer Institute, NIH, Bethesda, USA
Reinhard Kofler, Medical University, Innsbruck, Austria
Udo Kontny, University Children’s Hospital Freiburg, Germany
Ernst Kriehuber, Dept. Dermatology, Medical University,
Vienna, Austria
Elizabeth Lawlor, University of Michigan, Ann Arbor, USA
Isidro Machado and Antonio Llombart-Bosch, Dept. Pathology,
University of Valencia, Spain
Katia Scotlandi, Rizzoli Institute, Bologna, Italy
For further reading
[1] Raphaela Schwentner PhD thesis: Mechanisms of target
regulation by the chimeric oncogene EWS-FLI1 in Ewing’s sarcoma
[2] Stephan Niedan PhD thesis: Mechanisms of transcriptional
repression by EWS-FLI1 in Ewing sarcoma
[3] Ban, Bennani-Baiti et al. 2008
Fig. 1 – Immunohistochemical staining of a LCH lesion using an
antibody against full-length NOTCH1. Our data indicate that the
Notch signalling pathway could play an important role in the
pathogenesis of LCH.
46–47
Specific project
Specific project
Immunology
Tumour Immunology
Immunologie
Tumor-Immunologie
Group leader
Thomas Felzmann
[email protected]
Staff scientist
Alexander Dohnal
Postdoct. research fellow
Caterina Vizzardelli1
Diploma/MSc student
Melanie Floderer2
Natalie Knapp3
Klara Soukup4
Technician
Angela Halfmann
1 from Jan. 2011 to Dec. 2012
2 Jul. 2011 to Aug. 2013
3 since Jun. 2011
4 since Jul. 2012
During the last decades, dendritic cells (DC) have been
identified as the most important regulatory elements
in orchestrating immune responses. Studies using
primary mouse DC collected from lymphoid organs, skin
and other tissues, suggest that immunity is directed
by DC subsets, each separately executing a distinct
function. Confirmation of such DC subset-mediated
immune regulation in humans is complicated by the
fact that primary human tissue DC are not directly
available. The majority of information regarding human
DC is derived from DC differentiated in vitro from
monocytes. Such studies have revealed an interesting
phenomenon: rather than a differentiation into DC
subtypes, time-dependent changes of the DC function
were observed. This pattern of DC differentiation might
represent an additional layer of immune regulation.
DC respond to the notion of danger that comes in
different guises to initiate an activation or differentiation
process, conventionally referred to as maturation.
Maturation results from contact with pathogen- or
damage-associated molecular patterns. Binding of
microbial pattern molecules such as lipopolysaccha
rides (LPS) to Toll-like receptors (TLR) on DC signal
danger causing the DC to assume a potent immune
stimulatory phenotype for approximately one day.
LPS exposed DC trigger robust type 1 T-helper (Th1)
cell and cytotoxic T-lymphocyte (CTL) dominated
immune responses characterised by the release of
interleukin (IL) -12 for approximately one day.
The danger model is a helpful framework for understanding immune regulation, especially in the case of
anti-tumour immunity. DC reside in the entire organism,
where they continuously take up, process, and present
material mainly from dead cells to T-lymphocytes. As
long as the DC do not encounter a danger signal, they
present the material they took up in a tolerance maintaining fashion. This holds true for DC situated in the
tumour tissue as well. As opposed to microorganisms,
however, tumour cells – although representing a serious
threat – do not normally deliver danger signals to DC.
Immunregulation durch
die Krebsimmuntherapie
Thus, tumour antigens taken up by DC are presented
to T-cells in a tolerance inducing fashion, providing
a useful loophole for tumour cells to evade immune
surveillance.
Adding a TLR derived danger signal to the equation
has the capacity to tip the balance towards immune
stimulation against tumour antigens. Early DC cancer
vaccine designs advocated maturation of DC with a
cocktail comprised of pro-inflammatory cytokines.
More recent strategies for DC-based cancer immune
therapy utilise TLR agonists in order to trigger IL-12
secretion from DC causing differentiation of Th1 and
CTL priming human DC. In most of the early DC cancer
vaccine studies, DC were inoculated after at least
24 hours of in vitro exposure to a danger molecule such
as LPS. In contrast, in the design of our pre-clinical
model systems and clinical DC cancer immune therapy
trials, we elected to use the DC after only 6 hours of
in vitro exposure to LPS in order to take advantage of the
DC’s immune stimulatory time window characterised
by IL-12 release.
TLR engagement, however, induces not only proinflammatory activity but – with a delay of about one
day – a switch into an anti-inflammatory mode of action.
This switch plays a key role in priming regulatory
T-cell (Treg) mediated anti-inflammatory functions.
This feedback mechanism is a safeguard against
immune responses running out of control and
damaging healthy tissue. In cancer immune
therapy, this feedback signalling limits the power
of a DC-based cancer vaccine. Thus, our objective
in designing a next generation DC cancer immune
therapy is to circumvent DC-mediated negative
feedback regulation in order to enhance the potency
of a DC cancer vaccine.
Ludwig Boltzmann Institute for Cancer Research, Vienna, Austria
Hannover Medical School, Germany
Activartis Biotech GmbH, Vienna, Austria
Während der letzten Jahrzehnte wurden Dentritische
Zellen (DC) als wichtigstes regulatorisches Element in
der Koordinierung von Abwehrreaktionen identifiziert.
Studien in Mausmodellen mit DC aus lymphatischen
Organen, Haut und anderen Geweben weisen darauf
hin, dass spezielle Funktionen des Immunsystems
von unterschiedlichen DC-Untergruppen gesteuert
werden. Nachweise, dass auch im Menschen DC die
Immunregulation leiten, sind komplizierter zu erbringen,
da menschliches Gewebe für Experimente nur sehr
eingeschränkt zu Verfügung steht. Für Studien beim
Menschen müssen die DC in vitro aus Monozyten
differenziert werden. Derartige Untersuchungen
weisen auf ein interessantes Phänomen hin: Anstatt
einer Differenzierung in DC-Untergruppen kommt es
zu zeitabhängigen Funktionsveränderungen der DC.
Dieses Muster der Differenzierung von DC könnte einen
zusätzlichen Bereich der Immunregulation begründen.
DC reagieren auf Gefahrensignale unterschiedlicher
Erscheinung entweder mit Aktivierung oder Differen
zierung, was üblicherweise als Reifungsprozess
bezeichnet wird. Reifung erfolgt nach Kontakt mit
Pathogenen oder molekularen Mustern, die mit
Zellschädigung assoziiert sind. Die Bindung von mikro
biellen Molekülen wie z. B. den Lipopolysacchariden
(LPS) an Toll-like Rezeptoren (TLR) wird von den DC
als Gefahrensignal interpretiert. Die so aktivierten DC
lösen potente Immunreaktionen aus, die von Typ-1
T-Helfer Zellen (Th1) und von zytotoxischen T-Lymphozyten (CTL) ausgeführt werden. Diese DC sind durch
die Freisetzung von Interleukin (IL)-12 für etwa einen
Tag charakterisiert.
Das Gefahrenmodell ist ein nützliches Bezugs
system, um die Regulation der Antitumorimmunität zu
verstehen. DC besiedeln den gesamten Organismus
und präsentieren, solange sie kein Gefahrensignal
empfangen, aufgenommenes Material auf eine Weise,
dass die immunologische Toleranz aufrechterhalten
wird. Ähnliches gilt für DC im Tumorgewebe. Im
Gegensatz zu Mikroorganismen senden Tumorzellen,
obwohl sie natürlich eine große Gefahr darstellen,
keine für DC verwertbaren Gefahrensignale aus. Daher
schaffen Tumorantigene, die von DC aufgenommen
werden, für Tumorzellen ein Schlupfloch, durch das sie
der Immunüberwachung entkommen können. Werden
mikrobielle Gefahrenmoleküle zugeführt, kommt es zu
einer Aktivierung der DC und zur Präsentation von im
Tumorgewebe aufgenommenen Antigenen an T-Zellen.
In frühen Konzepten von Krebsimpfungen auf Basis
von DC wurden diese entweder gar nicht oder nur
eingeschränkt mit pro-inflammatorischen Zytokinen
aktiviert. Aktuelle Strategien verwenden TLR Agonisten
zur Aktivierung von menschlichen DC, welche über IL-12
Sekretion Th1 Zellen und CTL stimulieren. In den meisten
Studien mit DC-Tumorimpfstoffen werden die DC in
vitro für mindestens 24 Stunden Gefahr signalisierenden
Molekülen wie z. B. LPS ausgesetzt. Im Gegensatz
dazu werden in unseren präklinischen Modellen und
klinischen Studien die DC schon nach 6 stündiger
Aktivierung mit LPS eingesetzt, um das Zeitfenster
der IL-12 Sekretion optimal zu nutzen.
TLR Agonisten lösen in DC nicht nur pro-inflammatorische Prozesse aus. Mit etwa eintägiger Verzögerung
schalten die DC in einen anti-inflammatorischen
Modus um. Dieser Wechsel spielt eine Schlüsselrolle
in der Aktivierung von regulatorischen T-Zellen (Treg),
welche anti-inflammatorische Funktionen ausüben
und dient als Schutz vor Immunantworten, die sich
unkontrolliert gegen gesundes Gewebe richten. In der
Krebsimmuntherapie schwächt dieses Feedbacksignal
aber die Wirksamkeit einer Krebsimpfung. Unser Ziel
beim Design einer „Next Generation“ DC-Krebs
immuntherapie ist es, eine DC-vermittelte negative
Rückkopplung zu umgehen und somit die Wirksamkeit
der DC-Krebsimpfung zu verbessern.
In Kooperation mit
Ludwig Boltzmann Institut für Krebsforschung, Wien, Österreich
Medizinische Hochschule, Hannover, Deutschland
Activartis Biotech GmbH, Wien, Österreich
50–51
Immune regulation in
cancer immune therapy
Immunology
Research focus
An anti-inflammatory
DC master switch
DC coordinate innate and adaptive immunity to fight
infections and cancer (Fig. 1). Our observations reveal
that DC exposed to LPS in the presence of interferon-γ
(IFN-γ) acquire a continuously changing activation/
maturation phenotype [1]. The DC’s initial mode of
action is pro-inflammatory via up-regulation among
others the signalling molecule IL-12, which polarises
IFN-γ secreting Th1 cells. Within 24 hours, the same
DC switches from the pro- into an anti-inflammatory
phenotype. This is mediated by autocrine IL-10 release
and secretion of soluble IL-2 receptor alpha (sIL-2RA)
molecules. T-cells, when contacted with DC during
their anti-inflammatory phase lose their proliferative
capacity and develop Treg-like anti-inflammatory functions indicated by IL-10 secretion and elevated FoxP3
levels. Studying the kinetics of IL-12 and IL-10 expression
from LPS/IFN-γ activated myeloid DC on a single cell
level confirmed these observations. When T-cells are
separated from DC within 24 hours, they are spared
from the anti-inflammatory DC activity. We conclude
that, in addition to differentiation of DC into distinct
subsets, the observed sequential functional phases of
DC differentiation permit the fine-tuning of an immune
response. A better understanding of time-kinetic
DC features is required for optimally exploiting the
therapeutic capacity of DC in cancer immune therapy.
Having established that DC switch from a pro- into an
anti-inflammatory mode of action, we were interested
in the molecular mechanisms guiding that switch. DCmediated inflammation induced via LPS is promoted
by mitogen-activated protein kinase (MAPK)-activated
kinase (MK)-2, a substrate of p38 MAPK. We showed
that MK2 consolidates LPS-driven anti-inflammatory
DC functions by modulating p38- and Erk1/2-MAPK
and STAT3 signalling. In the primary LPS/p38/MK2 axis,
MK2-mediated feedback inhibits p38 and positively
cross-regulates Erk1/2 activity. Impairment of secondary autocrine IL-1α signalling by MK2 further decreases
the IL-1α/p38 but increases the anti-inflammatory
IL-10/STAT3 signalling route. Thus, blocking of MK2
activity enables DC to strengthen pro-inflammatory
effector mechanisms by promoting IL-1α-mediated
Th1 and Th17 but blocking Treg responses. Further,
MK2 deficient DC trigger enhanced cytotoxic activity.
These data suggest that MK2 exerts a profound antiinflammatory effect that prevents DC from prolonging
excessive effector T-cell functions.
For further reading
[1] Luger, Valookaran et al. 2012
Our observations will need to be taken into consideration
in designing future generations of DC-based cancer
vaccines.
Ludwig Boltzmann Institute for Cancer Research, Vienna, Austria
Hannover Medical School, Germany
Lymph node
52–53
Dodging anti-inflammatory
DC activity
Immunology
Specific project
adoptive
Cell Therapy
DC
priming
DC
cytotoxicity
DC
Tumor
Fig. 1 – Tumour-immune
system interactions. Antiinflammatory factors released
by tumour cells avoid priming
and activation of tumourspecific effector T-cells.
Adoptive transfer of ex vivo
manipulated DC break the
immune suppressive barrier
in tumours.
Specific project
Immunology
Clinical Cell Biology
& FACS Core Unit
Immunologie
Klinische Zellbiologie
& FACS Core Unit
Group leader
Gerhard Fritsch
[email protected]
Staff scientist
René Geyeregger
Technicians
Nelli Frank1
Christine Freimüller
Dieter Printz
Daniela Scharner
Julia Stemberger
Dijana Trbojevic
Elke Zipperer
Clinicians,
Heidrun Boztug
Susanne Matthes-Mann
Herbert Pichler
Volker Witt
1 since May 2012
Gruppe für „Klinische
Zellbiologie & FACS
Core Unit“
Forschung und FACS Core Unit
The main task of our diagnostics department is to provide cytometric cell analyses and cell sorting services,
primarily to the St. Anna Children’s Hospital, but also to
the CCRI and external research institutes. The number
of applications has increased and now also includes
analyses that address spherocytosis and evaluate
human sperm fertility. Every year, over 60,000 cytometric cell analyses and more than 2,000 cell sorts are
carried out. The main purpose of the latter is to control
the donor/recipient type of the different blood cells
using FISH or PCR methods in post allo-transplantation
patients. Internal quality control mechanisms are in
place, which conform to internal standard operating
procedures (SOPs) and to JACIE (Joint Accreditation
Committee-ISCT & EBMT) guidelines. In addition, we
participate in the regular Austrian and German quality
control trials, organised by ÖQUASTA (Austrian Society
for Quality Assurance and Standardisation of MedicalDiagnostics) and INSTAND (Society for Promotion of
Quality Assurance in Medical Laboratories). Our task
here is to measure subtypes of blood leukocytes,
stem cells, and residual leukocytes in blood products.
As a FACS (Fluorescence-activated cell sorting = flow
cytometry) core unit, our laboratory presently has at
its disposal 4 own flow cytometers for 6 to 16 parameter
cell measurements, one of which is a multi-colour
device (FACSAria) for sorting cells, the others being
a 4 and 13 colour device (FACSCalibur and LSRII) for
cell analyses. A fourth device (17-colour Fortessa) was
acquired in 2011 to replace the 16-year old FACSCalibur.
In close cooperation with clinicians, researchers of
the CCRI and other scientists, our laboratory offers
practical and theoretical support for all questions
regarding flow cytometry. Our research is focused
on the detection, quantification, enrichment and
selection of donor virus-specific T-cells that can
fight life-threatening complications after stem cell
transplantation. In this field, progress was achieved
by combining our T-cell expansion protocol with a
magnetic selection step to highly purify adenovirusspecific T-cells (Fig. 1: A–C). This will further reduce
patients’ risk for severe side effects. For those patients
for whom a matched donor with Adenovirus-specific
T cells is not available, we developed a method that
converts non-specific donor lymphocytes into
virus-specific T cells that can fight viral infections.
These two strategies could have a major impact on
future clinical treatment.
Der größte Bereich unserer Abteilung stellt im Rahmen
von Diagnostikaktivitäten durchflusszytometrische
Analysen sowie ein Zell-Sort Service zur Verfügung,
primär für das St. Anna Kinderspital, aber auch
für die St. Anna Kinderkrebsforschung und externe
Forschungsinstitute. Die Zahl der Anwendungen
hat sich ausgeweitet und umfasst neuerdings auch
Analysen zur Diagnose der Kugelzellanämie oder zur
Fruchtbarkeit humaner Spermien. Jährlich werden über
60.000 durchflusszytometrische Analysen und mehr
als 2.000 Zell-Sortierungen durchgeführt. Letztere
dienen dazu, bei fremd-transplantierten Patienten den
Spender/Empfängertyp der verschiedenen Blutzellen
mittels FISH- oder PCR-Methode zu überprüfen.
Unsere Arbeit unterliegt internen Qualitätskontrollen
sowie jenen von JACIE1. Wir nehmen regelmäßig an
Rundversuchen teil, die von ÖQUASTA2 und INSTAND3
organisiert werden. Dabei ist es unsere Aufgabe,
Leukozytenuntergruppen, Stammzellen und Rest
leukozyten in Blutprodukten zu messen.
Als FACS (Fluorescence-activated cell sorting =
Durchflusszytometrie)6 Core Unit verfügt unser
Labor derzeit über vier Durchflusszytometriegeräte
für 6 bis 16 Parameter Zellmessungen, von denen ein
Vielfarbengerät (FACSAria) dem Sortieren von Zellen
dient und ein 4- bzw. 13-Farbengerät (FACSCalibur
und LSRII) der Zellanalyse. Ein neues 17-Farbengerät
(Fortessa) wurde 2011 angeschafft, um den 16 Jahre
alten FACSCalibur zu ersetzen. In enger Zusammenarbeit mit Klinik und Forschung bietet unser Labor
Unterstützung für sämtliche Fragen der Durchfluss
zytometrie. Unsere Forschungsaktivitäten betreffen die
Messung, Quantifizierung, Anreicherung und Selektion
humaner Abwehrzellen gegen klinisch relevante Viren,
die im Rahmen der Stammzelltransplantation zu
lebensbedrohlichen Komplikationen führen können.
Hier wurden Erfolge erzielt, indem wir unsere Expansionsmethode mit einer zusätzlichen magnetischen
Aufreinigung kombinierten. Dadurch gelang es uns,
hochreine Adenovirus-spezifische Spenderzellen zu
isolieren, bei deren Infusion in den Patienten potentielle
Nebenwirkungen praktisch auszuschließen sind. Weil
es für einige Patienten keinen passenden Spender gibt,
der diese wichtigen Abwehrzellen im Blut hat, ent
wickelten wir eine Methode, um unspezifische Abwehrzellen so umzuprogrammieren, dass sie in der Lage sind,
Virusinfektionen zu erkennen und zu bekämpfen.
Beide Strategien könnten einen bedeutenden Einfluss
auf zukünftige klinische Fragestellungen haben.
103
104
Tetramer DSRed-A
105
100
AdV A01-CD3
50
100
AdV A01-CD3
50
102
Multimer
88C-A
150
Multimer
88C-A
150
88C-A
150
Streptamer
100
The second largest area encompasses clinical routine
and concerns the manipulation of all cells or blood
products that are administered to patients as part
of treatment. This takes place under sterile conditions
in our good manufacturing practice (GMP) facility.
After successful validation, we introduced a new
freezing medium for the storage of patient cells. The
preparation of mononuclear cells for clinical use in
extracorporeal photopheresis (“mini ECP”) proved
successful for treatment of Graft-versus-Host Disease
(GVHD) in very young patients. All these activities
require a valid accreditation under JACIE and certifi
cation of the laboratory by the AGES (Österreichische
Agentur für Gesundheit und Ernährungssicherheit)
PharmMed, which we were able to renew in June 2012.
50
Clinical routine
(x 1.000)
200
250
Diagnostik
(x 1.000)
200
250
Research, including FACS Core Unit
(x 1.000)
200
250
Diagnostics
102
103
104
105
Tetramer DSRed-A
Fig. 1: A–C – Magnetic selection to highly purify
adenovirus-specific T-cells.
102
103
104
Tetramer DSRed-A
105
Klinische Routine
Der zweitgrößte Bereich umfasst die klinische Routine.
Manipulation von Zellen oder Blutprodukten, die
Patienten zur Behandlung zugeführt werden, geschieht
unter sterilen Bedingungen in unserem dafür speziell
zertifizierten GMP 4-Labor. Zur Lagerung von Patienten
zellen verwenden wir ein neu validiertes Einfrier
medium. Die Reinigung und Verwendung von weißen
Blutzellen in der Extracorporalen Photopherese
(„Mini ECP“) erwies sich als erfolgreich zur Bekämpfung
der Graft-versus-Host-Erkrankung bei sehr jungen
Patienten. Diese Tätigkeiten erfordern eine gültige
Akkreditierung nach JACIE, die 2013 erneuert werden
muss, sowie die Zertifizierung des Labors durch
die Österreichische Agentur für Gesundheit und
Ernährungssicherheit (AGES) Pharmed5, welche wir
im Juni 2012 erneuern konnten.
1 JACIE: Joint Accreditation Committee-ISCT & EBMT.
Akkreditierungsprogramm
2 ÖQUASTA: Österreichische Gesellschaft für Qualitätssicherung
und Standardisierung medizinisch-diagnostischer Untersuchungen
3 INSTAND: Gesellschaft zur Förderung der Qualitätssicherung
in medizinischen Laboratorien
4 GMP: Good Manufacturing Practice
5 AGES Pharmed: Nationale Zulassungsstelle für Arzneimittel
6 FACS, FACS Core Unit: Zentrum für Durchflusszytometrie
56–57
Group of “Clinical
cell biology & FACS
Core Unit”
Immunology
Research focus
Improved magnetic selection
of Adenovirus-specific T cells
Based on our previously developed method to
increase purity and number of virus-specific T cells,
we further improved the purity of ADV-specific T cells
using magnetic cell selection. As shown in Fig. 1, the
purity significantly increased to almost 100%. The
combination of both methods could therefore further
reduce or even avoid potential side effects induced
by residual non ADV-specific T cells. This is a further
step towards safer T cell therapies for patients after
stem cell transplantation.
Impact of pre-transplant ATG
on immune reconstitution
Anti-thymocyte globulin (ATG) is widely used in the
conditioning regimen of children undergoing
haematopoietic stem cell transplantation (HSCT)
to prevent graft-versus-host-disease and rejection
of the graft. However, data on immune reconstitution
following ATG are scarce. We retrospectively analysed
the engraftment kinetics and clinical parameters
of 113 paediatric/adolescent patients (median age
11.9 years) undergoing HSCT following myeloablative
conditioning with or without in vivo T-cell depletion.
Patients with matched unrelated donor received either
ATG-Fresenius (n=45, group 1) or Thymoglobuline
Genzyme (n=30, group 2), while patients with matched
sibling donor (n=38, group 3) underwent SCT without
in vivo T-cell depletion. Immune cell subsets
(CD4, CD8, NK, CD19, naïve CD4) were determined
on day +50, +100, +180 and +360 post HSCT. Immune
reconstitution in the three groups was comparable in
most cell subsets (Table 1). Important differences
were lower average numbers of CD4+ cells following
ATG-Fresenius compared to Thymoglobuline
(p=0.049) and those not receiving ATG (p=0.002).
By day +180, median CD4 cells were similar in all
groups. Moreover, average naïve CD4+ cells were
lower in groups 1 and 2 compared to group 3 (p<0.001).
Concerning infectious complications, percentages of
CMV or Adenovirus reactivation in the peripheral blood
tended to be higher following ATG (group 1 and 2: 23/75,
31% vs. group 3: 3/38, 8%, p=0.29), while occurrence
of graft versus host disease grade 3 and 4 was similar
in the three groups (group 1 and 2: 10/75, 13% vs. group
3: 3/38, 8%, p=0.47).EBV lymphoproliferative disease
occurred only in patients following in vivo T-cell
depletion (group 1: 3/45, 7%, group 2: 3/30, 7%). Following myeloablative conditioning, immune reconstitution
of most cell subsets after day +50 is similar with or
without in vivo T-cell depletion. Occurrence of naïve
CD4 cells as a sign for thymic recovery is faster without
ATG. Apart from transiently lower CD4 cells following
ATG-Fresenius, immune reconstitution is not majorly
influenced by the ATG preparation used.
Neutrophil engraftment after
allogoeneic CD34 infusion
Neutrophil engraftment (NE) following myeloablative
conditioning was retrospectively analysed in 112 children
transplanted 2000–2012 in a single centre. Graft
source was bone marrow in 94 and peripheral blood
in 18 patients. 38 patients received grafts from
matched sibling donors (MSD), whereas 74 were
transplanted from matched unrelated donors (MUD).
The median donor age was 27.4 years and in 47 patients
the donor was <25 years. A total body irradiation
(TBI)-based conditioning was used in 72 patients,
whereas 39 received chemotherapy. Virus prophylaxis
consisted of acyclovir in 63 and gancyclovir in
32 patients. Engraftment was defined as the first of
three consecutive days with neutrophil counts
> 0.5 x 10^9/L peripheral blood. Median time to NE for
all 112 patients was 20 days (range: 10 – 40) after
receiving a median of 4.1 x 10^6/kg total CD34+ cells and
2.4 x 10^6/kg myeloid CD34+ cells respectively. Patients
given a low dose of total CD34+ cells (< 2 x 10^6/kg)
engrafted equally to those given 3 – 6 x 10^6/kg
CD34+ cells or more. Likewise, the number of infused
myeloid CD34+ cells did not correlate with NE (Fig. 2).
Neutrophil Engraftment in 112 Patients
40
35
30
25
20
15
10
5
0
0
5
10
15
all CD34+ cells [n x 10ˆ6/kg]
20
Stem cell source, donor age, conditioning, and antiviral
agents did not seem to have an impact on NE. Interestingly, patients transplanted from MSD engrafted earlier
than those from MUD (median 17.5 days vs. 21 days;
p=0.034). A subgroup analysis of MUD recipients moreover revealed a positive correlation between NE and
total CD34+ cell numbers but not for myeloid CD34+
subsets. The median NE in our cohort was significantly
faster for MSD recipients, whereas neither the graft
content of total CD34+ nor of the myeloid CD34+ cells
had an impact on engraftment kinetics. The evaluation
of donor age, stem cell source, conditioning or antiviral
agents revealed no significant influence on NE.
The RNA-Group of Niels Schaft at the Universitätsklinikum Erlangen,
Germany
Fig. 2 – In contrast to the widely accepted opinion that 2 – 10 x 10^6
CD34+ cells per kg patient are necessary to guarantee timely
engraftment, our data show no correlation between the number of
CD34+ cells infused and the time span to neutrophil engraftment
following allogeneic stem cell transplantation.
45
day ANC > 0.5 x 10ˆ9/L
Several weeks or months after haematopoietic stem
cell transplantation (HSCT) of children, an immune
system (including T cells that protect against viral
infections) does hardly exist. One option is to infuse
specific T cells into patients to overcome viral
infections. Unfortunately, in about 10% of all cases,
Adenovirus-specific T cells are not present in the
donor. Therefore, we developed a protocol to convert
normal non-specific T cells into T cells that can fight
against viral infections. To do so, we identified the T cell
receptor expressed on the surface of ADV-specific
T cells which they use to recognise and kill virusinfected cells. We transferred the genetic information
of this receptor into non-specific T cells, making these
T cells able to recognise and kill virus-infected cells.
25
Conversion of non-specific
T-cells into T-cells with
anti ADV specificity
58–59
Immunology
Specific project
Immunology
TransplantationImmunology
Immunologie
TransplantationsImmunologie
Group leader
Andreas Heitger
[email protected]
Ursula Hainz1
Markus Hölzl2
Birgit Jürgens
Postdoct. medical fellow
Sabine Heitzeneder3
PhD students
Sarah Ahmadi4
Barbara Dillinger5
Edda Veith6
Diploma/MSc students
Barbara Dillinger7
Mathias Hochgerner8
Margit Lanzinger9
Angelika Plach10
Stefanie Reinprecht11
Stefan Weinberger12
1
2
3
4
5
6
7
8
9
10
11
12
July 2010 – Dec. 2011
Oct. 2010 – Apr. 2012
Sept. 2008 – June 2012
since Sept. 2011
since Oct. 2012
Aug. 2007 – Apr. 2011
Jan. 2011 – Sept. 2012
since Jan. 2012
Oct. 2009 – Apr. 2011
Jan. 2012 – June 2012
July 2011 – Feb. 2012
since Oct. 2012
After hematopoietic stem cell transplantation (HSCT),
the emergence of immunological tolerance is
mandatory to ascertain the ultimate success of this
therapeutic procedure. The particular challenge
in HSCT is that donor-derived immune cells which are
part of the hematopoietic system accommodate in
the host and reconstitute immunocompetence against
infectious pathogens, a state termed allo-specific
tolerance. Human immune systems have evolved to
strictly discriminate “self” from “non-self” and
recognise microbiological invaders as “non-self”.
After HSCT, the donor derived immune cells have
to accept the host as a new self.
Despite careful selection of HSCT donors and
recipients for matching cellular components that are
believed to define “self”, i.e. major histocompatibility
(MHC) molecules, the transplanted immune cells often
recognise the host organism as “non-self” and begin
to mount immune reactions against HSCT recipients
which can manifest as graft-versus-host disease
(GvHD). GvHD occurs with an unpredictable grade
of severity and an unpredictable outcome. Still, GvHD
is the leading cause for transplant-related morbidity
and mortality resulting in a 30 – 50% death rate and
a debilitating course [1]. Additionally, the compromised
immune system leaves recipients highly susceptible
to otherwise harmless infectious pathogens and to
a complicated course of infections. Thus, an improved
understanding of the complex pathophysiology of
post-transplant immunity and tolerance is of high
demand to improve outcome and safety in HSCT.
Regarding our focus on tolerogenesis by augmenting
tryptophan metabolism, we recently showed that
this capacity is limited [2]. Therefore, we expandend
our efforts for a better understanding of allo-specific
tolerance by initiation of new collaborations and
projects with the aim to open new endeavours of
targeting key processes in allo-specific tolerance.
We continue to explore strategies of generating
allo-specific tolerized T cells ex-vivo, i.e. to educate
Umfassendes Verständnis
der Stammzelltransplantation
donor T cells ex-vivo to tolerate the recipient and thus
make them suitable to be adoptively transferred to
recipients in the post-transplant period to overcome
the susceptibility to infections but not induce GvHD. In
collaboration with the SME Effimune (Nantes, France)
and the Havard University (Boston, USA), we have
begun to investigate a unique combination of novel
compounds modulating co-signals which are essential
for T cell tolerance1. We also initiated an innovative
project studying the autoantibody repertoire after
HSCT. The auto-antibody repertoire (AAb repertoire)
is an essential compartment defining the “immune self”
[3]. Comparing the AAb-repertoire of HSCT donors and
recipients will provide insight, whether the ability
and the dynamics of restoring an appropriate AAb
repertoire contribute to overall immune reconstitution
and can serve as a biomarker predicting immune
failure or GvHD, especially chronic GvHD. Finally, we
concluded the study of the impact of mannan-binding
lectin in HSCT [4] and provide solid evidence that low
serum levels of MBL or MBL genotypes do not have any
impact on the outcome of HSCT, thus precluding that
patients with low MBL levels may profit from prophylactic MBL transfusion (manuscript in preparation). In
addition, on the basic research level, we have started
exploring the role of oxygen tension in governing
immune reactions. Accumulating evidence suggests
that low oxygen tension, as is found in most organ systems of the body, significantly impacts on the outcome
of immune reactions. As a disease model, we include
patients suffering from chronic granulomatous disease.
1 EU/FP-7 IAPP program “MODICELL”
Effimune, Nantes, France
Havard University, Boston, USA
The Weizmann’ Institute, Rehovot, Israel
Univ. Children’s Hospital Zürich, Switzerland
For further reading
[1] Blazar, Murphy et al. 2012
[2] Lanzinger, Jürgens et al. 2012
[3] Madi, Kenett et al. 2011
[4] Heitzeneder, Seidel et al. 2012
Die Ausbildung immunologischer Toleranz nach
hämatopoietischer Stammzelltransplantation (HSZT)
ist eine unabdingbare Voraussetzung für den Erfolg
dieser Therapiemaßnahme. Das humane Immun
system unterscheidet strikt zwischen „selbst“ und
„fremd“. Nach HSZT müssen spenderabgeleitete
Immunzellen lernen, den Empfänger-Organismus als
„selbst“ zu akzeptieren. Trotz einer genauen Spender
auswahl, bei der die molekularen Merkmale, die
hauptsächlich das immunologische „Selbst“ definieren
(major histocompatibility antigens, MHC Moleküle) mit
dem Empfänger übereinstimmen, kann es zu Unverträglichkeitsreaktionen kommen. Diese manifestieren
sich als Spender-gegen-Empfänger Krankheit (GvHD) in
unterschiedlichen Schweregraden und mit unsicherem
Ausgang. Die GvHD stellt immer noch die schwerste
Komplikation nach HSZT dar und führt bei 30 – 50%
der betroffenen Patienten zum Tod oder zu schweren
Beeinträchtigungen [1]. Zudem leiden Patienten bis
zur Wiederherstellung ihres Immunsystems an schwerer
Immundefizienz, die zu einer erhöhten Inzidenz
und Schwere von infektiösen Komplikationen nach
HSZT führt. Ein verbessertes Verständnis der postHSZT Immunität ist notwendig, um die HSZT zu einem
sicheren Therapieverfahren weiter zu entwickeln.
Bezüglich unseres Schwerpunktes der Toleranzin
duktion über den Tryptophanmetabolismus haben wir
kürzlich gezeigt, dass dessen Wirkung limitiert ist [2].
Deshalb haben wir neue Forschungsprojekte initiiert. In
Fortsetzung früherer Arbeiten forschen wir an Strategien
zur Generierung allo-spezifisch toleranter T Zellen.
T-Zellen sollen ex-vivo so ausgebildet werden, dass sie
nach adoptivem Transfer HSZT Empfänger mit effizienter Immunität gegen Infektionserreger versorgen,
ohne eine GvHD zu verursachen. In Zusammenarbeit
mit Effimmune (Nantes, Frankreich) und der Harvard
Universität (USA) testen wir eine innovative Kombination von Substanzen, die speziell in die Modulation
von Ko-Signalen eingreifen. Ko-Signale sind für die
endgültige Ausformung einer Immunantwort essentiell.
Die getesteten Substanzen blocken Ko-stimulation
über das T-Zell assoziierte CD28 Molekül einerseits und
stimulieren die Ko-inhibition über den PD-1/PD-L1 Weg
andererseits1. Des Weiteren erforschen wir die Rolle
des Autoantikörper (AAk)-Repertoires nach HSZT. Das
individuell gestaltete AAk-Repertoire stellt eine wesentliche Komponente der Definition des immunologischen
„Selbst“ dar [3]. Der Vergleich des AAk-Repertoires
von Spendern und Empfängern und der Verlauf
seiner Rekonstitution nach HSZT kann Aufschluss
darüber geben, ob und wie eine Veränderung des
AAk-Repertoires mit der allgemeinen Immunrekon
stitution assoziiert ist, und ob es bei der Entwicklung
einer GvHD eine Rolle spielt. Im vergangenen Jahr
haben wir die Studie über den Einfluss von Mannanbindenden Lektins (MBL) als Risikofaktor bei HSZT [4]
abgeschlossen. Die Ergebnisse zeigen deutlich dass
weder MBL Serum Spiegel noch MBL Genotypen den
Transplantationsverlauf negativ beeinflussen und somit
eine prophylaktische MBL Infusion keinen therapeutischen Wert bei pädiatrischer HSZT hat (Manuskript
in Vorbereitung). In einem ergänzenden Projekt
erforschen wir den Einfluss von Sauerstoffspannung
auf Immunreaktionen. Jüngste Forschungsergebnisse
zeigen, dass ein erniedrigter Sauerstoffgehalt, z. B. in
entzündetem Gewebe, Immunreaktionen wesentlich
beeinflussen kann. Als Krankheitsmodell inkludieren
wir hier Patienten mit chronischer Granulomatose.
1 EU/FP7 Programm, „MODICELL“
In Kooperation mit
Effimune, Nantes, Frankreich
Havard University, Boston, USA
Weizmann’ Institute, Rehovot, Israel
Universitätskinderklinik Zürich, Schweiz
Literaturangaben
[1] Blazar, Murphy et al. 2012
[2] Lanzinger, Jürgens et al. 2012
[3] Madi, Kenett et al. 2011
[4] Heitzeneder, Seidel et al. 2012
62–63
Understanding transplantation
immune tolerance across
multiple levels
Immunology
Research focus
The ongoing vision of the group “Transplantation
Immunology” is the improvement of the feasibility and
safety of paediatric hematopoietic stem cell transplantation (HSCT). We continue in our attempt to generate
allo-specific tolerant T cells ex-vivo which can be safely
transferred to HSCT recipients as a means to overcome
post-HSCT immune insufficiency without the risk of
inducing GvHD.
In our in-vivo studies of co-stimulation blockade
for inducing tolerance via amplified tryptophan
metabolism, we discovered that a blockade of the
costimulatory CD80/86 pathway by CTLA-4 fusion
proteins (CTLA4-Ig) does not induce amplified
tryptophan catabolism. Instead, CTLA4-Ig exerted a
powerful tolerogenic effect by selectively decreasing
allo-specific immune effector T cells while preserving
T regulatory cells (manuscript in preparation). This
prompted us to continue studying the tolerogenic
potential of co-signal modulation also in human cells.
The approach is based on the understanding that T cells
recognising antigens receive two signals by antigenpresenting cells (APC). The first signal, generated by
the stimulating antigen, activates T cells. The second
signal, the co-signal, shapes the type of reaction. An
ever-growing list of T cell surface molecules interacting with their ligands on APC have now been identified
to serve as co-signals with either co-stimulatory or
co-inhibitory function. The best studied co-stimulatory
pathway is via the interaction of CD80/86 on APC and
with CD 28 on T cells. CTLA4-Igs blocking this interaction have been pharmacologically designed to clinical
grade and are already being used as immunosupressants in solid organ transplantation. A human CTLA4-Ig,
belatacept [1], is currently probed in HSCT recipients
in a phase 1 study in the USA. However, a drawback of
CTLA4-Ig based treatments is a non-specific immunesuppressive effect, causing a generally immunosuppressed state in treated patients. In addition, CTLA4-Ig
potentially also blocks the immune regulatory CTLA-4
pathways.
In our laboratory, we found that belatacept is effective to induce a durable non-responsiveness in T cells
to allogeneic stimulation. The ex-vivo tolerization
approach chosen by us entails to exploit the effectiveness of belatacept but to spare patients from
the general immune compromise, as belatacept will
be removed from the cell product prior to adoptive
transfer. However, our results so far also revealed some
disadvantages of the CTLA4-Ig usage: In addition to
blocking the immune regulatory pathway through
CTLA-4 [2], we observed that CTLA4-Ig mainly affects
the CD4+ T helper cell population.
To overcome these disadvantages, we now focus on
two new components. First, we currently included
testing a newly developed specific CD28 antibody [2],
which directly targets the CD28 molecule and leaves
the counter-regulatory CTLA-4 pathway intact. Our
preliminary findings show the efficacy of specific CD28
blockade across multiple donor-responder pairs in
vitro. Like belatacept, however, direct CD28 blockade
mainly targets CD4+ cells. Thus, to also target cytotoxic
CD8+ cells, in future approaches we will include
stimulation of co-inhibitory PD-1 agonist antibody in our
ex-vivo cultures. This antibody is designed to stimulate
the co-inhibitory PD-1/PD-L1 pathway [3], which is particularly effective in CD8 cells and promotes deletional
tolerance of activated cytotoxic CD8 T cells [4]. Hence,
within the next years, we will intensively study whether
this innovative approach has the potential to stably
and robustly generate T cell tolerance for the benefit
of paediatric HSCT recipients.
Effimmune, Nantes, France
Harvard Medical University, Boston, USA.
For further reading
[1] Davies, Barbon et al. 2012
[2] Poirier, Mary et al. 2012
[3] Fife and Pauken 2011
[4] Keir, Butte et al. 2008
Chronic Granulomatous
Disease1
Hypoxia Impacts Adaptive
Immunity towards Fungal
Pathogens1
Immunology
Immune tolerance by
co-signal modulation
Specific project
Chronic granulomatous disease (CGD) is an inherited
immune disorder characterised by a disability to
produce reactive oxygen intermediates (ROI). It is
caused by a defect NADPH oxidase rendering patients
highly susceptible to uncontrolled fungal or mycobacterial infections emerging on the background of
hyperinflammation. Genetic defects may affect any
subunit (e.g. gp91phox or p47phox) of the NADPH
oxidase complex. In a murine p47phox-deficiency
model, lethal pulmonary aspergillosis was reported to
rely on a superoxide-dependent step in tryptophan
metabolism, leading to dominant production of IL-17,
defective regulatory T-cell activity and acute inflammatory lung injury [1]. In sharp contrast to these data, we
showed that tryptophan metabolizing activity was fully
preserved in human CGD [2]. We thus started examining whether an exaggerated Th17 response would
underlie the hyperinflammatory phenotype in CGD
patients. We confirmed published data showing that
the induction of first-line pro-inflammatory cytokines
IL-1 β, IL-6 and TNF- α was preserved in CGD patients.
In a next step, we induced a Th17 response by using
Th17-polarizing cytokine cocktails. Peripheral blood
mononuclear cells (PBMC) from CGD patients showed
decreased release of IL-17A and IFN-γ under polyclonal
Th17-polarizing conditions. Furthermore, CGD patients
showed genotype-dependent skewing of T helper cell
response towards Th1 (gp91phox deficiency) or Th17
(p47phox deficiency) under polyclonal Th17-polarizing
conditions. We are currently investigating the impact of
CGD genotype on adaptive immune responses towards
fungal pathogens. First evidence suggests that the
genotype indeed influences the balance of T helper cell
subsets (manuscript in preparation).
Recent studies have shown that oxygen tensions in different tissues are often low (0.5 – 3% oxygen) and could
be considered hypoxic. Furthermore, recent findings
suggest that hypoxic conditions significantly affect
immune responses [1]. Of note, most of our knowledge
comes from experiments performed under normoxic
conditions (21% oxygen). We designed a study to
examine the impact of low oxygen tension on adaptive
immune responses with emphasis on fungal infections.
Our data show that low oxygen tension delays but does
not inhibit immune response of human PBMC upon
polyclonal stimulation. In contrast, antigen-specific
stimulation is severely affected by hypoxia. Low oxygen
culture conditions inhibit activation and proliferation
of T cells upon challenge with C.albicans. Interestingly,
hypoxia does not influence uptake and processing of
C.albicans nor subsequent activation of APC but exerts
its inhibitory potential only during following co-culture
of such APC and T cells (Diploma thesis Mathias Hochgerner; manuscript in preparation). Moreover, hypoxia
noticeably decreases the amount of CD4+IL-17A+ and
CD4+IFN-γ+ T cells along with decreased release of
IL-17A and IFN-γ upon stimulation with fungal pathogens
in contrast to culturing under normoxic conditions.
Current experiments focus on the direct impact of
hypoxia-inducible factor 1 α on both, activation and
cytokine release upon fungal stimulation. In a second
approach, we investigate the influence of low oxygen
tension on the balance of regulatory T cells and effector
T-cell subsets (Diploma thesis Stefan Weinberger).
64–65
Specific project
Janine Reichenbach, University Children’s Hospital Zürich,
Switzerland
Ronnie Chee, Royal Free Hospital & University College London, UK
Gregor Dückers, HELIOS Klinikum Krefeld, Germany
For further reading
[1] Romani, Fallarino et al. 2008
[2] Jürgens, Fuchs et al. 2010
1 Coordinated by Birgit Jürgens
For further reading
[1] Nizet and Johnson, 2009
Specific project
Immunology
Development of
Cellular Therapeutics
Immunologie
Entwicklung zellulärer
Therapeutika
Group leader
Wolfgang Holter1
[email protected]
Staff scientist
Manfred Lehner1
PhD Student
Julia Proff1
1 since Apr. 2012
The research of the group focuses on the development
of adoptive therapies based on T cells, modified with
chimeric antigen receptors (CARs). First CARs directed
against antigens expressed on the surface of malignant
and virally infected cells were developed and published [1][2] at the Children’s Hospital of the University
Hospital Erlangen, Friedrich-Alexander Universität
Erlangen-Nürnberg, where the group worked until
they moved to the CCRI in April 2012. The study of the
biology of dendritic cells (DCs) with the aim of generating optimised vaccines was also part of the research
in Erlangen and was recently published, too [3].
CAR modified T cells
In clinical studies, it has been recently proven that
adoptive immunotherapy based on the transfer
of T cells modified with CARs mediates impressive
regression and even cure of tumours. Thus, the
approach is currently one of the most promising
forms of cancer immunotherapy.
Our prime goal is to establish new immuno-therapeutic
options based on CAR modified T cells for several
paediatric malignancies, including Ewing Sarcoma,
glioma and certain types of leukaemia. Additionally,
we hypothesise that the CAR-T cell approach could
also be adapted for the treatment of infections with
Cytomegalovirus (CMV), which is still an important
complication in transplant patients.
Entwicklung zellulärer
Therapeutika
Regulation of the survival of DCs
The optimisation of the differentiation and maturation
of DCs has been considered a key issue in the field of
DC vaccination for a long time. Strong co-stimulatory
potential of the DCs as well as the capacity for migration into the lymph nodes in conjunction with the ability
to produce cytokines supporting the differentiation
of Th1 T cells were defined as key criteria for this
optimisation. In our recent research, we focused on the
characterisation of conditions and factors regulating
the lifespan of DCs, which might also be important for
the efficacy of the vaccines.
For further reading
[1] Lehner, Götz et al. 2012
[2] Full, Lehner et al. 2010
[3] Lehner, Kellert et al. 2012
Der Forschungsschwerpunkt der Gruppe liegt auf der
Entwicklung adoptiver Therapieansätze basierend
auf T-Zellen, welche mit chimären Antigenrezeptoren
(CARs) modifiziert werden. Bis zum Umzug an das CCRI
im April 2012 arbeitete die Gruppe an der Kinderklinik
des Universitätsklinikums Erlangen, Friedrich-Alexander
Universität Erlangen-Nürnberg. Dort wurden erste
CARs mit Spezifität für Antigene, welche auf malignen
Zellen bzw. Virus-infizierten Zellen exprimiert
werden, entwickelt und publiziert [1][2]. Darüber hinaus
war in Erlangen auch die Untersuchung der Biologie
Dendritischer Zellen (DCs) mit dem Ziel der Herstellung
von optimierten DC-basierten Vakzinen im Fokus der
Forschung [3].
CAR modifizierte T-Zellen
scFv
NKG2D
lgG1
CM
CD28
CD3ζ
Fig. 1 – Domain structure of CARs with antigen binding domains
derived from either antibodies (scFv, single chain variable region
fragment directed against CMV-gB) or from the extracellular domain
of the receptor NKG2D. The receptors contain IgG1 as stabilizing
spacer and the signalling domains of CD3zeta and receptor CD28
(CM, cytoplasmic membrane).
In kürzlich durchgeführten klinischen Studien bewies
die adoptive Immuntherapie basierend auf dem Transfer von CAR-modifizierten T-Zellen eine beachtliche
Wirksamkeit mit einer beeindruckenden Rückbildung
bis hin zur Heilung von Tumoren. Dieser Ansatz zählt
daher gegenwärtig zu einer der vielversprechendsten
Formen der Tumor-Immuntherapie.
Das Hauptziel unserer Forschung ist die Etablierung
von neuen immuntherapeutischen Optionen für
verschiedene kindliche maligne Erkrankungen, u.a.
für Ewing Sarkome, Gliome und bestimmte Formen
der Leukämie. Zusätzlich postulieren wir, dass sich
CAR-modifizierte T-Zellen auch für die Behandlung
von Infektionen mit dem Cytomegalovirus (CMV),
welche immer noch eine bedeutende Komplikation in
Transplant-Patienten darstellen, eignen könnten.
Regulation der Lebensdauer von DCs
Lange Zeit galt im Feld der DC Impfung die Optimierung
der Differenzierung und Reifung von DCs als Schlüssel
thema. Dabei betrachtete man die Fähigkeit zur starken
Ko-stimulation sowie das Migrationspotential in die
Lymphknoten und die Produktion von Th1-differenzierenden Zytokinen als entscheidende Parameter.
Wir fokussierten uns zuletzt auf die Charakterisierung
von Bedingungen und Faktoren, welche die Lebensdauer von DCs bestimmen, die wiederum für die
Wirksamkeit von DC-Vakzinen essentiell sein dürfte.
Literaturangaben
[1] Lehner, Götz et al. 2012
[2] Full, Lehner et al. 2010
[3] Lehner, Kellert et al. 2012
68–69
Development of cellular
therapeutics
Immunology
Research focus
In order to redirect T cells to ES, we have generated
and characterised a CAR based on the ectodomain
of the activating NK cell receptor NKG2D.
NKG2D recognises eight ligands, which are expressed
on many transformed but not on most normal cells.
NKG2D-ligands (NKG2D-Ls) are expressed by various
tumours including ES and are important in tumour
immunosurveillance. We analysed in detail the
expression of MICA, MICB, ULBP-1, -2, and -3 in
fourteen ES cell lines. All tested cell lines expressed
at least one NKG2D-L. In order to retarget T cells
to these proteins, we designed a NKG2D-based
CAR of the second generation with an integrated
CD3z/CD28-derived co-stimulatory signaling domain.
Opposite transmembrane orientation of this signaling
domain and of NKG2D required inverse orientation
fusion of either of them. We hypothesised that the
extracellular domain of NKG2D, due to its particularly
located C-terminus, could be fused in inverse orientation and found indeed the resulting fusion protein to
specifically recognise NKG2D-Ls. High level surface
expression of the chimeric NKG2D (chNKG2D) in
recipient T cells was achieved by RNA transfection but
was followed by unexpected rapid down-modulation
upon target-cell recognition. In contrast, lentivirally
mediated chNKG2D expression was resistant to
receptor down-modulation and allowed sustained
ligand-dependent expansion of modified T cells.
Target cell death was induced by activated but not
by resting chNKG2D modified T cells. Importantly,
ES cells with even low NKG2D-L expression were
killed by both CD8pos and CD4pos cells (Fig. 2).
Our data suggest that an adoptive T cell approach
based on chNKG2D should be further pursued to
target ES and possibly other malignomas. Thus,
our next step will be the in vivo investigation of the
approach in an ES xenotransplantation model.
For further reading
Lehner, Götz et al. 2012
% specific lysis
Ewing sarcoma (ES) is the second most frequent type
of primary bone cancer in children and young adults.
Survival rates for localised ES have improved to about
70% with aggressive chemotherapy and local control.
Long-term survival rates achieved by conventional
cytotoxic chemotherapy in patients with metastatic
or recurrent disease, however, have reached a plateau
of around 20% over the last 20 years.
effector/target ratio
� chNKG2D
� αCMV-gH-CAR
control d1 control d4
Fig. 2 – The diagram shows the
lytic activity of CD3 activated
CD8 and CD4 positive T cells
transfected with mRNA for
either the chNKG2D or a similar
CMV-specific control receptor.
Shown is the specific lysis of
the Ewing sarcoma cell line
STA-ET-11, characterized by lowlevel expression of NKG2D-Ls,
at the indicated effector:target
ratio (mean ± S.D. of triplicates).
gB-CAR CEA-CAR
chNKG2D
no CAR
Fig. 3 – Supernatants from CAR expressing T cells inhibit CMV infection.
Human fibroblasts were infected with CMV (encoding GFP) in presence
or absence of supernatants. Progression of infection was monitored
by counting green fluorescent fibroblasts one (d1) and four days (d4)
later (white bars: no supernatant; gray bars: supernatant from T cell
co-culture with non-infected fibroblasts; black bars: supernatants from
T cell co-culture with infected fibroblasts).
Retargeting T cells to viral
glycoproteins for adoptive
therapy of CMV
TNF is critical for
the survival of human
dendritic cells
Immunology
Targeting Ewing sarcoma
by T cells expressing
a chimeric NKG2D receptor
Specific project
Cellular immunity is required for controlling human
CMV infections in vivo. Despite close monitoring
followed by preemptive therapy, reactivation of viral
disease in immunocompromised patients can cause
life-threatening complications. An attractive treatment
strategy is the application of virus specific cytotoxic
T cells isolated from blood of CMV positive donors
and expanded in vitro for adoptive immunotherapy.
However, this therapy is not applicable in the high
risk subgroup of stem cell transplant patients characterised by donor CMV sero-negativity and patient CMV
sero-positivity. Together with Prof. Dr. Armin Enssers
group at the Institute for Clinical and Molecular Virology
in Erlangen, we constructed a CAR targeting the CMV
glycoprotein B (gB) which shall allow to develop an
adoptive CMV directed immunotherapy. Activated
T cells engrafted with the construct by electroporation
of in vitro transcribed RNA showed specific effector
functions like degranulation and cytotoxic activity
after co-incubation with gB-expressing target cells.
In addition, redirected T cells specifically released
IFN-γ and TNF after stimulation with CMV infected
human foreskin fibroblasts (HFF) and conferred
inhibition of CMV replication by soluble factors as
supernatants of co-cultures of T cells and infected
HFFs inhibited CMV infection (Fig. 3). In ongoing
experiments, we examine differentially activated
T cells as well as NK cells engaged with our CAR
for their potential to lyse infected HFF to identify the
most suitable vehicles for CMV directed adoptive
immunotherapy.
The lifespan of DCs is determined by the balance of
pro- and anti-apoptotic proteins. Using serum-free
culture of human monocyte-derived DCs, we showed
that DCs following TLR stimulation with poly(I:C) or
LPS underwent apoptosis which was correlated with
low TNF production. Apoptosis was prevented by
the addition of exogenous TNF or by concomitant
stimulation with R-848, which strongly amplified
endogenous TNF production. In the contrary,
neutralisation of TNF confirmed that DC survival
was mediated by autocrine TNF induced either by
stimulation with R-848 or also by ligation of CD40.
70–71
Specific project
Prof. Armin Ensser, Institut für Klinische und Molekulare Virologie,
Universitätsklinikum Erlangen, Friedrich-Alexander-Universität
Erlangen-Nürnberg, Germany
Notably, DCs stimulated by poly(I:C) or IFN- α, another
known inducer of DC apoptosis, were characterised
by high levels and activation of the pro-apoptotic
protein BAK. Further investigation showed that the
ratio of anti-apoptotic BCL-2 to BAK correlated best
with the survival of activated DCs. In line with this
observation, the addition of TNF increased this ratio,
but had little effect on BAX and XIAP. Knockdown
experiments using siRNAs confirmed that the survival
of activated and also of immature DCs was regulated
by BAK and showed that TNF was protective only in
the presence of FLIPL.
Our study demonstrates that the survival of DCs during
differentiation and activation strongly depends on
autocrine TNF and that the inhibition of BAK plays an
important role in this process.
Prof. Eckhart Kämpgen, Department of Dermatology, Universitäts
klinikum Erlangen; Prof. Martin Leverkus, Department of Dermatology,
Venereology, and Allergology, Medical Faculty Mannheim, University
of Heidelberg, Germany
For further reading
Full, Lehner et al. 2010
Lehner, Kellert et al. 2012
Specific project
Molecular Microbiology and
Molecular Microbiology
Molekulare Mikrobiologie und
Molekulare Mikrobiologie
Group leader
Thomas Lion
[email protected]
Staff scientist
Reinhard Klein
Renate Kastner1
PhD students
Mirza Ibrisimovic2
Doris Kneidinger
Diploma/MSc students
Nikola Vidovic3
Florian Bellutti4
Technicians
Helga Daxberger5
Elisabeth Geiger
Dragana Jugovic5
Michaela Neßlböck5
Sandra Preuner
Sophie Renner5
Clinicians,
Sabine Breuer
Philipp Eickhoff
Tamás Fazekas
Andreas Vécsei
1 since Jan. 2012
2 until Dec. 2012
3 since Jan. 2012
4 until Sept. 2012
5 Dept. Molecular Genetic
Analysis and Molecular
Microbiology, LabDia
LabDia
Labordiagnostik
GmbH
The SME LabDia Labordiagnostik GmbH was established in the year 2006 (with Prof. Thomas Lion, MD,
PhD, as Medical Director) as a non-profit subsidiary of
the St. Anna Children’s Cancer Research Organisation,
with the aim to promote development and performance of innovative diagnostics. The main areas of our
activity include haematology/oncology and infection.
Das Ambulatorium LabDia Labordiagnostik GmbH
wurde im Jahr 2006 (mit Prof. DDr. Thomas Lion als
ärztlichem Direktor) als gemeinnütziges Tochter
unternehmen der St. Anna Kinderkrebsforschung
mit dem Ziel gegründet, neue diagnostische Verfahren
zu entwickeln und anzubieten. Die Schwerpunkte
unserer Tätigkeit liegen in den Bereichen Hämatologie/
Onkologie und Infektiologie.
In close collaboration with the St. Anna Children’s
Cancer Research Institute (CCRI) and other national
and international research centres, we continuously
establish new assays and introduce them into clinical
diagnostics.
In enger Zusammenarbeit mit dem CCRI und anderen
Forschungseinrichtungen werden laufend neue
Methoden etabliert, validiert und in die klinische
Diagnostik eingeführt.
All diagnostic tests provided are based on our own
developments, some of which have been patented.
We actively participate in national and international
boards focusing on the standardisation of different
diagnostic methodologies, and have coordinated
European activities in the area of diagnostic development in EU-funded projects. LabDia offers the newest
technical know-how and high competence in the areas
of diagnostics provided. In addition to our activities as
a diagnostic reference centre for national and international therapeutic trials, LabDia also offers its services
to individual patients treated at various centres in
Austria and other countries. The most important
diagnostic methodologies currently offered by LabDia
include cytogenetic analyses, fluorescence in situ
hybridization (FISH) assays, qualitative and quanti
tative PCR tests, pharmacokinetic analyses, flow
cytometry and cell sorting by FACS, and investigation
of DNA mutations by various techniques. LabDia is a
European-certified reference centre for comprehensive diagnostics of chronic myeloid leukaemia, which is
an important focus within our spectrum of diagnostic
activities. The current panel of diagnostic tests offered
by individual divisions of LabDia Labordiagnostik is
displayed on our website (www.labdia.at).
Unsere diagnostischen Leistungen beruhen auf
eigenen, zum Teil patentierten Entwicklungen.
Wir arbeiten in nationalen und internationalen Gremien
an der Standardisierung verschiedener diagnostischer Verfahren mit und haben auch europäische
Aktivitäten in der Diagnostikentwicklung im Rahmen
von EU-Projekten koordiniert. Das Ambulatorium
LabDia verfügt über neuestes technisches Know-how
und eine hohe fachliche Kompetenz in den Bereichen
der angebotenen Diagnostik. Neben unserer Tätigkeit
als diagnostisches Referenzzentrum in nationalen
und internationalen Studien, bietet LabDia sein
Diagnostikspektrum auch für individuelle Patienten
aus dem In- und Ausland an. Unsere wichtigsten
diagnostischen Methoden umfassen zytogene
tische Analysen, Fluoreszenz in situ Hybridisierungs
(FISH)-Assays, q
ualitative und quantitative PCR
Testmethoden, pharmakokinetische Bestimmungen,
Durchfluss-Zytometrie und Zellsortierung mittels
FACS sowie Analysen von DNA-Mutationen. LabDia ist
unter anderem ein europäisch zertifiziertes Referenz
zentrum für Diagnostik bei chronisch myeloischer
Leukämie (CML). Aktuelle Leistungen der LabDia Labordiagnostik GmbH können der Website (www.labdia.at)
entnommen werden.
Medical Director
Thomas Lion
[email protected]
Vice Director
Oskar A. Haas
Cytogenetics and
Molecular Cytogenetics
Gudrun Divoky1
Ulrike Engel
Brigitte Grimm
Susanna Koskela
Margit König
Bettina Nocker
Gertrud Pass
Eva Winkler
Wohlmacher Sven
Pharmacological Diagnostics
Ulrike Kastner
Clinical Cell Biology
& FACS Core Unit 2
Gerhard Fritsch
Christine Freimüller
René Geyeregger
Dieter Printz
Julia Stemberger
Dijana Trbojevic
Elke Zipperer
MRD Diagnostics2
Susanne Fischer
Andrea Inthal1
Ruth Joas
Solid Tumour Diagnostics2
Peter Ambros
Ingeborg Ambros
Bettina Brunner
Andrea Ziegler
Immunological Diagnostics2
Michael Dworzak
Angela Schumich
Susanne Suhendra
Molecular Genetic Analysis
and Molecular Microbiology2
Helga Daxberger
Elisabeth Geiger
Dragana Jugovic
Michaela Neßlböck
Sandra Preuner
Sophie Renner
1 maternity leave
2 CCRI groups that contribute to
the LabDia diagnostic activities.
74–75
LabDia
Labordiagnostik
GmbH
Research, development (R&D) and diagnostics in
the field of microbiology and chronic myeloid
leukaemia (CML) are the major areas of activity in
our division. The exploitation of diagnostic assays
emanating from our R&D work and the performance
of specific developmental tasks were transferred
to LabDia Labordiagnostik, a non-profit institution
established in 2006 as a subsidiary of the
St. Anna Children’s Cancer Research Organisation.
Our work is primarily focused on infectious problems
in oncological patients undergoing allogeneic stem
cell transplantation (allo-SCT) or chemotherapy.
In addition to bacteria, viral and fungal pathogens
frequently cause life-threatening infections in severely
immunocompromised children. Early and reliable
diagnosis is essential for successful therapy. We have
therefore developed and, in part, patented quantitative
molecular detection assays for many clinically relevant
viruses and fungal species. We were able to demonstrate that the clinical implementation of various
methods developed in our division permits early assess
ment of impending infectious complications [1][2].
Our new diagnostic approaches facilitate improved
treatment of life-threatening infections in immuno
compromised patients. Moreover, we have developed
the basis for novel therapeutic strategies against
invasive infections with adenoviruses which
are particularly dangerous for children undergoing
allogeneic stem cell transplantation [3][4][5]. An
additional focus of our activities is the diagnostic
monitoring of patients after allo-SCT. In a European
project coordinated by our group, we have established
a standardised methodology for quantitative analysis
of patient- and donor-derived cells (chimerism) [6].
The patented technique is already commercially
available as a diagnostic kit. Moreover, another recently
developed diagnostic approach to early prediction
of graft rejection will facilitate timely therapeutic
interventions [7].
The recent development and clinical implementation
of a molecular technique for the surveillance of mutant,
therapy-resistant subclones in patients with CML [8]
provided important information on clonal evolution
of the disease, timely detection of resistance and
clone-specific responses to treatment [9]. These new
insights will facilitate the advancement of research to
improve our understanding of the dynamics of CML.
We will pursue this task in a recently granted long-term
project (Special Research Area Program funded by
the Austrian Science Fund).
In addition to our R&D program, we provide services as
a reference laboratory for molecular diagnostics within
national and international therapy trials in the fields of
infectious diseases and leukaemia (ALL-BFM, AML-BFM,
CML-Ped, CML-11-CELSG, BFM-ALL-SCT, EWING,
ENEST-1st, ENEST-Path, CARRAFLU), and we perform
clinical studies addressing infectious disease-related
issues.
For further reading
[1] Landlinger, Preuner et al. 2010
[2] Lion, Kosulin et al. 2010
[3] Kneidinger, Ibrišimović et al. 2012
[4] Ibrišimović, Kneidinger et al. 2012
[6] Lion, Watzinger et al. 2012
[7] Breuer, Preuner et al. 2012
[8] Preuner, Denk et al. 2008
[9] Preuner, Mitterbauer et al. 2012
Die Tätigkeitsschwerpunkte unserer Abteilung liegen
im Bereich der mikrobiologischen Forschung und Entwicklung sowie der chronisch myeloischen Leukämie
(CML). Die Entwicklung neuer diagnostischer Verfahren
und deren klinische Implementierung wurden in das
gemeinnützige Ambulatorium LabDia Labordiagnostik
GmbH übertragen, das 2006 als Tochterunternehmen
der St. Anna Kinderkrebsforschung gegründet wurde.
Im Bereich der Mikrobiologie beschäftigen wir uns vor
allem mit infektiologischen Problemen von allogen
knochenmarktransplantierten und onkologischen
Patienten unter Chemotherapie. Bei schwer immun
geschwächten Kindern stellen neben bakteriellen
Infektionen insbesondere Virus- und Pilzinfektionen
eine sehr ernste Bedrohung dar. Eine frühzeitige und
verlässliche Diagnostik ist eine wichtige Voraussetzung
für erfolgreiche Therapien. Wir haben daher quanti
tative molekulare Detektionsmethoden für viele
pathogene Viren und klinisch relevante Pilzformen
etabliert und zum Teil patentiert. Wir konnten bereits
zeigen, dass der klinische Einsatz unserer Methoden
eine frühzeitige Vorhersage drohender infektiologischer
Komplikationen ermöglicht [1][2]. Diese neuen diagnos
tischen Verfahren tragen maßgeblich zur Behandlungsverbesserung lebensbedrohlicher Infektionen von
schwer immungeschwächten Patienten bei. Darüber
hinaus wurden für invasive Infektionen mit Adenoviren,
die insbesondere bei Kindern nach allogener Stammzelltransplantation zu schweren Erkrankungen führen
können, Grundlagen für neue Therapiestrategien
erarbeitet [3][4][5]. Ein weiterer wichtiger Bereich
unserer Tätigkeiten ist die diagnostische Überwachung
von Patienten nach allogener Stammzelltransplantation.
Im Rahmen eines von uns koordinierten europäischen
Projektes haben wir eine standardisierte Methodik
für das quantitative Monitoring von Spender- und
Empfängerzellen (Chimärismus) etabliert [6]. Das
patentierte Verfahren steht bereits als kommerziell
erhältlicher, diagnostischer Kit zur Verfügung. Darüber
hinaus haben wir eine Methodik für die frühzeitige
Vorhersage von Transplantatabstoßungen entwickelt,
die künftig als Grundlage für zeitgerechte Therapiemaßnahmen dienen wird [7].
Die rezente Entwicklung und klinische Anwendung
eines molekularen Verfahrens zur Überwachung
mutierter Subklone bei Patienten mit CML, die mit
Therapieresistenz einhergehen [8], hat es uns ermöglicht,
wichtige Erkenntnisse über die klonale Evolution der
Erkrankung, die frühzeitige Resistenzerkennung und das
klon-spezifische Therapieansprechen zu gewinnen [9].
Auf der Grundlage dieser Erkenntnisse planen wir
weiterführende Forschungsprojekte, die zu einem
besseren Verständnis der Dynamik der CML beitragen
sollen. Diese Untersuchungen werden im Rahmen eines
kürzlich bewilligten Sonderforschungsbereichs
(SFB-FWF) durchgeführt.
Neben unserem Forschungs- und Entwicklungs
programm stellen wir als Referenzlabor für molekulare
Infektions- und Leukämiediagnostik Serviceleistungen
für nationale und internationale Therapiestudien
(ALL-BFM, AML-BFM, CML-Päd, BFM-ALL-SCT, EWING,
ENEST-1st, ENEST-Path, CARRAFLU) zur Verfügung
und führen eigene klinische Studien mit infektiologischen Fragestellungen durch.
Literaturangaben
[1] Landlinger, Preuner et al. 2010
[6] Lion, Watzinger et al. 2012
[7] Breuer, Preuner et al. 2012
76–77
Research focus
We hypothesised that sensitive detection of mutant
subclones and monitoring of their proliferation kinetics
could permit assessment of their biological behavior
and resistance to ongoing treatment. To address this
question, we have developed a quantitative PCR-based
technique (LD-PCR), permitting the detection of
individual mutations with a detection limit of ≥1%, and
facilitating highly reproducible and accurate
assessment of changes in the size of mutant subclones
exceeding ± 5% [1]. We have subsequently investigated
prospectively collected serial blood specimens derived
from CML patients displaying a variety of mutations.
The results provide new insights into the evolution of
point-mutated subclones during the course of therapy.
Our data demonstrate that the quantitative LD-PCR
approach permits early detection of relevant mutant
subclones, and enables accurate surveillance of
subclone evolution. Mutant subclones may appear and
expand rapidly after onset or change of TKI-therapy,
and subclone-specific response to treatment can
be readily documented [2]. The rapid appearance of
mutant cells following changes of treatment supports
the notion that CML is composed of various subclones.
Many of them carry point mutations in the TKD, indi
cating a non-linear, branching clonal architecture of the
disease. Detection of expanding mutant subclones by
LD-PCR mostly paralleled, but occasionally preceded
the rise in BCR-ABL transcripts by several weeks.
Quantitative monitoring of mutant subclones during
treatment with TKIs provided information on their
actual responsiveness to therapy, and facilitated early
detection of imminent evolution of resistant disease.
Implementation of quantitative approaches to the
monitoring of mutant subclones in the surveillance
of CML patients can therefore improve our current
options for timely treatment decisions. This may help
optimise disease management in patients displaying
point mutations in the BCR-ABL TKD or other sites of
potential relevance. The observations made in patients
with CML may serve as a paradigm for similar studies
in patients with other haematologic neoplasias to
assess the role of mutant subclone formation and
kinetics in the evolution of resistant disease. Further
research into phenomena related to the subclonal
architecture of specific leukaemias will be pursued
with financial support of a recently awarded SFB grant
from the Austrian Science Fund.
Adenoviruses (AdVs) are a major threat to severely
immunocompromised patients, including particularly
children undergoing allogeneic stem cell trans
plantation (allo-SCT). We have shown that AdVs can
persist in several tissues in a latent state after primary
infection. Reactivations in immunosuppressed patients
can lead to life-threatening disseminated disease.
We have demonstrated that invasive infections in
the allo-SCT setting almost invariably originate from
the gastrointestinal tract. Patients at risk of invasive
infections need timely therapeutic intervention.
We have therefore established diagnostic strategies
facilitating early onset of treatment [1]. However,
currently available therapy options do not always
permit control of the disease, and novel approaches
are therefore urgently needed. The current dilemma
in treating AdV-mediated infections in severely
immunocompromised patients prompted us to
explore alternative treatment strategies.
Small interfering RNAs (siRNAs) and artificial microRNAs
(amiRNAs) are a class of synthetic RNAs resembling
cellular miRNAs. Similar to their natural relatives, they
can mediate the knockdown of endogenous gene
expression. This process, termed RNA interference,
can be harnessed to target and potentially silence
both cellular and viral genes. We determined that
silencing of the expression of several adenoviral genes
that are essential for viral DNA synthesis can efficiently
inhibit virus replication, and consequently, the
generation of virus progeny in vitro [2][3]. Furthermore,
a combination with conventional antiviral drugs such
as cidofovir increased the cumulative antiviral effect.
These insights suggest that a combination of two
very distinct approaches may permit improved control
of viral infections.
In a second approach [4], we demonstrated that introduction of the herpes simplex virus thymidine kinase
(HSV-TK) gene and its selective expression within
AdV-infected cells can inhibit the replication of human
AdV in vitro, when cells are concomitantly treated with
the prodrug ganciclovir. This strategy of selectively
sensitising AdV-infected cells to treatment with
ganciclovir may constitute another way of controlling
life-threatening AdV infections in patients with severely
impaired immune response.
For further reading
Fig. 1 – Surveillance of the evolution and expansion of leukaemic
subclones. Proliferation kinetics of three different mutant subclones
in a patient with chronic myeloid leukaemia (CML) in response to
different treatment modalities. The differential kinetics indicates
the presence of mutations within the BCR-ABL tyrosine kinase domain
in separate subclones, and reveals the evolution of resistant disease.
The monitoring of drug-resistant mutant subclones provides a
potentially important diagnostic tool in the surveillance of patients
with CML and other malignant disorders.
Mut clone, BCR-ABL/ABL1 (%)
Recent observations in different types of leukaemia
revealed their composition of multiple subclones,
carrying divergent genetic alterations which may
mediate differential responses to treatment. Subclones resistant to the applied treatment can expand
and give rise to disease recurrence or progression.
Neoplasias in which activated tyrosine kinases (TKs)
represent important driver mutations can be amenable
to treatment with specific tyrosine kinase inhibitors
(TKIs). However, the occurrence of subclones with
mutations in the TK gene conferring TKI resistance
can present a serious impediment to successful
therapy. We use chronic myeloid leukaemia (CML) as
a model disease to study this phenomenon. CML is
characterised by the BCR-ABL fusion gene encoding
a constitutively activated tyrosine kinase, and was the
first disease for which treatment with a specific TKI has
been developed. Despite the tremendous success of
this therapeutic approach, the development of resistance remains an important problem. Point mutations
within the BCR-ABL tyrosine kinase domain (TKD) are
currently regarded as the most important mechanism
of resistance to TKIs, and more than 100 different
mutations have been described to date. The screening
for resistant mutations is most commonly performed
by direct sequencing of the entire BCR-ABL TKD upon
amplification by PCR. This approach, however, does
not reveal the presence of mutant subclones constituting less than 10 – 20% of the entire leukaemic cell pool.
Despite the modest detection limit of this approach,
the clinical relevance of mutations within individual
subclones identified by this technique can be difficult
to predict.
Strategies to combat
Adenovirus infections in the
immunocompromised host
T315l
E255K
G250E
BCR-ABL1/ABL1
For further reading
HU
Dasa (100 mg)
HU
post SCT/no
HU
Nilo
(800 mg)
Nilo
(? mg)
Nilo (200 mg)
78–79
Surveillance of the
evolution and expansion
of leukaemic subclones
Specific Project
Specific Project
Clinical Research
S2_IRP: Studies & Statistics
for Integrated Research
and Projects
Klinische Forschung
S2_IRP: Studien & Statistik
Group leader
Ruth Ladenstein
[email protected]
Scientific assistant
Claudia Zeiner
Statisticians
Evgenia Glogova
Ulrike Pötschger, senior
International trials
Alisa Alspach
Inge Hirsch1
Ingrid Pribill
Marlene Reisinger2
Elfriede Thiem
Leukaemia & Lymphoma studies
Saelde Baumgartner2,3
Corinne Grafl
Susanne Karlhuber1,3,4
Nora Mühlegger3
Dasa Janousek5
Marek Nykiel6
Solid Tumour studies
Eva Sorz3
Project manager, O.K.ids
Andrea Mikolasek
1 Also: Stem Cell
Transplantation studies
2 Quality Management
3 Phase I/II/III Pharmaceutical
Industry studies for the
4 Also Haematology studies
5 Data Management Support
6IT-support
Senior Statistician
U. Pötschger, MSc
Statistician
E. Glogova, MSc
Langerhans Cell Histiocytosis
Assoc. Prof. M. Minkov, MD
RSA*
E. Thiem, MSc
EBMT/IBFM SCT Trials
Assoc. Prof. S. Matthes-Leodolter, MD
Prof. C. Peters, MD
RSA*
I. Hirsch, MSc
SIOPEN Neuroblastoma Studies
Assoc. Prof. R. Ladenstein, MD, MBA, cPM
RSA*
A. Alsbach, BSc
I. Pribill, PhD
M. Reisinger, BSc
Scientific Guidance, Study
Coordinators & Local Investigators
Assoc. Prof. A. Attarbaschi, MD
Assoc. Prof. M. Dworzak, MD
Assoc. Prof. G. Mann, MD
RSA*
N. Mühlegger, MSc
C. Grafl
S. Karlhuber, PhD
N. Mühlegger, MSc
M. Nykiel
Assoc. Prof. L. Kager, MD
RSA*
E. Sorz
Prof. R. Ladenstein, MD, MBA, cPM
A. Lawitschka, MD
Assoc. Prof. S. Matthes-Leodolter, MD
Prof. C. Peters, MD
V. Witt, MD
RSA*
I. Hirsch, MSc
S. Karlhuber, PhD
Assoc. Prof. M. Minkov, MD
Assoc. Prof. L. Kager, MD
RSA*
S. Karlhuber, PhD
International & National Paediatric
Haemato-Oncology Groups
International Groups
International Trials
Leukaemia & Lymphoma
Task Team
Solid Tumour
Task Team
Stem Cell Transplantation
Task Team
Haematology
Task Team
O.K.ids
Project Manager
A. Mikolasek
Phase I/II/III Pharma
Industry with St. Anna
Children‘s Hospital
RSA*
S. Baumgartner, MSc
S. Karlhuber, PhD
N. Mühlegger, MSc
E. Sorz
Quality Management
S. Baumgartner, MSc
M. Reisinger, BSc
Data Management
Support
IT Support
RSA*
D. Janousek, MSc
M. Nykiel
Fig. 1 – Complete organisation chart,
including clinical study leaders and
research and study assistants.
*RSA: Research and Study Assistant
AIEOP: Associazione Italiana
Ematologia Oncologia Pediatrica;
www.aieop.org
ANZCHOG: Australia and
New Zealand Children’s
Haematology/Oncology Group;
www.anzchog.org
BSPHO: Belgian Society of
Paediatric Haemato-Oncology;
www.bspho.be
CCLG: Children’s Cancer
and Leukaemia Group, UK;
www.cclg.org.uk
COG: Children’s Oncology
Group, USA
DCOG-ECTC: Dutch Childhood
Oncology Group – Early Clinical
Trial Consortium
DFCI: Dana-Farber Cancer
Institute, USA;
www.dana-farber.org
EBMT: The European Group
for Blood and MarrowTrans
plantation; www.ebmt.org
EnPREMA: European Medicines
Agency; www.emea.europa.eu
FRALLE: French Acute Lymphoblastic Leukaemia Group
GPOH: German Group of
Paediatric HaematoOncology;
www.kinderkrebsinfo.de/gpoh
HSPHO: Hellenic Society of
Paediatric HaematologyOncology
I BFM SG: The International
BFM Study Group;
www.bfm-international.org
ICORG: The All Ireland
Cooperative Oncology Research
Group; www.icorg.ie
ISPHO: Israeli Society of Paediatric Haematology Oncology
ITCC: Innovative Therapies
for Children with Cancer;
www.itcc-consortium.org
LCH: HS Histiocyte Society
MRC: Medical Research Council,
UK; www.mrc.ac.uk
NOPHO: Nordic Society for
Paediatric Haematology and
Oncology, Denmark, Finland,
Iceland, Norway, Sweden;
www.nopho.org
PINDA: National Chilean
Pediatric Oncology Group
PPLLSG: Polish Pediatric
Leukemia Lymphoma
Study Group
SEOP: Spanish Society of
Paediatric Oncology
SFCE: Société Française
des Cancers et Leucémies de
l’Enfant et de l’Adolescent;
http://sfce1.sfpediatrie.com
SAKK: Swiss Group for
Clinical Cancer Research;
http://sakk.ch
SIOP: International Society
of Paediatric Oncology,
(SIOP Office c/o Kenes
Associations Worldwide,
Switzerland);
www.siop-online.org
SIOP EUROPE: European Society
for Paediatric Oncology
(SIOPE Office Brussels, Belgium);
www.siope.eu
SIOPEN: International Society
of Paediatric Oncology European
Neuroblastoma Study Group;
www.siopen-r-net.org
National Groups
AGPHO: Austrian Group of
Paediatric HaematoOncology
ÖGKJ: Austrian Society for
Paediatrics
(Österreichische Gesellschaft für
Kinder- und Jugendheilkunde);
www.docs4you.at
Clinical Research
Statistics
Coordinating Operative And Research Assistant
C. Zeiner, MSc
82–83
Operative & Strategic Management
Networks and Groups
S2IRP: Klinische Forschung
bei internationalen Studien
The Unit for Studies & Statistics for Integrated Research
& Projects (S2IRP) has an important role at the interface
of laboratory research activities at the CCRI and the
clinical application of trials at the hospital site of the
St. Anna Children’s hospital. As such the S2IRP has a
practical agenda to facilitate international randomised
trials and to foster clinical research in paediatric
oncology. Prospective trials in paediatric-haemato
oncology have enabled to advance the field and to
improve the outcome for children and young people
suffering of cancer over the last 50 years, now
achieving survival chances of up to 80% and even
more in some more favourable cancer types. These
trials are also the backbone for associated translational
research questions and may implement new stratifying
diagnostics, which support optimised, risk-adapted
treatments ultimately leading to improved overall
survival at a lower treatment cost with regard to long
term treatment side effects and quality of life.
S2IRP as a centre of expertise for studies and statistics
runs prospective trials pursuant to EU directives,
ICH guidelines and the Austrian Pharmaceuticals
Act (AMG). The team offers advice and expertise
protocol development and case report form design,
database design support and appropriate use of trial
management software, gaining ethical and regulatory
approvals and the necessary reporting follow-up
procedures, trial co-ordination, randomisation
procedures, support in patient recruitment and data
management. Quality assurance procedures are in
place. Austrian haemato-oncologists and the team
of research and study assistants are trained annually
according to GCP requirements.
The statistical group supports clinical investigators of
the St. Anna Children’s Hospital and researchers of the
CCRI with all statistical issues. This includes the statistical consultation at the design stage (statistical section
of the protocol), the data analysis and the statistical
support of the final publication. Within the established
national and international networks of investigators
in paediatric hemato-oncology (e.g. I-BFM, SIOPEN,
Histiocyte-Society and EBMT) statistical groups have
been formed to allow a close collaboration between
statisticians in all issues related to statistical methodology. In these statistical collaborations, the CCRIstatistics is an active member. According to the requirements of the EU directive 2001 and its implementation
to the Austrian Pharmaceuticals Act (AMG) in 2004,
the St. Anna Kinderkrebsforschung e.V. has undertaken
major efforts to secure the continuation of academicinvestigator driven trials. The St. Anna Kinderkrebsforschung e.V. assumed the responsibility as sponsor
on the international and national level for these trials.
Since September 2005, the research institute holds
a master policy for Austria to cover trial insurance for
patients on trials and investigators involved.
The S2IRP is a coordinating clinical trials unit, administering registered data of more than 10.000 patients
from ongoing and past national and international trials.
Current AGM-conform academic trials are run in the
areas of Langerhans Cell Histiocytosis (2), Leukaemias
& Lymphomas (6), Solid Tumours (7), and Stem Cell
Transplantation (4). In addition a number of registries
are supported: Leukaemias & Lymphomas (6), Solid
Tumours (4), Haematology (5) and Stem Cell Transplantation (4). A limited number of industry sponsored trials
– partly (3), completely (2) – are run with help of the unit.
International trials with the St. Anna Kinderkrebsforschung e.V. in the sponsor role focus on the fields
of Langerhans’ cell disease (international study centre
of LCH trials I, II, III), Neuroblastoma (international
study centre of the European High Risk Neuroblastoma
trial HR-NBL-1/SIOPEN and the LTI /ch14.18/CHO
SIOPEN trial) and Stem Cell Transplantation for Acute
Lymphoblastic Leukaemia (international study centre
of the international ALL-SCT Study). The cumulative
patient numbers within these three international
studies are now: 2.893 patients in LCH trials (I, II and III),
2144 patients in the HR-NBL-1/SIOPEN trial and 1.106
patients in the ALL-SCT trials.
Die Abteilung für Studien und Statistik (S2IRP) ist ein
wichtiges Bindeglied zwischen dem Forschungsinstitut
und dem St. Anna Kinderspital. Kernaufgaben sind die
Durchführung von internationalen, randomisierten
Studien und die Förderung der klinischen Forschung
in der pädiatrischen Hämato-Onkologie. Prospektive
Studien ermöglichten es, die Überlebenschancen
für Kinder und Jugendliche mit Krebserkrankungen in
den letzten 50 Jahren entscheidend zu verbessern.
Die Überlebenschancen liegen bei etwa 80%, bei
einigen Krebsarten mit günstigerer Prognose sogar
höher. Diese Studien sind auch die Basis für assoziierte
translationale Forschungsfragen. Neue, stratifizierende Diagnostik kann so implementiert werden und
ermöglicht optimierte, risiko-adaptierte Therapien,
welche letztendlich zu einem verbessertem Gesamtüberleben mit einer reduzierten Nebenwirkungsrate
und verbesserter Lebensqualität führen.
S2IRP ist ein Koordinierungszentrum (KKS) für Studien
und Statistik und führt prospektive Studien gemäß
EU-Direktiven, ICH Richtlinien und dem österreichischen
Arzneimittelgesetz (AMG) durch. Das Team stellt
Beratung und Expertise bei Protokollentwicklung und
Erhebungsbögen zur Verfügung und unterstützt
bei Datenbankdesign sowie Anwendung von Studien
managementsoftware. Es führt Einreichungen bei
Ethikkommissionen und Behörden durch und betreut
die Datenerhebung, das Datenmanagement, Randomi
sierungen und die Nachverfolgung. Außerdem unterstützt das Team bei der Patientenrekrutierung. Weiters
verfügt das S2IRP über ein Qualitätsmanagement
system. Österreichische Hämato-Onkologen und die
Forschungs- und StudienassistentInnen werden einmal
jährlich gemäß GCP-Auflagen fortgebildet. Die Statistik
gruppe unterstützt Kliniker am St. Anna Kinderspital
und Wissenschaftler der St. Anna Kinderkrebsforschung
in allen statistischen Belangen. Dazu zählen statistische
Beratungen in der Designphase (Statistikteil des Pro
tokolls), Datenanalyse und statistische Unterstützung
bei der finalen Publikation.
Innerhalb national und international etablierter Netzwerke von Wissenschaftlern in der pädiatrischen
Hämato-Onkologie (z. B. I-BFM, SIOPEN, Histiocyte
Society und EBMT) wurden Statistikgruppen gegründet,
die eine enge Zusammenarbeit im Bereich der statistischen Methodologie pflegen. Die Statistikgruppe
ist aktives Mitglied dieser Kooperationen im Statistik
bereich. Gemäß den Auflagen der EU-Direktive 2001
und deren Umsetzung im AMG im Jahr 2004, setzte
sich die St. Anna Kinderkrebsforschung dafür ein, die
Durchführung von akademischen Studien sicherzu
stellen. Die St. Anna Kinderkrebsforschung übernahm
die Sponsorverantwortung für diese Studien auf nationaler und internationaler Ebene. Seit September 2005
verfügt das Forschungsinstitut über eine Rahmen
versicherung für österreichische Studienpatienten und
Prüfärzte. S2IRP ist Koordinierungszentrum für klinische
Studien und betreut Daten von mehr als 10.000 Patienten aus laufenden und geschlossenen nationalen und
internationalen Studien. Derzeit laufen akademische
AMG Studien in folgenden Gebieten: Langerhanszell
Histiocytose (2), Leukämien & Lymphome (6), Solide
Tumoren und Stammzelltransplantation (4). Zusätzlich
werden folgende Register betreut: Leukämien &
Lymphome (6), Solide Tumoren (4), Hämatologie (5)
und Stammzelltransplantation (4). Einige Industrie
studien (5) werden durch das S2IRP unterstützt.
Für drei internationale Studien hat die St. Anna Kinderkrebsforschung e.V. die Sponsorrolle übernommen,
nämlich für die Bereiche der Langerhanszell Histiozytose (internationale Studienzentrale der LCH-Studien
I, II,III), dem Neuroblastom (internationale Studien
zentrale der Europäischen Hochrisiko Neuroblastomstudie HR-NBL-1/SIOPEN und der LTI/ch14.18/CHO
SIOPEN Studie) sowie für die Stammzelltransplantation
für akute lymphoblastische Leukämie (internationale
Studienzentrale der internationalen ALL-SCT Studie).
Die kumulative Anzahl der Patienten dieser drei Studien
beträgt derzeit: 2.893 Patienten in den LCH-Studien
(I, II, III), 2.144 Patienten in der HR-NBL-1/SIOPEN Studie
und 1.106 Patienten in den ALL-SCT Studien.
84–85
S IRP Clinical Research
in International Trials
Clinical Research
2
Research Focus
Langerhans Cell Histiocytosis
Study Reference Centre
EHN (EuroHistioNet)
project 2008–2011
Stem Cell Transplantation
Study Centre
Main recent tasks of the Langerhans Cell Histiocytosis
(LCH)-study reference centre were the planning,
designing and organising of the study protocol for the
next International LCH-IV trial which opened in Austria
in December 2012. Other international participating
subcentres are expected to follow in 2013. The results
of the LCH-III data analysis showed no benefit for
patients treated with Prednisone, Vinblastine and
additional Methotrexate as compared to the standard
treatment arm with Prednisone, Vinblastine and
6-Mercaptopurine when referring to time to non-active
disease, overall response or numbers of reactivation.
In low-risk patients treated for 12 months with low-risk
treatment, the reactivation rate was significantly
lower than in patients treated for only 6 months.
A manuscript describing the results of the LCH-III
study is in preparation.
Euro-Histio-Net was brought up to join efforts in
increasing the knowledge of the disease LCH, improving
quantity and quality of knowledge exchange and
producing guidelines. Any new information will be
distributed by a web portal for LCH professionals,
doctors and patients at www.eurohistio.net.
Acute lymphoblastic leukaemia (ALL) is the most
frequent haemato-oncological disease in children.
In spite of excellent survival rates of more than 80%
after risk adapted front line studies according to
BFM strategies, some children will suffer a relapse.
Allogeneic stem cell transplantation (SCT) is a
therapeutic option opening a second chance for
long-term survival.
The LCH study reference centre is supported i.a. by the
Histiocytosis Association of America (HAA), a grant
of the Österreichische Nationalbank (ÖNB) and by
the European Commission. The current LCH-IV study
protocol committee (chair, Assoc. Prof. Milen Minkov,
MD; team, B. Fahrner, MD, Prof. Gardner, MD, C. Hutter,
MD, PhD, L. Ronceray, MD) agreed upon diagnostic
guidelines for LCH-IV patients. Especially, criteria
for the diagnostic and follow-up evaluations of
LCH lung disease, bone or central nervous system
involvement were revised. On the laboratory side,
Dr. Caroline Hutter investigates pathogenetic
mechanisms in LCH (for details, see report of the
Molecular Biology group).
The associated partners of the project are four
European research institutes from Austria,
France, Italy and Spain and one patient association
from Great Britain.
The project aimed to merge information in an
international database which can serve as a common
platform for many different groups collecting data
on LCH and associated syndromes, on paediatric and
adult patients. Several people and teams worldwide
produced guidelines for diagnosis, treatment
and follow-up of LCH. One of the ambitious goals
of Euro-Histio-Net was to review these efforts, to
generate a synthesis of this information and to provide
guidelines after approval of many involved specialists.
In several European countries, patient associations
have developed websites dealing with histiocytic
diseases. Euro-Histio-Net aimed to coordinate these
existing resources and to extend this network. The
European patient associations will decide jointly about
the exact contents of this part of the Euro-Histio-Net
web portal (Patients Web Site).
The ALL SZT BFM 2003 became the biggest prospective multinational trial (Germany, Austria, and
Switzerland) on the value of allogeneic haematopoietic
stem cell transplantation for children and adolescents.
This was made possible through the establishment,
for the first time, of common strategies for conditioning regimen, donor recruitment, GVHD-prophylaxis
and methods of T-cell depletion according to a given
indication for an allogeneic transplantation. From 2003
until 2011, 624 patients with a median age of 9 years at
diagnosis were entered on trial. It was demonstrated
that with the given measures, unrelated matched
donor-HSCT is equivalent to allogeneic HSCT from
HLA identical siblings. Incidence of severe acute
and chronic GVHD is low and the treatment-related
mortality after one year is below 9% in both groups.
To confirm these excellent results, the protocol is
running since 2007 in 11 other European countries
(ALL-SCT BFM international). So far 482 patients have
been registered on this GCP study. Data collection
runs on an internet-based remote data entry system
developed in collaboration with the Austrian Research
Centre Seibersdorf (AIT).
Both studies (ALL-SZT BFM 2003 and ALL SCT BFM
international) closed their patient registration in 2011.
The results of these two studies are currently analysed
and the publication is in preparation.
Clinical Research
Specific project
The successor trial ALL SCTped 2012 focuses on a
new approach to reduce toxicity and late effects after
TBI. It has been approved by the ethics committee
and the consent of the Austrian competent authority
by Feb. 11th 2013 and will open soon.
Since March 2008, Prof. C. Peters has been elected
chair of the Paediatric Disease Working Party of the
European Marrow and Bone Transplantation Group
(EBMT). This is an important strategic position to
further enhance international collaboration for patientoriented clinical research and studies in the field of
stem cell transplantation to ultimately improve survival
and quality of life in children subject to stem cell transplantation. Many activities have taken place in these
last years, mainly focusing on education and consensus
treatment for rare diseases. Experts from different
fields were brought together and new projects and
studies were launched. Since autumn 2012, Prof. Peters
is also responsible for education within EBMT, and
became part of the EBMT Executive Board.
86–87
Specific project
Specific project
Clinical Research
The mission of the European International Society for
Pediatric Oncology Neuroblastoma Group (SIOPEN)
is to ultimately improve survival in children suffering
from high-risk neuroblastoma (NBL) with improved
treatment concepts including immunotherapy after
intensive induction and stem cell transplanation.
Moreover, futherfinetuning of risk-adapted therapy
advances treatment deescalation strategies and thus,
assurance of quality of life in children with inter
mediate- and, in particular, low-risk disease. Supported
by the European Commission1, the SIOPEN-R-NET
research network (www.siopen-r-net.org) was built.
An internet and web-based centralised remote data
entry database and communication system was
developed by AIT Austrian Institute of Technology
GmbH, Austria (www.ait.ac.at) serving as common trial
platform for SIOPEN. The SIOPEN Association was
established in 2009 with a registered office in Vienna
to foster Neuroblastoma research and enables the
long-term sustainability of the group. The liaison office
at the S2IRP acts as the members’ contact point.
(http://membership.siopen-r-net.org). In 2012 the
SIOPEN Association has active members in
27 countries including currently Australia, Austria,
Belgium, Croatia, Czech Republic, Denmark, Finland,
France, Germany, Greece, Hong Kong, Hungary, Ireland,
Israel, Italy, Japan, New Zealand Norway, Poland,
Portugal, Serbia, Singapour, Slovakia, Spain, Sweden,
Switzerland and United Kingdom.
This network application to the Austrian Ministry of
Health and Pharmaceutical Industry was successfully
approved and announced at the Forum Alpach/Tyrol
in 2012 in a press conference by the minister of health
(BM Alois Stöger) and, based on a privat-public funding
partnership, will be supported over 3 years. The
expertise of 5 coordinating clinical trial units of Austrian
universities and institutes will network with paediatric
experts and clinical sites to foster drug development
and approval of medicines for children and adolescents
in close collaboration with industry but will equally
support and enhance academic research trials to
establish treatment guidelines. These efforts were
driven by the vision to support solutions to overcome
off-label use in clinical trials for children and
adolescents. O.K.ids aims to become part of EnprEMA,
the European Network of paediatric networks. The
web page is currently under construction and will
be available soon for further, more detailed information
on aims and progress: www.Okids-net.at
88–89
European SIOPEN
Neuroblastoma Study
Reference Centre
Specific project
O.K.ids – Medicines for
Children Research
Organisation – Austria
In 2002, SIOPEN launched the HR-NBL-1 (1.5)/
SIOPEN trial for the treatment of high-risk neuro
blastoma under the leadership of Prof. R. Ladenstein
as international chair. In 2010, the use of G-CSF during
induction was published as newly established treatment standard reducing significantly side effects as
a first result of the trial [1]. At the ASCO Annual meeting
in 2011, the randomisation result on 598 patients
comparing two high-dose treatment (HDT) approaches
followed by stem cell reinfusion was awarded by a
presentation in the plenary session. A significant
difference in favour of the European approach with
Busulfan-Melphalan (Bu-Mel) high dose chemotherapy
over the North American (Children’s Oncology group)
regimen carboplatin, etoposide, melphalane was
observed (3-years event free survival 49% vs 33%;
3-years overall survival 60% vs 48%, p=0.004).
This relapse and progression incidence was significantly lower with BuMel (48% vs 60%, p<0.001) as
well as the severe toxicity rate [2]. The BuMel regime
is now the recommended new treatment standard.
The data is now matured, results maintained and final
publication in preparation.
The trial also enables access to immunotherapy
for children with High-Risk NBL in Europe based on
a purely investigator-driven effort and a European
wide SIOPEN funding campaign managed by
Prof. R. Ladenstein to allow for the ch14.18/CHO
anti-GD2 antibody development. Having been given
Scientific Advice as Academia at the European Medical
Agency (EMA) in 2010, a small biotech company
APEIRON (http://www.apeiron-biologics.com) has
shown interest and contracted in 2011 with the CCRI
and the SIOPEN group to develop the antibody for the
market. As a result of this cooperation with APEIRON,
the ch14.18/CHO antibody has been granted Orphan
Status in 2012, both in Europe by the EMA and in the
US by the FDA. The part of the trial is expected to
answer the randomised immunotherapy question
towards the end of 2013 having accrued already
342 patients out of a planned sample of 400 patients.
To allow antibody access also for patients with primary
refractory or relapsing NBL, the “Long-term Infusion
Study (LTI)” using the antibody in combination
with subcutaneous interleukin 2 with Prof. H. Lode
(Greifswald/Germany) and Prof. R. Ladenstein as main
investigators was developed as a phase I/II trial with
support of the S2IRP team and is run by the unit with
the St. Anna Kinderkrebsforschung e.V. as a sponsor.
1 EC FP5 Grant No. QLRI-CT-2002–01768
(project coordination: Prof. Ladenstein, MD, MBA, cPM)
For further reading
[1] Ladenstein, Valteau-Couanet et al. 2010,
J Clin Oncol. 28(21):3516–24
[2] Ladenstein, Poetschger, et al., J Clin Oncol 29: 2011
(suppl; abstr 2)
Specific project
This European Commission-funded project run
through the CCRI is coordinated by Prof. R. Ladenstein
and was established during her presidency of
SIOPE (2010–2012). Starting in 2011 with 33 European
partner institutions in 16 countries, it will deliver
80 milestones and manifold associated tasks within
18 work packages over 4 years.
ENCCA aims are to
• Ensure children and adolescents in Europe receive
the best treatment and caring support.
• Establish a durable, integrated clinical and
translational research infrastructure for Europe.
• Improve access to new anti-cancer drugs for
children/adolescents in Europe through a
biology-driven drug development strategy.
• Address inequalities by addressing the differences
in terms of ethics, attitudes to experimental
therapies and quality of care for children with cancer.
• Build for the future through investment in the
cancer control workforce, methodology and
infrastructure and enhancement of relationships
between industry and clinical research.
• Improve substantially the quality of life of children
and adolescents with cancer and cancer survivors
through improved, risk-adapted treatment strategies
and enhanced, personalised guidance through
risk-adapted long term follow up and care.
• Enhance communication and increase under
standing among childhood cancer patients,
their families and the research community.
Major milestones have been achieved since the Kickoff
in January 2011 and what follows is just to highlight a few.
A European Clinical Research Council (ECRC) was
founded as a stakeholder platform of presidents
of national paediatric haemato-oncological Societies
and chairs of European clinical paediatric haematooncological trials, dealing with common burning issues
of the field (i.e. revision of the European Clinical Trial
Directive and of suggested possible solutions for its
revision, engagement with the European Parliament
with specific events held there on invitation). The
founded Parent-Patient Advocacy Committee (PPAC)
will engage in close collaboration with the ECRC in
the policy agenda of burning needs of children and
young people with cancer. ENCCA key awareness
actions include the establishment of strong relations
with policy makers (DG Sanco, EMA, members of the
European Parliament – 3 events in the EU Parliament).
A strategy for the development of new drugs in four
malignancies (Rhabdomyosarcoma, Acute Myeloid
Leukaemia, Hepatoblastoma and Medulloblastoma)
was established. A prototype of the Survivorship
Passport originating from the idea of childhood cancer
survivors was developed with the FP7 partner Project
PanCareSurFup (www.pancaresurfup.eu) and support
of the International Confederation of Childhood Cancer
Parent Organizations ICCPO (http://icccpo.org), to
provide a detailed and up-to-date description of the
tumour type, treatment, complication during treatment
and any other relevant data required for counseling
or assessing health need. A European steering group
to develop a Teenage Young Adult (TYA) Oncology
framework with key leaders and stakeholders identified
in 18 Countries, dealing with early diagnosis and better
healthcare models to improve the currently poor
outcome of the TYA population was established.
For detailed information on activities and results please
visit the ENCCA web page: www.encca.eu
One ENCCA work package on “Standardised and innovative methodology for clinical trial design and analysis”
addresses the needs for innovative approaches to
meet the requirements and unsolved methodological
challenges of trials in Paediatric Haemato-Oncology.
Ulrike Pötschger, the senior S2IRP statistician, has her
main statistical research focus in the evaluation of
long-term outcome. Her tasks within this work package
complement an ongoing collaboration on this topic
with the Institute of Clinical Biometry (Medical
University of Vienna) that started in 2008. Although
Cox-regression and the Log-Rank test are commonly
applied, neither method directly addresses longterm outcome. Cure models and the pseudo value
regression technique are promising approaches and
in many situations, these non-standard methods
clearly outperform the commonly used approaches.
Innovative study designs based on these models have
been recently implemented in the new LCH-IV study.
A European Network for
Cancer Research in Children
and Adolescents: ENCCA
90–91
Clinical Research
Specific project
Services,
Administration,
PR and
Donations Department
Service-Einrichtungen,
Administration,
PR und
Spendenabteilung
Bioinformatics
Maximilian Kauer
Science communication
Sandra Brezina-Krivda
Lisa Glenk1
Research Support
Barbara Brunmair
Nuno Andrade
Antonella Chiucchiuini2
Ivona Brasnjevic3
Finance & Administration Director
Karla Valdés Rodríguez4
Raoul Lavaulx-Vrécourt5
Secretaries
Marion Zavadil (CCRI Research)
Brigitte Glatz (LabDia)
Claudia Zeiner (S 2–IRP)6
IT Department
Group leader
Johann Kalhs
Team
Navid Kamalejan
Florian Kromp
Mark Rossiwall
Lukas Schneider
PR and Donations Department
Group leader
Andrea Prantl
Team
Elisabeth Tax
Katarina Krizanac11
Anja Fennes12
Bettina Nistler
Beatrix Wanek13
Personnel office & accounting
Caroline Schmid
Accountancy,
book-keeping,
balance-sheet accounting
& controlling
Monika Beran7
Claudia Hochweis8
Alexandra Lidy
Shideh Karvandi
Quality management
Michaela Artwohl
Sandra Weinzettl9
Facility management and pursuing
Corvin Fiala10
Klaus Kienzer
1
2
3
4
5
6
7
8
9
10
11
12
13
since Aug. 2012
since June 2012
until Sept. 2012
until Nov. 2011
since Apr. 2012
coordinating operative and
research assistant S2IRP
since Nov. 2011
until Dec. 2011
since Febr. 2012
since Nov. 2012
on maternity leave
since Oct. 2012
Jan. – Dec. 2012
James R. Downing
Scientific Director. Associate Director of
Basic Research. Cancer Center. Co-Leader.
Haematological Malignancies Laboratory.
Departments: Administration, Pathology,
St. Jude Children’s Research Hospital,
Memphis, USA
Lee J. Helman
Scientific Director for Clinical Research.
Head of the Molecular Oncology Section
Department: Paediatric Oncology Branch.
National Cancer Institute (NCI),
Bethesda, USA
Stephan Ladisch
Vice Chair for External Affairs. Principal
investigator. Department of Paediatrics;
Children’s Research Institute; Center for
Cancer and Immunology Research (CCIR);
Children’s National Medical Center,
Washington, USA.
Professor Biochemistry and Molecular
Biology, George Washington University, USA
Crystal L. Mackall
Chief Pediatric Oncology Branch/Head of
Immunology Section. Paediatric Oncology
Branch National Cancer Institute (NCI),
Bethesda, USA
Andrew Pearson
Chairman, Paediatric Oncology Section.
Head of Paediatric Research Team, Section
of Paediatric Oncology, Institute of Cancer.
The Royal Marsden Hospital, Sutton, UK
Gregory H. Reaman
Principal Investigator: Center for Cancer
and Immunology Research (CCIR),
Children’s National Medical Center,
Washington, USA
Maria Grazia Valsecchi
Professore Ordinario, Biostatistics
Centre Department of Clinical
Medicine and Prevention
University of Milano-Bicocca, Italy
94–95
Klaus-Michael Debatin
Medical Director. University of Ulm,
Clinic for Paediatrics and Adolescent
Medicine, Germany
St. Anna Kinderkrebsforschung /CCRI Scientific Report 2011–2012
Appendix
Anhang
Scientific Advisory Board
Wissenschaftlicher Beirat
Caroline Hutter
was awarded the 2012 Dr. Jon Pritchard
Award organised by The Histiocyte Society.
Hutter, C. et al. Blood 2012. Volume 120,
Issue 26, Pages 5199–5208. Notch is active
in Langerhans cell histiocytosis and confers
pathognomonic features on dendritic cells.
Heinrich Kovar
was awarded the 2011 Science Award of
the Austrian Paediatric Society.
Le Deley et al. 2010: Journal of Clinical
Oncology: Volume 28, Issue 12,
Pages 1982–1988. Impact of EWS-ETS fusion
type on disease progression in Ewing’s
sarcoma/peripheral primitive neuroecto
dermal tumor: prospective results from
the cooperative Euro-E.W.I.N.G. 99 trial.
Lilian Kuster
was awarded the 2011 first prize for the
best work in paediatric oncology in Austria
Kuster, L. et al. 2011. Blood: Volume 117,
Issue 9, Pages 2658–2667. ETV6/RUNX1positive relapses evolve from an ancestral
clone and frequently acquire deletions of
genes implicated in glucocorticoid signaling.
Ruth Ladenstein
was awarded the 2011 Science Award of
the Working Party Science and Research
of the Austrian Society for Paediatrics,
Section: Haematological – Oncological
Research
Ladenstein, R. et al, Journal of Clinical
Oncology 2010. Volume 28, Issue 20,
Pages 3284–3291. Primary Disseminated
Multifocal Ewing Sarcoma: Results of
the Euro-EWING 99 Trial
Ladenstein, R. et al, Journal of Clinical
Oncology 2010. Volume 28, Issue 21,
Pages 3516–3524. Randomized Trial
of Prophylactic Granulocyte ColonyStimulating Factor During Rapid COJEC
Induction in Pediatric Patients With
High-Risk Neuroblastoma: The European
HR-NBL1/SIOPEN Study
Stephan Niedan
was awarded the 2012 AACR-Pezcoller
Foundation Scholar in Training Award.
Niedan, S. et al. 2012. Re-activation of
EWS-FLI1 suppressed FOXO1 expression
as a novel therapeutic strategy for
Ewing’s sarcoma
Raphaela Schwentner
was awarded the 2012 AACR Woman
in Cancer Research (WICR) Scholar in
Cancer Research Award.
Schwentner, R. et al. 2012. A functional
ETS/E2F module in cancers expressing
ETS fusion genes
Florian Bellutti
Molecular interactions between
adenoviral RNAs and the cellular
RNA interference machinery.
Supervised by Reinhard Klein, PhD.
Diploma thesis
Ulrike Kaindl
Modulation of the p53 pathway
by ETV6/RUNX1 in childhood
acute lymphoblastic leukemia.
Supervised by Prof. Renate
Panzer-Grümayr, MD. PhD Thesis
Dagmar Denk
The role of PAX5 fusion genes in childhood
Supervised by Sabine Strehl, PhD.
PhD Thesis
Stephan Niedan
Mechanisms of transcriptional repression
by EWS-FLI1 in Ewing sarcoma.
Supervised by Prof. Heinrich Kovar, PhD.
PhD Thesis
Barbara Dillinger
The of IDO induced tryptophan depletion
and kynurenine accumulation in inhibition
of translation initiation and apoptotic
decline in activated human T-cells.
Supervised by Assoc.
Prof. Andreas Heitger, MD.
MSc Diploma Thesis
Angelika Plach
Donor T cells – Prevention of rejection
through induction of immunological
tolerance.
Supervised by Assoc. Prof. Andreas
Heitger, MD. BSc Bachelor Thesis
Friedrich Erhart
The molecular biology of dendritic cells
and their application in cell therapy.
Supervised by Alexander M. Dohnal, PhD.
MD Thesis
Nelli Frank
Analysis of senescence markers and
impact of drug-induced senescent
neuroblastoma cells on tumour cell
proliferation and immunomodulation.
Supervised by Sabine Taschner-Mandl, PhD,
and Assoc. Prof. Peter Ambros, PhD.
MSc Thesis
Gerhard Fuka
Exploring ETV6/RUNX1 function in
childhood acute lymphoblastic leukemia.
Supervised by Prof. Renate
Panzer-Grümayr, MD. PhD Thesis
Mirza Ibrisimovic
Alternative approaches for the
treatment of adenovirus infections.
Supervised by Reinhard Klein, PhD.
PhD Thesis
Stefanie Reinprecht
The influence of reactive oxygen
intermediates on the polarization
and behaviour of human T-cells.
Supervised by Birgit Jürgens, PhD.
BSc Bachelor Thesis
Lucia Riedmann
The effect of EWS and its oncogenic
derivative EWS-FLI1 on transcriptional
and post-transcriptional gene regulation
in Ewing sarcoma.
PhD Thesis
Raphaela Schwentner
Mechanisms of target regulation
by the chimeric oncogene
EWS-FLI1 in Ewing’s sarcoma.
PhD Thesis
Sneha Valookaran
Importance of timing in immune
modulation: A study on toll like
receptor-4 activated dendritic cells.
MSc Thesis
Edda Veith
Immune-regulation of allogeneic
stimulation by CTLA-4Ig:
Propagating CD4+CD25+FOXP3 +
T cells in an IDO independent fashion.
Supervised by Assoc. Prof. Andreas
Heitger, MD. PhD Thesis
Sarah Vittori
Characterization of a putative
immunosuppressive MAPKAP kinase
family member in human Dendritic Cells.
MSc Thesis
96–97
Jozef Ban
was awarded the 2012 Best Basic Science
Paper of the European Muscoskeletal
Oncology Society Award.
Ban, J. et al. 2012. Deacetylase SIRT1 links
tumor suppressive NOTCH signaling to
p53 in Ewing sarcoma.
Completed MSc Diplomas
and PhD Theses
Abgeschlossene Diplomarbeiten
und Dissertationen
Awards
Preise
High-resolution genomic profiling of
disseminated tumour cells for a refined risk
assessment. Coordinated by Peter Ambros.
Project no. 151141.
Clinical and biological relevance of novel
genomic aberrations affecting the glucocorticoid response in childhood leukemia.
Coordinated by Renate Panzer-Grümayer.
Project no. 14500
Period covered: 01.01.2012 – 31.12.2012
Interference of immunoregulatory
tryptophan metabolism with translation
initiation. Coordinated by Birgit Jürgens,
group leader Andreas Heitger.
Project no. 14225
Period covered: 01.01.2011 – 30.06.2012
Clinical and biological relevance of the
P2RY8-CRLF2 fusion gene in childhood
Coordinated by Maria Morak, group leader
Renate Panzer-Grümayer.
Project no. 14129
Period covered: 01.01.2011 – 30.06.2012
Mannan-binding lectin deficiency
in pediatric stem cell transplantation.
Coordinated by Andreas Heitger.
Project no. 13075
Period covered: 01.07.2008 – 30.04.2012
Control of Ewing sarkoma stem cell growth.
Coordinated by Heinrich Kovar.
Project no. 13349
Period covered: 01.01.2009 – 30.06.2011
Origin of relapses from childhood
hyperdiploid B-lineage leukemias.
Coordinated by Georg Mann, principle
investigator Renate Panzer-Grümayer.
Project no. 13881
Period covered: 01.08.2010 – 31.12.2011
Differential expression of chromatin
fractions in aggressive/benign neuro
blastomas. Coordinated by Eva Bozsaky,
group leader Peter Ambros.
Project no. 13835
Period covered: 01.08.2010 – 30.09.2012
Therapeutic targeting of the tumor
suppressor p53 in Ewing’s sarcoma family
tumors. Coordinated by Dave Aryee,
group leader Heinrich Kovar.
Project no 14205
Period covered: 01.01.2011 – 30.06. 2012
Evaluation of novel potential predictive
biomarkers in pediatric acute lymphoblastic
leukemia. Coordinated by Karin Nebral,
group leader Sabine Strehl.
Project no. 14133
Period covered: 01.01.2011 – 30.07.2012
The role of CDKN1B-p27KIP1 deregulation
in the pathogenesis of pediatric T-cell
Coordinated by Sabine Strehl.
Project no. 14444
Period covered: 01.12.2011 – 30.11.2013
Exploring the genetic base of relapsing
high-hyperdiploid acute lymphoblastic
leukemia in children with a genome-wide
next generation sequencing approach.
Coordinated by Georg Mann, principal
investigator Renate Panzer-Grümayer.
Project no. 14850.
Period covered: 01.07.2012 – 31.12.2013
The role of hypoxia in ETV6/RUNX1 positive
childhood leukemia: implications for
biology and clinic. Coordinated by Gerd Krapf,
group leader Renate Panzer-Grümayer.
Project no. 13665.
Period covered: 01.01.2010 – 31.12.2011
Uncovering Clinical Markers for Langerhans
Cell Histiocytosis. Coordinated by
Caroline Hutter, group leader Heinrich Kovar.
Project no. 13434
Period covered: 01.07.2009 – 31.12. 2011
Austrian Academy of Sciences
Österreichische Akademie der
Wissenschaften (ÖAW)
Austrian Science Fund
Fonds zur Förderung der wissen
schaftlichen Forschung (FWF)
Mechanisms of target regulation by the
chimeric oncogene EWS-FLI1 in Ewing’s
sarcoma. Applicant Raphaela Schwendtner,
DOC-fFORTE fellowship no. 22882
Period covered: 01.01.2010 – 31.12.2012
Allo-antigen-specific-T-cell-tolerance
induced by human dendritic cells
expressing the tryptophan metabolizing
enzyme indoleamine 2,3-dioxygenase.
Coordinated by Andreas Heitger.
Stand alone project no. P20865-B13.
Period covered: 01.08.2008 – 31.12.2011.
Austrian Research Promotion Agency
Österreichische Forschungsförderungsgesellschaft (FFG)
ADENOTAG – Validation of a novel
powerful technology for clinical-scale
isolation of T-cells for adenovirus therapy.
Coordinated by René Geyeregger,
group leader Gerhard Fritsch.
Eurostars/FFG project no. E! 5744/829495.
Period covered: 01.06.2011 – 31.03.2013
INTERACCT – Integrating Entertainment
and reaction assessment into child therapy.
Coordinated by Helmut Hlavacs (Research
Group Entertainment Computing, University
of Vienna, Austria), partner Anita Lawitschka
Bridge project no. 8385941
In SITU DC-CIT – Pre-clinical development
of an Off-the-Shelf individualised cancer
immune therapy. Coordinated by
Wolfgang Schöfberger (Institute for
Anorganic Chemistry, Johannes Kepler
University Linz, Austria), partner Alexander
Dohnal, group leader Thomas Felzmann.
Bridge project no. 836532
Period covered: 01.10.2012 – 30.09.2015
1 Operational start in 2013
A phase I/IIdose schedule finding study of
ch14.18/CHO continuous infusion combined
with subcutaneous IL-2 in patients with
primary refractory or relapsed neuro
blastoma. A SIOPEN study. Coordinated by
Ruth Ladenstein.KLI project no. 175-B00.
Period covered: 01.08.2011 – 31.07.2013
Elucidating the role of PAX5 chimeric proteins
in the pathogenesis of childhood B-cell
precursor acute lymphoblastic leukemia.
Coordinated by Sabine Strehl.
Stand alone project no. P 21554-B19
Period covered: 01.11.2009 – 31.07.2013
Mechanisms of transcriptional control by
EWS-FLI.1 Coordinated by Heinrich Kovar.
Stand alone project no. 22328-B09
Period covered: 04.01.2010 – 30.06.2013
MK2/3 in immune regulation by dendritic
cells – DC feedback loops. Coordinated
by Alexander Dohnal, group leader
Thomas Felzmann.
Stand alone project no. P 23271-B11
Period covered: 01.04.2011 – 31.03.2014
Oncogenic fusion proteins & epigenome:
partners in crime? Applicant Eleni Tomazou,
Lise Meitner fellowship no. M 1448-B13.
Period covered: 01.10.2012 – 30.09.2014
PROVABES – Prospective validation of
Biomarkers in Ewing Sarcoma for
personalised translational medicine.
Coordinated by Uta Dirksen (University
Hospital Münster, Pediatric Haematology
and Oncology, Germany), partner
Heinrich Kovar.
EraNET-TRANSCAN 2012
project no. I225-B191.
RNA interference-mediated inhibition of
adenoviruses. Coordinated by Reinhard Klein,
group leader Thomas Lion.
Translational research project no. L665-B13.
Period covered: 01.09.2009 – 31.12.2012.
Subclone expansion and evolution in CML
and other MPNs. Coordinated by Thomas
Lion, Subproject of the SFB-F47 research
cluster Myeloproliferative neoplasmspathogenesis and development of new
therapeutic strategies (Coordinator Peter
Valent, Medical University of Vienna).
SFB-project no. F4705-B201.
Role of EWS-FLI1 in post-transcriptional gene
regulation Coordinated by Heinrich Kovar.
Stand alone project no. 20665-B12.
Period covered: 01.03.2008 – 30.09.2011
The role of microRNAs in the gene
regulatory network of Ewing’s sarcoma
Coordinated by Heinrich Kovar.
Stand alone project no. 24708-B21
Period covered: 01.08.2012 – 31.07.2015
Timing of genetic “second hit” alterations
in childhood acute lymphoblastic leukemia.
Coordinated by Renate Panzer-Grümayer.
Stand alone project no. 22073-B19.
Period covered: 01.01.2010 – 31.06.2013
TRANSCALL – Translational Research in
Childhood Acute Lymphoblastic Leukemia.
Coordinated by Martin Stanulla
(University Hospital Schleswig Holstein,
Dept. Pediatrics, Kiel, Germany),
partner Renate Panzer-Grümayer.
EraNET-TRANSCAN 2012
project no. I226-B191.
Herzfelder’sche Familienstiftung
Analysis of the secretory profile and
immunogenicity of drug-induced senescent
tumor cells in aggressive neuroblastoma.
Coordinated by Sabine Taschner-Mandl,
group leader Peter Ambros.
Period covered: 01.07.2012 – 30.06.2014.
Liddy Shriver Sarcoma Initiative
Liddy Shriver Sarkom Initiative
Molecular mechanisms and pharmacologic
inhibition of bone sarcoma metastasis
Coordinated by David Loeb (Sidney Kimmel
Comprehensive Cancer Center,
Johns Hopkins University, Baltimore)
partner Heinrich Kovar.
Period covered: 01.11.2012 – 31.10.2014
Anniversary Fund of the Österreichische
Nationalbank (Austrian National Bank)
Jubiläumsfonds der Österreichischen
Nationalbank (OeNB)
98–99
External Grants
and Research Funding Bodies
Fremdgeförderte Projekte
und Fördergeber
ASSET – Analysing and stryking the
sensitivities of embryonal tumors
Coordinated by Walter Kölch (Systems
Biology, Univ. College Dublin, Ireland).
Partner Heinrich Kovar.
FP7-Health-2010 project no. 259348
Period covered: 01.11.2010 – 31.10.2015
ENCCA – European network for cancer
research in children and adolescents.
Coordinated by Ruth Ladenstein in collab
oration with Peter Ambros, Heinrich Kovar,
Michael Dworzak and external partners.
Period covered: 01.01.2011 – 31.12.2014
EURO-HISTIO-NET – A reference network for
Langerhans Cell Histiocytosis and associated
syndromes. Coordinated by Milen Minkov.
Public Health Executive Agency (PHEA).
Project no. 2007120
Period covered: 01.01.2008 – 31.08.2011
IntReALL – International study for treatment
of childhood relapsed ALL2010 with standard
therapy, systematic integration of new
agents and establishment of standardized
diagnostic and research.
Coordinated by Arend von Stackelberg
(Charité-Universitätsmedizin Berlin,
Germany), partner Andishe Attarbaschi.
Period covered 01.10.2011 – 30.09.2016
MODICELL – Targeted modulation of
immune-system responses in cell therapies.
Coordinated by Andreas Heitger in
collaboration with Thomas Felzmann
and external partners.
FP7-People-2011 project no. 2858751
Vienna Science and Technology Fund
Wiener Wissenschafts-, Forschungs-,
und Technologiefonds (WWTF)
Flow cytometric signal typing for
therapy response prediction in pediatric
myeloid leukaemia. Coordinated by
Michael N. Dworzak.
Life Sciences 2007 project no. LS07–037.
Period covered: 01.03.2008 – 31.09.2013
Wilhelm Sander foundation, Munich
Wilhelm Sander-Stiftung, München
Chimäre T-Zellrezeptoren für die
CMV-Therapie nach Stammzell
transplantation in Hochrisikopatienten.
Coordinated by Wolfgang Holter,
Armin Ensser, Manfred Lehner.
Project 2009.002.1
Period covered: 08.2009 – 07.2011
Histiocytosis association,
New Jersey, USA
Histiozytose-Gesellschaft,
New Jersey, USA
Analysis of Jagged2 Signaling in
Langerhans Cell Histiocytosis.
Coordinated by Caroline Hutter,
Histiocytosis association grant 20121.
AIEOP-BFM ALL 2009, PEG-Asparaginase
(Oncaspar®).Coordinated by Georg Mann.
Medac Gesellschaft für klinische Spezial
präparate GmbH, Germany
Period covered 2010–2012
ALL-SZT-Studies. Coordinated by
Christina Peters. DKMS Germany
Annual contribution. Umbrella organisation of
the Austrian Childhood Cancer Aid (Dachverband der österreichischen Kinderkrebshilfe)
Period covered: 2011–2012
EBMT-PDWP. Coordinated by
Christina Peters.
OKIDS GmbH, O.K.ids – Medicines for
Children Research Organisation.
Coordinated by Ruth Ladenstein.
“Federal Ministery of Health” grant approval:
summer 2012, “Health aims” grant approval:
November/December 2012, Pharmig’ grant
approval December 2012.
SIOPEN Association to foster Neuroblastoma Research (SIOP Europe
Neuroblastoma Group), Vienna.
Coordinated by Ruth Ladenstein.
Fundraising SIOPEN Association.
2 Group leader of the S2IRP Coordinating
Centre for Paediatric-Oncological Traials
at the CCRI, Ruth Ladenstein
Seventh Framework Programme (FP7)
of the European Commission
7. Rahmenprogramm
der Europäischen Union
100–101
National and International Trusts
for Funding Clinical Trials2
Nationale und internationale
Fördergeber für klinische Studien2
Andreou, D., Bielack, S.S., Carrle, D., Kevric, M.,
Kotz, R., Winkelmann, W., Jundt, G.,
Werner, M., Fehlberg, S., Kager, L., Kuhne, T.,
Lang, S., Dominkus, M., Exner, G.U., Hardes, J.,
Hillmann, A., Ewerbeck, V., Heise, U.,
Reichardt, P. & Tunn, P.U. (2011). The
influence of tumor- and treatment-related
factors on the development of local
recurrence in osteosarcoma after
adequate surgery. An analysis of
1355 patients treated on neoadjuvant
Cooperative Osteosarcoma Study Group
protocols. Ann Oncol. 22(5), 1228–35.
Angelini, P., London, W.B., Cohn, S.L.,
Pearson, A.D., Matthay, K.K., Monclair, T.,
Ambros, P.F., Shimada, H., Leuschner, I.,
Peuchmaur, M., Irwin, M.S. & Baruchel, S.
(2012). Characteristics and outcome of
patients with ganglioneuroblastoma,
nodular subtype: a report from the INRG
project. Eur J Cancer. 48(8), 1185–91.
Attarbaschi, A., Mann, G., Rosolen, A.,
Williams, D., Uyttebroeck, A., Marky, I.,
Lamant, L., Horibe, K., Wrobel, G.,
Beishuizen, A., Wossmann, W., Reiter, A.,
Mauguen, A., Le Deley, M.C., Brugieres, L.,
European Intergroup for Childhood
Non-Hodgkin Lymphoma AT. (2011).
Limited stage I disease is not necessarily
indicative of an excellent prognosis in
childhood anaplastic large cell lymphoma.
Blood. (Multicenter Study Research Support,
Non-U.S. Gov’t). 117(21), 5616–9.
Attarbaschi, A., Morak, M., Cario, G.,
Cazzaniga, G., Ensor, H. M., Te Kronnie, T.,
Bradtke, J., Mann, G., Vendramini, E.,
Palmi, C., Schwab, C., Russell, L. J.,
Schrappe, M., Conter, V., Mitchell, C. D.,
Strehl, S., Zimmermann, M., Pötschger, U.,
Harrison, C. J., Stanulla, M., PanzerGrümayer, R., Haas, O. A., & Moorman, A. V.;
on behalf of the Associazione Italiana di
Ematologia ed Oncologia Pediatrica
(AIEOP)-Berlin-Frankfurt-Münster (BFM)
Study Group and National Cancer Research
Institute (NCRI)-Children’s Cancer and
Leukaemia (CCLG) Study Group. (2012).
Treatment outcome of CRLF2 -rearranged
childhood acute lymphoblastic leukaemia:
a comparative analysis of the AIEOP-BFM
and UK NCRI-CCLG study groups.
Br J Haematol, 158(6), 772–777.
Ban, J., Jug, G., Mestdagh, P., Schwentner, R.,
Kauer, M., Aryee, D.N., Schaefer, K.L.,
Nakatani, F., Scotlandi, K., Reiter, M.,
Strunk, D., Speleman, F., Vandesompele, J.
& Kovar H. (2011). Hsa-mir-145 is the top
EWS-FLI1-repressed microRNA involved in a
positive feedback loop in Ewing’s sarcoma.
Oncogene. 30(18), 2173–80.
Beier, R., Albert, M.H., Bader, P., Borkhardt, A.,
Creutzig, U., Eyrich, M., Ehlert, K., Gruhn, B.,
Greil, J., Handgretinger, R., Holter, W.,
Klingebiel, T., Kremens, B., Lang, P., MauzKorholz, C., Meisel, R., Muller, I., Peters, C.,
Reinhardt, D., Sedlacek, P., Schulz, A.,
Schuster, F.R., Schrauder, A., Strahm, B.,
Sykora, K.W., Wossmann, W., Zimmermann, M.
& Sauer, M.G. (2012). Allo-SCT using BU,
CY and melphalan for children with AML
in second CR. Bone Marrow Transplant.
(Epub ahead of print)
Bennani-Baiti, I.M., Aryee, D.N., Ban, J.,
Machado, I., Kauer, M., Mühlbacher, K.,
Amann, G., Llombart-Bosch, A. & Kovar, H.
(2011). Notch signaling is off and
is uncoupled from HES1 expression
in Ewing’s sarcoma.
J. Pathol., 225(2), 353–63.
Bennani-Baiti, I.M., Machado, I., LlombartBosch, A. & Kovar H. (2012). Lysine-specific
demethylase 1 (LSD1/KDM1A/AOF2/BHC110)
is expressed and is an epigenetic drug target
in chondrosarcoma, Ewing’s sarcoma,
osteosarcoma, and rhabdomyosarcoma.
Human. Pathol. 43, 1300–7.
Booth, C., Gilmour, K.C., Veys, P.,
Gennery, A.R., Slatter, M.A., Chapel, H.,
Heath, P.T., Steward, C.G., Smith, O.,
O’Meara, A., Kerrigan, H., Mahlaoui, N.,
Cavazzana-Calvo, M., Fischer, A.,
Moshous, D., Blanche, S., Pachlopnik
Schmid, J., Latour, S., de Saint-Basile, G.,
Albert, M., Notheis, G., Rieber, N., Strahm, B.,
Ritterbusch, H., Lankester, A., Hartwig, N.G.,
Meyts, I., Plebani, A., Soresina, A., Finocchi, A.,
Pignata, C., Cirillo, E., Bonanomi, S., Peters, C.,
Kalwak, K., Pasic, S., Sedlacek, P., Jazbec, J.,
Kanegane, H., Nichols, K.E., Hanson, I.C.,
Kapoor, N., Haddad, E., Cowan, M., Choo, S.,
Smart, J., Arkwright, P.D. & Gaspar, H.B. (2011).
X-linked lymphoproliferative disease due to
SAP/SH2D1A deficiency: a multicenter study
on the manifestations, management and
outcome of the disease. Blood, 117(1), 53–62.
Boztug, H., Karitnig-Weiss, C., Ausserer, B.,
Renner, E.D., Albert, M.H., SawalleBelohradsky, J., Belohradsky, B.H., Mann, G.,
Horcher, E., Rummele-Waibel, A.,
Geyeregger, R., Lakatos, K., Peters, C.,
Lawitschka, A. & Matthes-Martin, S. (2012).
Clinical and immunological correction
of DOCK8 deficiency by allogeneic
hematopoietic stem cell transplantation
following a reduced toxicity conditioning
regimen. Pediatric hematology and oncology.
29(7), 585–94.
Breuer, S., Preuner, S., Fritsch, G.,
Daxberger, H., Koenig, M., Poetschger, U.,
Lawitschka, A., Peters, C., Mann, G., Lion, T.
& Matthes-Martin. S. (2012). Early recipient
chimerism testing in the T- and NK-cell
lineages for risk assessment of graft rejection
in pediatric patients undergoing allogeneic
stem cell transplantation. Leukemia.
26(3), 509–19.
Breuer, S., Rauch, M., Matthes-Martin, S.
& Lion, T. (2012). Molecular diagnosis and
management of viral infections in hema
topoietic stem cell transplant recipients.
Mol Diagn Ther. 16(2), 63–77.
Burkhardt, B., Oschlies, I., Klapper, W.,
Zimmermann, M., Woessmann, W.,
Meinhardt, A., Landmann, E., Attarbaschi, A.,
Niggli, F., Schrappe, M. & Reiter, A. (2011).
Non-Hodgkin’s lymphoma in adolescents:
experiences in 378 adolescent NHL patients
treated according to pediatric NHL-BFM
protocols. Leukemia. 25(1), 153–60.
Coenen, E. A., Raimondi, S. C., Harbott, J.,
Zimmermann, M., Alonzo, T. A., Auvrignon, A.,
Beverloo, H. B., Chang, M., Creutzig, U.,
Dworzak, M. N., Forestier, E., Gibson, B.,
Hasle, H., Harrison, C. J., Heerema, N. A.,
Kaspers, G. J., Leszl, A., Litvinko, N.,
Lo Nigro, L., Morimoto, A., Perot, C.,
Reinhardt, D., Rubnitz, J. E., Smith, F. O.,
Stary, J., Stasevich, I., Strehl, S., Taga, T.,
Tomizawa, D., Webb, D., Zemanova, Z.,
Pieters, R., Zwaan, C. M., & van den HeuvelEibrink, M. M. (2011). Prognostic significance
of additional cytogenetic aberrations in
733 de novo pediatric 11q23/MLL-rearranged
AML patients: results of an international
study. Blood, 117(26), 7102–7111.
Collins, M., Wilhelm, M., Conyers, R.,
Herschtal, A., Whelan, J., Bielack, S.
& Kager, L. (2012). Benefits and adverse
events in younger versus older patients
receiving (neo)-adjuvant chemotherapy
for osteosarcoma. Clin-Oncol.
Corbacioglu, S., Cesaro, S., Faraci, M.,
Valteau-Couanet, D., Gruhn, B., Rovelli, A.,
Boelens, J.J., Hewitt, A., Schrum, J.,
Schulz, A.S., Muller, I., Stein, J., Wynn, R.,
Greil, J., Sykora, K.W., Matthes-Martin, S.,
Fuhrer, M., O’Meara, A., Toporski, J.,
Sedlacek, P., Schlegel, P.G., Ehlert, K.,
Fasth, A., Winiarski, J., Arvidson, J.,
Mauz-Korholz, C., Ozsahin, H., Schrauder, A.,
Bader, P., Massaro, J., D’Agostino, R.,
Hoyle, M., Iacobelli, M., Debatin, K.M.,
Peters, C. & Dini, G. (2012). Defibrotide
for prophylaxis of hepatic veno-occlusive
disease in paediatric haemopoietic
stem-cell transplantation: an open-label,
phase 3, randomised controlled trial.
Lancet. 379(9823), 1301–9.
Creutzig, U., van den Heuvel-Eibrink, M.M.,
Gibson, B., Dworzak, M.N., Adachi, S.,
de Bont, E., Harbott, J., Hasle, H.,
Johnston, D., Kinoshita, A., Lehrnbecher, T.,
Leverger, G., Mejstrikova, E., Meshinchi, S.,
Pession, A., Raimondi, S.C., Sung, L., Stary, J.,
Zwaan, C.M., Kaspers, G.J. & Reinhardt, D.;
on behalf of the AML Committee of the
International BFM Study Group (2012).
Diagnosis and management of acute
myeloid leukemia in children and
adolescents: recommendations from
an international expert panel.
Blood. 120(16), 3187–3205.
Creutzig, U., Zimmermann, M., Bourquin, J.P.,
Dworzak, M.N., Fleischhack, G.,
von Neuhoff, C., Sander, A., Schrauder, A.,
von Stackelberg, A., Ritter, J., Starý, J.
& Reinhardt, D. (2011). CNS irradiation in
pediatric acute myleoid leukemia:
Equal results by 12 or 18 Gy in studies
AML-BFM98 and 2004. Pediatr Blood
Cancer. 57(6), 986–92.
Dabritz, J., Attarbaschi, A., Tintelnot, K.,
Kollmar, N., Kremens, B., von Loewenich, F.D.,
Schrod, L., Schuster, F., Wintergerst, U.,
Weig, M., Lehrnbecher, T., Groll & A.H. (2011).
Mucormycosis in paediatric patients:
demographics, risk factors and outcome
of 12 contemporary cases. Mycoses.
[Case Reports, Research Support,
Non-U.S. Gov’t]. 54(6), e785–8.
Decock, A., Ongenaert, M., Hoebeeck, J.,
De Preter, K., Van Peer, G., Van Criekinge, W.,
Ladenstein, R., Schulte, J.H., Noguera, R.,
Stallings, R.L., Van Damme, A., Laureys, G.,
Vermeulen, J., Van Maerken, T., Speleman, F.
& Vandesompele, J. (2012). Genome-wide
promoter methylation analysis in neuroblastoma identifies prognostic methylation
biomarkers. Genome Biol. 13(10), R95.
Denk, D., Nebral, K., Bradtke, J., Pass, G.,
Möricke, A., Attarbaschi, A., & Strehl, S. (2012).
PAX5-AUTS2: a recurrent fusion gene in
childhood B-cell precursor acute lymphoblastic leukemia. Leuk Res, 36(8), e178–181.
Ellinghaus, E., Stanulla, M., Richter, G.,
Ellinghaus, D., te Kronnie, G., Cario, G.,
Cazzaniga, G., Horstmann, M.,
Panzer-Grümayer, R., Cavé, H., Trka, J.,
Cinek, O., Teigler-Schlegel, A., ElSharawy, A.,
Häsler, R., Nebel, A., Meissner, B., Bartram, T.,
Lescai, F., Franceschi, C., Giordan, M.,
Nürnberg, P., Heinzow, B., Zimmermann, M.,
Schreiber, S., Schrappe, M. & Franke A. (2012).
Identification of germline susceptibility
loci in ETV6-RUNX1-rearranged childhood
acute lymphoblastic leukemia. Leukemia.
26(5), 902–9.
Fazekas, T., Attarbaschi, A., Lawitschka, A.,
Seidel, M., Potschger, U., Peters, C., Mann, G.,
Gadner, H. & Matthes-Martin, S. (2012).
Lethal pulmonary complications after
pediatric allogeneic hematopoietic stem
cell transplantation. The Pediatric infectious
disease journal. 31(2), 115–9.
Fazekas, T., Eickhoff, P., Lawitschka, A.,
Knotek, B., Potschger, U. & Peters, C. (2012).
Exhaled nitric oxide and pulmonary
complications after paediatric stem cell
transplantation. European journal of
pediatrics. 171(7), 1095–101.
Fazekas, T., Eickhoff, P., Pruckner, N.,
Vollnhofer, G., Fischmeister, G., Diakos, C.,
Rauch, M., Verdianz, M., Zoubek, A.,
Gadner, H. & Lion, T. (2012). Lessons learned
from a double-blind randomised placebocontrolled study with a iota-carrageenan
nasal spray as medical device in children
with acute symptoms of common cold.
BMC Complement Altern Med. 12(1), 147.
Ambros, I.M., Brunner, B., Aigner, G.,
Bedwell, C., Beiske, K., Benard, J., Bown, N.,
Combaret, V., Couturier, J., Defferrari, R.,
Gross, N., Jeison, M., Lunec, J., Marques, B.,
Martinsson, T., Mazzocco, K., Noguera, R.,
Schleiermacher, G., Speleman, F., Stallings, R.,
Tonini, G.P., Tweddle, D.A., Valent, A., Vicha, A.,
Roy, N.V., Villamon, E., Ziegler, A., Preuner, S.,
Drobics, M., Ladenstein, R., Amann, G.,
Schuit, R.J., Potschger, U. & Ambros, P.F.
(2011). A multilocus technique for risk
evaluation of patients with neuroblastoma.
Clin Cancer Res. 17(4), 792–804.
102–103
Publications
Publikationen
Fazekas, T., Pruckner, N., Lawitschka, A.,
Seidel, M.G., Eickhoff, P., Potschger, U.,
Szepfalusi, Z., Gadner, H. & Peters, C. (2012).
Non-atopic IgE and eosinophil cationic
protein after allogeneic hematopoietic
stem cell transplantation in children.
Annals of hematology. 91(6), 949–56.
Fišer, K., Sieger, T., Schumich, A., Wood, B.,
Irving, J., Mejstříková, E. & Dworzak, M.N.
(2012). Detection and monitoring of normal
and leukemic cell populations with hierarchical clustering of flow cytometry data.
Cytometry A 81(1), 25–34.
Friesenbichler, W., Lakatos, K., Funovics, P.
& Kager, L. (2012). Knochentumoren und
tumorähnliche Knochenläsionen der
Extremitäten im Kindes- und Jugendalter.
Teil 1: Maligne Knochentumoren. Pädiatrie
und Pädologie. 47(4), 20–3.
Fritsch, G., Witt, V., Pichler, J., Spengler, H.P.,
Scharner, D., Zipperer, E., Trbojevic, D.,
Stemberger, J., Geyeregger, R., Tea M.K.
& Printz, D. (2012). Robust multi-parameter
single-platform quantification of myeloid
and B-lymphoid CD34 progenitor cells in
all clinical CD34 cell sources and in thawed
PBSC. Ped Hematol. 29 (7), 595–610.
Fuka, G., Kantner, H.P., Grausenburger, R.,
Inthal, A., Bauer, E., Krapf, G., Kaindl, U.,
Kauer, M., Dworzak, M.N., Stoiber, D.,
Haas, O.A. & Panzer-Grümayer, R. (2012).
Silencing of ETV6/RUNX1 abrogates
PI3K/AKT/mTOR signaling and impairs
reconstitution of leukemia in xenografts.
Leukemia. 26(5), 927–33.
Gaipa, G., Cazzaniga, G., Valsecchi, M.G.,
Panzer-Grümayer, R., Buldini, B., Silvestri, D.,
Karawajew, L., Ratei, R., Benetello, A., Sala, S.,
Schumich, A., Schrauder, A., Villa, T.,
Veltroni, M., Ludwig, W.-D., Conter, V.,
Schrappe, M., Biondi, A., Dworzak, M.N.
& Basso, G. (2012). Time Point-Dependent
Concordance of Flow Cytometry and
RQ-PCR in Minimal Residual Disease
Detection in Childhood ALL: The Experience
of the AIEOP-BFM ALL MRD Study Group.
(MND and GB share the senior authorship of
this paper). Haematologica 97(10), 1582–93.
Gaze, M.N., Boterberg, T., Dieckmann, K.,
Hormann, M., Gains, J.E., Sullivan, K.P. &
Ladenstein, R. (2012). Results of a Quality
Assurance Review of External Beam Radiation Therapy in the International Society of
Paediatric Oncology (Europe) Neuroblastoma Group’s High-risk Neuroblastoma Trial:
A SIOPEN Study. Int J Radiat Oncol Biol Phys.
Epub 2012 Jun 30.
Heitger, A. (2011). Regulation of expression
and function of IDO in human dendritic cells.
Curr Med Chem. 18(15), 2222–33.
Heitzeneder, S., Seidel, M., Förster-Waldl, E.
& Heitger, A. (2012). Mannan-binding lectin
deficiency – Good news, bad news, doesn’t
matter? Clin Immunol. 143(1), 22–38.
Hirabayashi, S., Flotho C., Moetter J.,
Heuser M., Hasle H., Gruhn B., Klingebiel T.,
Thol F., Schlegelberger B., Baumann I.,
Strahm B., Stary J., Locatelli F., Zecca M.,
Bergstraesser E., Dworzak M., van den
Heuvel-Eibrink M.M., De Moerloose B.,
Ogawa S., Niemeyer C.M. & Wlodarski M.W.
(2012). Spliceosomal gene aberrations are
rare, coexist with oncogenic mutations, and
are unlikely to exert a driver effect in childhood MDS and JMML. Blood 119(11), e96-e99.
Hutter, C., Kauer, M., Simonitsch-Klupp, I.,
Jug, G., Schwentner, R., Leitner, J., Bock, P.,
Steinberger, P., Bauer, W., Carlesso, N.,
Minkov, M., Gadner, H., Stingl, G., Kovar
& H., Kriehuber, E. (2012). Notch is active
in Langerhans cell histiocytosis and
confers pathognomonic features on
dendritic cells. Blood. 120(26), 5199–208.
Ibrišimović, M., Kneidinger, D., Lion,
T. & Klein, R. (2012). An adenoviral vectorbased expression and delivery system
for the inhibition of wild-type adenovirus
replication by artificial microRNAs.
Antiviral Res. 97(1), 10–23.
Ibrišimović, M., Nagl, U., Kneidinger, D.,
Rauch, M., Lion, T. & Klein, R. (2012). Targeted
expression of herpes simplex virus thymidine
kinase in adenovirus-infected cells reduces
virus titers upon treatment with ganciclovir
in vitro. J Gene Med. 14(1), 3–19.
Inthal, A., Zeitlhofer, P., Zeginigg, M.,
Morak, M., Grausenburger, R., Fronkova, E.,
Fahrner, B., Mann, G., Haas, O.A. & PanzerGrümayer, R. (2012). CREBBP HAT domain
mutations prevail in relapse cases of high
hyperdiploid childhood acute lymphoblastic
leukemia. Leukemia. 26(8), 1797–803.
Kager, L. & Bielack, S. (2012). Mifamurtid.
Internistische Praxis. 51, 899–901.
Kager, L. & Evans, W. (2012). Pharma
cogenomics and Hematologic Diseases.
Hematology: Basic Principles and Practise. 6
Klusmann, J.H., Reinhardt, D.,
Zimmermann, M., Kremens, B.,
Vormoor, J., Dworzak, M., Creutzig,
U. & Klingebiel, T. (2012) The role
of matched sibling donor allogeneic
stem cell transplantation in pediatric
high-risk acute myeloid leukemia:
results from the AML-BFM 98 study.
Haematologica. 97(1), 21–9.
Kneidinger, D., Ibrišimović, M., Lion, T.
& Klein, R. (2012). Inhibition of adenovirus
multiplication by short interfering RNAs
directly or indirectly targeting the viral
DNA replication machinery.
Antiviral Res. 94(3), 195–207.
Lanzinger, M., Jürgens, B., Hainz, U.,
Dillinger, B., Raberger, J., Fuchs, D. &
Heitger A. (2012). Ambivalent effects of
dendritic cells displaying prostaglandin
E2-induced indoleamine 2,3-dioxygenase.
Eur J Immunol. 42(5), 1117–28.
Kovar, H. (2011). Dr. Jekyll and Mr. Hyde,
the two faces of the FUS/EWS/TAF15 protein
family. Sarcoma 2011: 837474.
Lawitschka, A., Ball, L., & Peters, C. (2012).
Nonpharmacologic treatment of chronic
graft-versus-host disease in children and
adolescents. Biol Blood Marrow Transplant.
[Review]. 18(1 Suppl), 74–81
Kovar, H., Alonso, J., Aman, P., Aryee, D.N.,
Ban, J., Burchill, S.A., Burdach, S., De Alava, E.,
Delattre, O., Dirksen, U., Fourtouna, A.,
Fulda, S., Helman, L.J., Herrero-Martin, D.,
Hogendoorn, P.C., Kontny, U., Lawlor, E.R.,
Lessnick, S.L., Llombart-Bosch, A.,
Metzler, M., Moriggl, R., Niedan, S., Potratz, J.,
Redini, F., Richter, G.H., Riedmann, L.T.,
Rossig, C., Schafer, B.W., Schwentner, R.,
Scotlandi, K., Sorensen, P.H., Staege, M.S.,
Tirode, F., Toretsky, J., Ventura, S., Eggert, A.
& Ladenstein, R. (2012). The first European
interdisciplinary ewing sarcoma research
summit. Front Oncol. 2:54.
Kuster, L., Grausenburger, R., Fuka, G.,
Kaindl, U., Krapf, G., Inthal, A., Mann, G.,
Kauer, M., Rainer, J., Kofler, R., Hall, A.,
Metzler, M., Meyer, L.H., Meyer, C., Harbott, J.,
Marschalek, R., Strehl, S., Haas & O.A., PanzerGrümayer, R. (2011). ETV6/RUNX1-positive
relapses evolve from an ancestral clone
and frequently acquire deletions of genes
implicated in glucocorticoid signaling.
Blood. 117(9), 2658–67.
Ladenstein, R., Potschger, U., Siabalis, D.,
Garaventa, A., Bergeron, C., Lewis, I.J.,
Stein, J., Kohler, J., Shaw, P.J., Holter, W.,
Pistoia, V. & Michon, J. (2011). Dose finding
study for the use of subcutaneous
recombinant interleukin-2 to augment
natural killer cell numbers in an outpatient
setting for stage 4 neuroblastoma after
megatherapy and autologous stem-cell
reinfusion. J Clin Oncol. 29(4), 441–8.
Lakatos, K., Friesenbichler, W., Funovics, P.
& Kager, L. (2012). Knochentumoren und
tumorähnliche Knochenläsionen der
Extremitäten im Kindes- und Jugendalter,
Teil 2: Benigne Knochentumoren. Pädiatrie
und Pädologie. 47(5), 22–6.
Lehner, M., Götz, G., Proff, J., Schaft, N.,
Dörrie, J., Full, F., Ensser, A., Muller, Y.A.,
Cerwenka, A., Abken, H., Parolini, O.,
Ambros, P.F., Kovar, H. & Holter, W. (2012).
Redirecting T cells to Ewing’s sarcoma family
of tumors by a chimeric NKG2D receptor
expressed by lentiviral transduction or mRNA
transfection. PLoS One. 7(2), e31210.
Lehner, M., Kellert, B., Proff, J., Schmid, M.A.,
Diessenbacher, P., Ensser, A., Dörrie, J.,
Schaft, N., Leverkus, M., Kämpgen, E.
& Holter, W. (2012). Autocrine TNF is critical
for the survival of human dendritic cells
by regulating BAK, BCL-2, and FLIPL.
J Immunol. 188(10), 4810–8.
Lessnick, S.L., Kovar, H. & Houghton, P. (2011).
The Molecular Basis of Sarcoma. Sarcoma.
2011, 864130.
Lion, T. (2012). Molecular monitoring
after HSCT: Chimerism. Chapter 16.2.
The EBMT Handbook, 6th Editors: J. Apperley,
E. Carreras, E. Gluckmamn, T. Maszi; Forum
Service Editore 2012; 280–287.
Lion, T., Watzinger, F., Preuner, S.,
Kreyenberg, H., Tilanus, M., de Weger, R.,
van Loon, J., de Vries, L., Cavé, H.,
Acquaviva, C., Lawler, M., Crampe, M.,
Serra, A., Saglio, B., Colnaghi, F., Biondi, A.,
van Dongen, J.J., van der Burg, M.,
Gonzalez, M., Alcoceba, M., Barbany, G.,
Hermanson, M., Roosnek, E., Steward, C.,
Harvey, J., Frommlet, F. & Bader, P. (2012).
The EuroChimerism concept for a stand
ardized approach to chimerism analysis
after allogeneic stem cell transplantation.
Leukemia. 26(8), 1821–8.
London, W.B., Castel, V., Monclair, T.,
Ambros, P.F., Pearson, A.D., Cohn, S.L.,
Berthold, F., Nakagawara, A., Ladenstein, R.L.,
Iehara, T. & Matthay, K.K. (2011). Clinical and
biologic features predictive of survival after
relapse of neuroblastoma: a report from
the International Neuroblastoma Risk Group
project. J Clin Oncol. 29(24), 3286–92.
Luger, R., Valookaran, S., Knapp, N.,
Vizzardelli, C., Dohnal, A.M. & Felzmann, T.
(2013). Toll-like receptor 4 engagement
drives differentiation of human and murine
dendritic cells from a pro-into an antiinflammatory mode. PLoS One. 8(2), e54879.
Matthes-Martin, S., Potschger, U., Barr, R.,
Martin, M., Boztug, H., Klingebiel, T.,
Attarbaschi, A., Eibler, W. & Mann, G. (2012).
Costs and cost-effectiveness of allogeneic
stem cell transplantation in children are
predictable. Biol Blood Marrow Transplant.
18(10), 1533–9.
Matutes, E., Pickl, W.F., Van’t Veer, M.,
Morilla, R., Swansbury, J., Strobl, H.,
Attarbaschi, A., Hopfinger, G., Ashley, S.,
Bene, M.C., Porwit, A., Orfao, A., Lemez, P.,
Schabath, R. & Ludwig, W.D. (2011).
Mixed-phenotype acute leukemia: clinical
and laboratory features and outcome in
100 patients defined according to the WHO
2008 classification. Blood, 117(11), 3163–71.
Mikkelsen, T.S., Thorn, C.F., Yang, J.J.,
Ulrich, C.M., French, D., Zaza, G.,
Dunnenberger, H.M., Marsh, S., McLeod, H.L.,
Giacomini, K., Becker, M.L., Gaedigk, R.,
Leeder, J.S., Kager, L., Relling, M.V., Evans, W.,
Klein, T.E. & Altman, R.B. (2011). PharmGKB
summary: methotrexate pathway.
Pharmacogenet Genomics. 21(10), 679–86.
Morak, M., Attarbaschi, A., Fischer, S.,
Nassimbeni, C., Grausenburger, R.,
Bastelberger, S., Krentz, S., Cario, G.,
Kasper, D., Schmitt, K., Russell, L.J.,
Pötschger, U., Stanulla, M., Eckert, C.,
Mann, G., Haas, O.A. & Panzer-Grümayer, R.
(2012). Small sizes and indolent evolutionary
dynamics challenge the potential role of
P2RY8-CRLF2-harborin g clones as main
relapse-driving force in childhood ALL.
Blood. 120(26), 5134–42.
104–105
Fuka, G., Kauer, M., Kofler, R., Haas, O.A.
& Panzer-Grümayer, R. (2011). The
leukemia-specific fusion gene ETV6/RUNX1
perturbs distinct key biological functions
primarily by gene repression. PLoS One.
6(10), e26348.
Fazekas, T., Eickhoff, P., Rauch, M., Verdianz, M.,
Attarbaschi, A., Dworzak, M., Peters, C.,
Hammer, K., Vecsei, A., Pötschger, U.
& Lion, T. (2012). Prevalence and clinical
course of viral upper respiratory tract
infections in immunocompromised pediatric
patients with malignancies or after
hematopoietic stem cell transplantation.
J Pediatr Hematol Oncol. 34(6), 442–9.
Nebral, K., Krehan, D., & Strehl, S. (2011).
Expression of PAX5 splice variants: a
phenomenon of stress-induced, illegitimate
splicing? Br J Haematol, 155(2), 277–280.
Passweg, J.R., Baldomero, H., Gratwohl, A.,
Bregni, M., Cesaro, S., Dreger, P., de Witte, T.,
Farge-Bancel, D., Gaspar, B., Marsh, J.,
Mohty, M., Peters, C., Tichelli, A., Velardi, A.,
de Elvira, C.R., Falkenburg, F., Sureda, A.,
Madrigal, A. & European Group for B,
Marrow T. (2012). The EBMT activity survey:
1990–2010. Bone Marrow Transplant,
47(7), 906–23.
Pellett, P.E., Ablashi, D.V., Ambros, P.F.,
Agut, H., Caserta, M.T., Descamps, V.,
Flamand, L., Gautheret-Dejean, A.,
Hall, C.B., Kamble, R.T., Kuehl, U., Lassner, D.,
Lautenschlager, I., Loomis, K.S., Luppi, M.,
Lusso, P., Medveczky, P.G., Montoya, J.G.,
Mori, Y., Ogata, M., Pritchett, J.C., Rogez, S.,
Seto, E., Ward, K.N., Yoshikawa, T.
& Razonable, R.R. (2012). Chromosomally
integrated human herpesvirus 6: questions
and answers. Rev Med Virol. 22(3), 144–55.
Peters C. (2012). The EBMT Paediatric
Disease Working Party: Current Concepts
and Future Aims. memo – Magazine of
European Medical Oncology. 2(3), 178–181.
Rubie, H., De Bernardi, B., Gerrard, M.,
Canete, A., Ladenstein, R., Couturier, J.,
Ambros, P., Munzer, C., Pearson, A.D.,
Garaventa, A., Brock, P., Castel, V.,
Valteau-Couanet, D., Holmes, K., Di Cataldo, A.,
Brichard, B., Mosseri, V., Marquez, C.,
Plantaz, D., Boni, L. & Michon, J. (2011).
Excellent outcome with reduced treatment in
infants with nonmetastatic and unresectable
neuroblastoma without MYCN amplification:
results of the prospective INES 99.1.
J Clin Oncol. 29(4), 449–55.
Postel-Vinay, S., Veron, A.S., Tirode, F.,
Pierron, G., Reynaud, S., Kovar, H., Oberlin, O.,
Lapouble, E., Ballet, S., Lucchesi, C.,
Kontny, U., González-Neira, A., Picci, P.,
Alonso, J., Patino-Garcia, A.,
de Paillerets, B.B., Laud, K., Dina, C., Froguel, P.,
Clavel-Chapelon, F., Doz, F., Michon, J.,
Chanock, S.J., Thomas, G., Cox, D.G.
& Delattre, O. (2012). Common variants
near TARDBP and EGR2 are associated
with susceptibility to Ewing sarcoma.
Nature genetics, 44(3), 323–7.
Ruggeri, A., Michel, G., Dalle, J.H., Caniglia, M.,
Locatelli, F., Campos, A., de Heredia, C.D.,
Mohty, M., Hurtado, J.M., Bierings, M.,
Bittencourt, H., Mauad, M., Purtill, D.,
Cunha, R., Kabbara, N., Gluckman, E.,
Labopin, M., Peters, C. & Rocha, V. (2012).
Impact of pretransplant minimal residual
disease after cord blood transplantation
for childhood acute lymphoblastic leukemia
in remission: an Eurocord, PDWP-EBMT
analysis. Leukemia. 26(12), 2455–61.
Preuner, S., Mitterbauer, G., Mannhalter, C.,
Herndlhofer, S., Sperr, W.R., Valent, P.
& Lion, T. (2012). Quantitative monitoring
of BCR/ABL1 mutants for surveillance
of subclone-evolution, -expansion, and
-depletion in chronic myeloid leukaemia.
Eur J Cancer. 48(2), 233–6.
Pulverer, W., Wielscher, M.,
Panzer-Grümayer, R., Plessl, T., Kriegner, A.,
Vierlinger, K. & Weinhäusel, A. (2012). The
stem cell signature of CHH/CHG methylation
is not present in 271 cancer associated 5’UTR
gene regions. Biochimie. 94(11), 2345–52
Roos-Weil, D., Dietrich, S., Boumendil, A.,
Polge, E., Bron, D., Carreras, E.,
Iriondo Atienza, A., Arcese, W., Beelen, D.W.,
Cornelissen, J.J., Kroger, N., Milone, G.,
Rossi, G., Jardin, F., Peters, C., Rocha, V.,
Sureda, A., Mohty, M. & Dreger, P. (2012).
Stem cell transplantation can provide
durable disease control in Blastic
plasmacytoid dendritic cell neoplasm
(BPDCN). Blood. Epub 2012 Nov 30.
Ruperto, N., Eichler, I., Herold, R., Vassal, G.,
Giaquinto, C., Hjorth, L., Valls-i-Soler, A.,
Peters, C., Helms, P.J. & Saint Raymond, A.
(2012). A European Network of Paediatric
Research at the European Medicines Agency
(Enpr-EMA). Arch Dis Child. 97(3), 185–8.
Salzer, E., Daschley, S., Choo, S., Gombert, M.,
Valente, E., Ginzel, S., Schwendinger, M.,
Haas, O.A., Fritsch, G., Pickl, W.F.,
Borkhardt, A., Boztug, K., Bienemann, K.
& Seidel, M.G. (2012). Combined
Immunodeficiency with Life-Threatening
EBV-Associated Lymphoproliferative
Disorder in Patients Lacking Functional CD27.
Haematologica. (Epub ahead of print).
Schleiermacher, G., Michon, J., Ribero, A.,
Pierron, G., Mosseri, V., Rubie, H., Munzer, C.,
Benard, J., Auger, N., Combaret, V., JanoueixLerosey, I., Pearson, A., Tweddle, D.A.,
Brown, N., Gerrard, M., Wheeler, K., Noguer, R.,
Villamon, E., Canete, A., Castel, V.,
Marques, B., De Lacerda, A., Tonini, G.P.,
Mazzocco, K., Defferrari, R., De Bernardi, B.,
Di Cataldo, A., Van Roy, N., Brichard, B.,
Ladenstein, R., Ambros, I., Ambros, P.,
Beiske, K., Delattre, O. & Couturier, J. (2011).
Segmental chromosomal alterations lead
to a higher risk of relapse in infants with
MYCN-non amplified localised unresectable/
disseminated neuroblastoma (a SIOPEN
collaborative study). British Journal of
Cancer. 105(12), 1940–8.
Schleiermacher, G., Mosseri, V., London, W.B.,
Maris, J.M., Brodeur, G.M., Attiyeh, E.,
Haber, M., Khan, J., Nakagawara, A.,
Speleman, F., Noguera, R., Tonini, G.P.,
Fischer, M., Ambros, I., Monclair, T.,
Matthay, K.K., Ambros, P., Cohn, S.L. &
Pearson A.D. (2012). Segmental chromosomal alterations have prognostic impact
in neuroblastoma: a report from the INRG
project. Br J Cancer. 107(8), 1418–22.
Schmidt, W.M., Uddin, M.H., Dysek, S.,
Moser-Thier, K., Pirker, C., Höger, H.,
Ambros, I.M., Ambros, P.F., Berger, W. &
Bittner, R.E. (2011). DNA damage,
somatic aneuploidy, and malignant sarcoma
susceptibility in muscular dystrophies.
PLoS Genet. 7(4), e1002042.
Schrappe, M., Valsecchi, M.G., Bartram, C.R.,
Schrauder, A., Panzer-Grümayer, R.,
Möricke, A., Parasole, R., Zimmermann, M.,
Dworzak, M., Buldini, B., Reiter, A., Basso, G.,
Klingebiel, T., Messina, C., Ratei, R.,
Cazzaniga, G., Koehler, R., Locatelli, F.,
Schäfer, B.W., Aricò, M., Welte, K.,
van Dongen, J.J., Gadner, H., Biondi, A.
& Conter, V. (2011). Late MRD response
determines relapse risk overall and in
subsets of childhood T-cell ALL: results
of the AIEOP-BFM-ALL 2000 study.
Blood. 118(8), 2077–84.
Seidel, M.G., Rami, B., Item, C., Schober, E.,
Zeitlhofer, P., Huber, W.D., Heitger, A.,
Bodamer, O.A. & Haas O.A. (2012).
Concurrent FOXP3- and CTLA4-associated
genetic predisposition and skewed
X chromosome inactivation in an
autoimmune disease-prone family.
Eur J Endocrinol. 167(1), 131–4.
Seto, D., Chodosh, J., Brister, J.R., Jones, M.S.
& Members of the Adenovirus Research
Community. (2011). Using the whole-genome
sequence to characterize and name human
adenoviruses. J Virol. 85(11), 5701–2.
Snowden, J.A., Saccardi, R., Allez, M.,
Ardizzone, S., Arnold, R., Cervera, R.,
Denton, C., Hawkey, C., Labopin, M.,
Mancardi, G., Martin, R., Moore, J.J.,
Passweg, J., Peters, C., Rabusin, M.,
Rovira, M., van Laar, J.M., Farge, D.,
Party EADW & Paediatric Diseases Working
P. (2012). Haematopoietic SCT in severe
autoimmune diseases: updated guidelines
of the European Group for Blood and Marrow
Transplantation. Bone Marrow Transplant.
47(6), 770–90.
Steiner, M., Hochreiter, D., Kasper, D.C.,
Kornmuller, R., Pichler, H., Haas, O.A.,
Potschger, U., Hutter, C., Dworzak, M.N.,
Mann, G. & Attarbaschi, A. (2012). Asparagine
and aspartic acid concentrations in bone
marrow versus peripheral blood during
Berlin-Frankfurt-Munster-based induction
therapy for childhood acute lymphoblastic
leukemia. Leuk Lymphoma. (Research
Support, Non-U.S. Gov’t). 53(9), 1682–7.
Strehl, S., König, M., Boztug, H., Cooper, B. W.,
Suzukawa, K., Zhang, S. J., Chen, H. Y.,
Attarbaschi, A., & Dworzak, M. N. (2012).
All-trans retinoic acid and arsenic trioxide
resistance of acute promyelocytic leukemia
with the variant STAT5B-RARA fusion gene.
Leukemia. 2012 Dec 28. (Epub ahead of print).
Styczynski, J., Balduzzi, A., Gil, L.,
Labopin, M., Hamladji, R.M., Marktel, S.,
Yesilipek, M.A., Fagioli, F., Ehlert, K.,
Matulova, M., Dalle, J.H., Wachowiak, J.,
Miano, M., Messina, C., Diaz, M.A.,
Vermylen, C., Eyrich, M., Badell, I.,
Dreger, P., Gozdzik, J., Hutt, D., Rascon, J.,
Dini, G., Peters, C., European Group for B,
Marrow Transplantation & Pediatric Diseases
Working P. (2012). Risk of complications
during hematopoietic stem cell collection
in pediatric sibling donors: a prospective
European Group for Blood and Marrow
Transplantation Pediatric Diseases Working
Party study. Blood. 119(12), 2935–42.
Szczepanski, T., van der Velden, V.H.,
Waanders, E., Kuiper, R.P., Van Vlierberghe, P.,
Gruhn, B., Eckert, C., Panzer-Grümayer, R.,
Basso, G., Cavé, H., Stadt, U.Z., Campana, D.,
Schrauder, A., Sutton, R., van Wering, E.,
Meijerink, J.P. & van Dongen J.J. (2011).
Late recurrence of childhood T-cell acute
lymphoblastic leukemia frequently
represents a second leukemia rather than
a relapse: first evidence for genetic
predisposition. J Clin Oncol. 29(12), 1643–9.
Taggart, D.R., London, W.B., Schmidt, M.L.,
DuBois, S.G., Monclair, T.F., Nakagawara, A.,
De Bernardi, B., Ambros, P.F., Pearson, A.D.,
Cohn, S.L. & Matthay K.K. (2011). Prognostic
value of the stage 4S metastatic pattern
and tumor biology in patients with metastatic
neuroblastoma diagnosed between birth
and 18 months of age. J Clin Oncol. 29(33),
4358–64.
106–107
Petzer, A.L., Fong, D., Lion, T., Dyagil, I.,
Masliak, Z., Bogdanovic, A., Griskevicius, L.,
Lejniece, S., Goranov, S., Gercheva, L.,
Stojanovic, A., Peytchev, D., Tzvetkov, N.,
Griniute, R., Stanchev, A., Grubinger, T.,
Kwakkelstein, M., Schuld, P., Gastl, G.
& Wolf D. (2012). High-dose imatinib
induction followed by standard-dose main
tenance in pre-treated chronic phase chronic
myeloid leukemia patients-final analysis
of a randomized, multicenter, phase III trial.
Haematologica. 97(10), 1562–9.
Moroz, V., Machin, D., Faldum, A., Hero, B.,
Iehara, T., Mosseri, V., Ladenstein, R.,
De Bernardi, B., Rubie, H., Berthold, F.,
Matthay, K.K., Monclair, T., Ambros, P.F.,
Pearson, A.D., Cohn, S.L. & London W.B.
(2011). Changes over three decades in
outcome and the prognostic influence
of age-at-diagnosis in young patients
with neuroblastoma: a report from the
International Neuroblastoma Risk Group
Project. Eur J Cancer. 47(4), 561–71.
Thiel, U., Wolf, P., Wawer, A., Blaeschke, F.,
Grunewald, T. G., von Lüttichau, I. T.,
Klingebiel, T., Bader, P., Borkhardt, A.,
Laws, H. J., Handgretinger, R., Lang, P.,
Schlegel, P. G., Eyrich, M., Gruhn, B.,
Ehninger, G., Koscielniak, E., Klein, C.,
Sykora, K. W., Holler, E., Mauz-Körholz, C.,
Woessmann, W., Richter, G. H., Schmidt, A. H.,
Peters, C., Dirksen, U., Jürgens, H., Bregni, M.
& Burdach, S. (2012). Human Leukocyte
Antigen Distribution in German Caucasians
with Advanced Ewing’s Sarcoma. Klin Padiatr.
224(6), 353–8.
Valent, P., Gastl, G., Geissler, K., Greil, R.,
Hantschel, O., Lang, A., Linkesch, W., Lion, T.,
Petzer, A. L., Pittermann, E., Pleyer, L.,
Thaler, J., Wolf, D. (2012). Nilotinib as frontline
and second-line therapy in chronic myeloid
leukemia: open questions. Crit Rev Oncol
Hematol. 82(3), 370–7.
Veal, G.J., Nguyen, L., Paci, A., Riggi, M.,
Amiel, M., Valteau-Couanet, D., Brock, P.,
Ladenstein, R. & Vassal, G. (2012).
Busulfan pharmacokinetics following
intravenous and oral dosing regimens in
children receiving high-dose myeloablative
chemotherapy for high-risk neuroblastoma
as part of the HR-NBL-1/SIOPEN trial.
Eur J Cancer. 48(16), 3063–72.
Wachowiak, J., Sykora, K.W., Cornish, J.,
Chybicka, A., Kowalczyk, J.R., Gorczynska, E.,
Choma, M., Grund, G., Peters, C. & EBMT
Pediatric Diseases Working Party (2011).
Treosulfan-based preparative regimens
for allo-HSCT in childhood hematological
malignancies: a retrospective study on behalf
of the EBMT pediatric diseases working party.
Bone Marrow Transplant. 46(12), 1510–8.
Warsch, W., Kollmann, K., Eckelhart, E.,
Fajmann, S., Cerny-Reiterer, S., Hölbl, A.,
Gleixner, K.V., Dworzak, M., Mayerhofer, M.,
Hoermann, G., Herrmann, H., Sillaber, C.,
Egger, G., Valent, P., Moriggl, R. & Sexl V. (2011).
High STAT5 levels mediate imatinib
resistance and indicate disease
progression in chronic myeloid leukemia.
Blood. 117(12), 3409–20.
Winter, G. E., Rix, U., Lissat, A., Stukalov, A.,
Müllner, M. K., Bennett, K. L., Colinge, J.,
Nijman, S. M., Kubicek, S., Kovar, H.,
Kontny, U. & Superti-Furga, G. (2011).
An integrated chemical proteomic
approach identifies specific vulnerability
of Ewing’s sarcoma to combined inhibition
of Aurora kinases A and B. Mol. Cancer
Therapeutics, 10(10), 1846–56.
Witt, V., Beigelböck, E. & Fritsch, G. (2011).
Bone marrow processing with the AMICUSTM
separator system. J Clin Apher. 26(4), 195–9.
Wlodarski, M.W., Mötter, J., Gorr, T.A.,
Olk-Batz, C., Hasle, H., Dworzak, M.,
Niemeyer, C.M. & Flotho, C. (2011). Abnormal
promoter DNA methylation in juvenile
myelomonocytic leukemia is not caused by
mutation in DNMT3A. Blood. 118, 4490–1.
Zimmermann, C., Potschger, U., Amann, G.,
Horcher, E., Dieckmann, K., Lakatos, K.,
Urban, C., Lackner, H., Höllwarth, M.,
Meister, B., Crazzolara, R., Ebetsberger, G.,
Fink, F., Jones, N., Gamper, A., Moser, R.,
Kerbl, R., Jauk, B., Pobegen, W., Henkel, M.,
Ausserer, B., Friesenbichler, W., Böhm, J.,
Graf, N., Holter, W., Gadner, H., Zoubek, A.,
Kager, L. Results of children with renal
tumors treated on the Austrian-Hungarian
Wilms Tumor Study 1989 and the
international Society of Pediatric Oncology
SIOP 93-01/GPOH trial in Austria.
Memo – Magazine of European Medical
Oncology. 5(4), 289–295.
108–109
Van der Burg, M., Kreyenberg, H., Willasch, A.,
Barendregt, B.H., Preuner, S., Watzinger, F.,
Lion, T., Roosnek, E., Harvey, J., Alcoceba, M.,
Díaz, M. G., Bader, P. & van Dongen, J.J.;
EU-supported EuroChimerism Consortium
project QLRT-2001–01485 (2011). Standardization of DNA isolation from low cell numbers
for chimerism analysis by PCR of short
tandem repeats. Leukemia. 25(9), 1467–70.
Thiel, U., Wawer, A., Wolf, P., Badoglio, M.,
Santucci, A., Klingebiel, T., Basu, O.,
Borkhardt, A., Laws, H.J., Kodera, Y.,
Yoshimi, A., Peters, C., Ladenstein, R.,
Pession, A., Prete, A., Urban, E.C.,
Schwinger, W., Bordigoni, P., Salmon, A.,
Diaz, M.A., Afanasyev, B., Lisukov, I.,
Morozova, E., Toren, A., Bielorai, B.,
Korsakas, J., Fagioli, F., Caselli, D.,
Ehninger, G., Gruhn, B., Dirksen, U., AbdelRahman, F., Aglietta, M., Mastrodicasa, E.,
Torrent, M., Corradini, P., Demeocq, F., Dini, G.,
Dreger, P., Eyrich, M., Gozdzik, J., Guilhot, F.,
Holler, E., Koscielniak, E., Messina, C.,
Nachbaur, D., Sabbatini, R., Oldani, E.,
Ottinger, H., Ozsahin, H., Schot,s R., Siena, S.,
Stein, J., Sufliarska, S., Unal, A., Ussowicz, M.,
Schneider, P., Woessmann, W., Jurgens, H.,
Bregni, M. & Burdach. S. (2011). No improvement of survival with reduced-versus highintensity conditioning for allogeneic stem
cell transplants in Ewing tumor patients.
Ann Oncol. 22(7), 1614–21.
Publisher
St. Anna Kinderkrebsforschung e.V.
Zimmermannplatz 10, A-1090 Vienna
www.ccri.at
Herausgeber
Zimmermannplatz 10, A-1090 Vienna
www.ccri.at
Contact
Marion Zavadil
CCRI Secretariat
[email protected]
Kontakt
Marion Zavadil
CCRI Sekretariat
[email protected]
We would like to express our gratitude to
the numerous private donors who have
been loyal in their support for many years,
as well as the following national and inter
national funding bodies and organisations:
EU 7th Research Programme (FP7)
Austrian Science Fund (FWF)
Austrian Research Promotion Agency (FFG)
Responsibility for
scientific content
Heinrich Kovar
Scientific Director
[email protected]
Verantwortlich für den
wissenschaftlichen Inhalt
Heinrich Kovar
Scientific Director
[email protected]
Text Editor & Project Management
Lisa Glenk
Science Communication
[email protected]
[email protected]
Redaktion & Projektmanagement
Lisa Glenk
Science Communication
[email protected]
[email protected]
Vienna Science and Technology Fund (WWTF)
Federal Ministry of Health
Austrian National Bank (OeNB)
Austrian Academy of Sciences
Art Direction & Design
Lichtwitz Leinfellner visuelle Kultur KG
Konzeption & Gestaltung
Lichtwitz Leinfellner visuelle Kultur KG
Photography
Gerhard Wasserbauer
Fotografie
Gerhard Wasserbauer
Print
Ueberreuter Print GmbH
Paper
Maxioffset 120/130 g
Font
Omnes Pro
Druck
Ueberreuter Print GmbH
Papier
Maxioffset 120/130 g
Schrift
Omnes Pro
SIOPEN Association (Society of
Paediatric Oncology European
Neuroblastoma Network)
Liddy Shirver Sarcoma Initiative
EMBT (European Group for Blood
and Marrow Transplantation)
Wilhelm Sander Foundation, Munich
Umbrella organisation of the
Austrian Childhood Cancer Aid
Working Party Science and Research
of the Austrian Society for Paediatrics,
Section: Haematological-Oncological
Research
Medac Gesellschaft für klinische
Spezialpräparate GmbH, Germany
Download of the Scientific
Report as pdf file:
http://science.ccri.at
Download des Forschungsberichtes
als PDF-Datei:
DKMS, Germany
Support
Unterstützung
Wir möchten uns gerne an dieser Stelle
bei unseren zahlreichen privaten Spendern
und Spenderinnen bedanken, die uns
seit vielen Jahren treu unterstützen.
Des Weiteren bedanken wir uns bei den
folgenden nationalen und internationalen
Fördergebern und Organisationen:
7. Forschungsrahmenprogramm der
Europäischen Kommission (FP7)
Fonds zur Förderung der wissenschaftlichen
Forschung (FWF)
Bank Account
Bank Austria, BLZ 12000
Konto: 00 656 166 600
IBAN: AT 79 12000 00656166600
BIC: BKAUATWW
Further details:
Page 92–93
www.ccri.at
Österreichische Forschungsförderungsgesellschaft (FFG)
Wiener Wissenschafts-, Forschungsund Technologiefonds (WWTF)
Bundesministerium für Wissenschaft
und Forschung (BMWF)
Österreichische Nationalbank (OeNB)
Bankverbindung
Bank Austria, BLZ 12000
Konto: 00 656 166 600
IBAN: AT 79 12000 00656166600
BIC: BKAUATWW
Akademie der Wissenschaften
SIOPEN Association (Internationale Gesell.
des Europäischen pädiatrisch-onkolog.
Neuroblastom-Forschungsnetzwerks)
Liddy Shirver Sarcoma Initiative
EMBT (Europäische Gruppe für
Blut- und Knohenmarktransplantation)
Wilhelm Sander Foundation, München
Dachverband der österr. Kinderkrebshilfe
AG Wissenschaft und Forschung der
Österr. Gesell. für Kinder- und Jugend
heilkunde ÖGKJ im Bereich der hämatoonkolog. Forschung – Arbeitsgruppe für
Wissenschaft und Forschung der Österr.
Gesell. für Kinder- und Jugendheilkunde,
Sektion: Hämatolog.-onkolog. Forschung
Medac Gesellschaft für klinische
Spezialpräparate GmbH, Deutschland
DKMS, Deutschland
Weitere Details:
Seite 92–93
www.ccri.at
110–111
Acknowledgements
Danksagung
Imprint
Impressum
Reflections on the past 25 years of children’s cancer research
point to a promising future: However, there are still many open
questions that still need to be answered.
Die Rückschau auf 25 Jahre Kinderkrebsforschung lässt uns
hoffnungsvoll in die Zukunft blicken: Aber es sind auch noch
sehr viele Fragen offen.
www.ccri.at

- Children`s Cancer Research Institute

Transcription

Similar documents

Anatomically shaped foil for hair coloring

Swiss Food Tech Day, Photonics 4 Food

(Das) Werk Band (Jahr): 61 (1974) - E

1512-1594 Gerardus Mercator, or Gerard Kremer as he was called

Johann Sebastian Bach`s Performance Environment

mancher Wortarten (Adverbien, Präpositionen), die Syntax

LOS Sekundar Issue No. 4 - German

Advance by Satellite-System

Booklett 21078

magazine by Alfabet students • June, 2014