the netherlands cancer institute scientific annual report ... - NKI / AvL

THE
NETHERLANDS
CANCER
INSTITUTE
SCIENTIFIC
ANNUAL
REPORT 2010

SCIENTIFIC ANNUAL REPORT 2010

4
Patron HM Queen Beatrix

SCIENTIFIC ANNUAL REPORT 2010
THE NETHERLANDS CANCER INSTITUTE
CANCER RESEARCH LABORATORY AND CANCER HOSPITAL
www.nki.nl
5

6copyright
COPYRIGHT
Scientifi c Annual Report 2010
Illustrations and unpublished data in these reports may not be
used without permission of the author.
Copyright ©
The Netherlands Cancer Institute
Antoni van Leeuwenhoek Hospital
Plesmanlaan 121
1066 CX Amsterdam
The Netherlands
Phone: +31 20 512 9111
Fax: +31 20 617 2625
ISSN 1387-8611

CONTENTS
Board Members 9
Research Divisions 10
Introduction 12
Education in Oncology 16
Division of Biochemistry 18
Division of Cell Biology I 22
Division of Cell Biology II 29
Division of Experimental Therapy 36
Division of Gene Regulation 47
Division of Division of Immunology 53
Division of Molecular Biology 61
Division of Molecular Carcinogenesis 73
Division of Molecular Genetics 79
Division of Psychosocial Research and Epidemiology 88
Division of Diagnostic Oncology 99
Division of Medical Oncology 114
Division of Radiotherapy 124
Division of Surgical Oncology 147
Biometrics Department 162
Clinical Trials 167
Invited Speakers 182
Projects 184
Personnel Index 202
7 contents

8colophon
COLOPHON
Coordinators
Suzanne Corsetto
Henri van Luenen
Monique Duyndam
Photograph HM The Queen Beatrix
Enquiries/permission:
Rijksvoorlichtingsdienst, afd Pers en Publiciteit/Foto
Postbus 20009
2500 EA Den Haag
Photo Ruud Taal/Capital Press
Copyright RVD
Photograph AJM Berns: Loek Zuijderduin
Other photographs and illustrations:
Audiovisual services
The Netherlands Cancer Institute/
Antoni van Leeuwenhoek Hospital
Printed by Drukkerij Badoux, Houten

BOARD MEMBERS
INTERNATIONAL SCIENTIFIC ADVISORY
BOARD
T De Lange, Leon Hess Professor, The Rockefeller
University, New York, USA
RA Flavell, Professor of Immunobiology, Yale
University School of Medicine, New Haven, USA
WGJ Hol, Professor of Biochemistry and Biological
Structure, University of Washington, Seattle, USA
J Mendelsohn, President MD Anderson Cancer Center,
University of Texas, Houston, USA
P Nurse, Professor of Microbiology, President of
The Rockefeller University, New York, USA
R Nusse, Professor of Developmental Biology, Stanford
University, Stanford, USA
HL Ploegh, Professor of Biology, Whitehead Institute
for Biomedical Research, Cambridge, USA
S Powell, Chairman, Department of Radiation Oncology,
Memorial Sloan-Kettering Cancer Center, New York,
USA
K Vousden, Director, Beatson Institute for Cancer
Research, Glasgow, UK
RA Weinberg, Professor of Biology, Massachusetts
Institute of Technology, Whitehead Institute for
Biomedical Research, Cambridge, USA
NATIONAL SCIENTIFIC ADVISORY BOARD
DD Breimer, Professor of Pharmacology, Leiden
University
JC Clevers, Professor of Clinical Immunology,
Hubrecht Institute, Utrecht
EGE De Vries, Professor of Medical Oncology,
University of Groningen
JHF Falkenburg, Professor of Experimental
Hematology, Leiden University
CG Figdor, Professor of Experimental Immunology,
Radboud University Nijmegen
JHJ Hoeijmakers, Professor of Molecular Genetics,
Erasmus University Rotterdam
P Lambin, Professor of Radiation Oncology, Maastricht
University
RH Medema, Professor of Experimental Oncology,
Utrecht University
CJH Van de Velde, Professor of Surgical Oncology,
Leiden University
President of Board of Governors W Kok
9
board members
BOARD OF DIRECTORS
AJM Berns, chairman and director of research
S Rodenhuis, director clinical research and development
WH Van Harten, director organization and management
BOARD OF GOVERNORS
W Kok, president
HCJ Van der Wielen, vice president
T De Swaan, treasurer
GH Blijham
FH Schröder
MC Smeets
EH Swaab
PC Van der Vliet
MJ Van Mourik
SCIENTIFIC ADVISORY COUNCIL
AJM Berns, chairman
JJ Neefjes, secretary
S Rodenhuis
B Van Steensel
M Verheij (until September 2010)
T Ruers (from September 2010)
J Borst (until December 2010)
J Jonkers (from December 2010)

10
research divisions
RESEARCH DIVISIONS
Biochemistry
Titia Sixma (head)
Anastassis Perrakis
Caroline Kapper (offi ce manager)
Cell Biology I
Arnoud Sonnenberg (head)
Wim van Blitterswijk
John Collard
Metello Innocenti
Kees Jalink
Wouter Moolenaar
Ed Roos
Patty Lagerweij (offi ce manager)
Cell Biology II
Jacques Neefjes (head)
Rob Michalides
Huib Ovaa
Peter Peters
Marieke van der Velde (offi ce manager)
Experimental Therapy
Adrian Begg (head)
Jan Schellens
Fiona Stewart
Marcel Verheij
Jelle Wesseling
Thea Eggenhuizen (offi ce manager)
Gene Regulation
Reuven Agami (head)
Jan-Hermen Dannenberg
Maarten Fornerod
Fred van Leeuwen
Bas van Steensel
Suzanne Corsetto (offi ce manager)
Immunology
Jannie Borst (head)
Christian Blank
John Haanen
Heinz Jacobs
Ton Schumacher
Florry Vyth-Dreese
José Overwater (offi ce manager)
Molecular Biology
Hein Te Riele (head)
Piet Borst (honorary staff member)
Jos Jonkers
Sabine Linn
Alfred Schinkel
Lodewyk Wessels
Tom de Knegt (offi ce manager)
Linda Römer (secretary)
Molecular Carcinogenesis
René Bernards (head)
Roderick Beijersbergen
Rob Wolthuis
Molecular Genetics
Maarten van Lohuizen (head)
Anton Berns
Jacqueline Jacobs
Anna-Pavlina Haramis
John Hilkens
Paul Krimpenfort
Daniel Peeper
Margriet Snoek
Erica Delwel (offi ce manager)
Marie Anne van Halem (secretary)
Psychosocial Research and Epidemiology
Flora van Leeuwen (head)
Neil Aaronson
Eveline Bleiker
Wim van Harten
Matti Rookus
Sanne Schagen
Marjanka Schmidt
Yvonne Driessen-Ruwaard
(offi ce manager)
Diagnostic Oncology
Laura van ’t Veer (head)
Tanja Alderliesten
Priscilla Axwijk
Philippe Baars
Olga Balague Ponz
Peter Besnard
Hans Bonfrer
Daphne de Jong
Kenneth Gilhuijs
Cees Hoefnagel
Frans Hogervorst
Irma Kluijt
Wim Koops
Robert Kröger
Charlotte Lange
Claudette Loo
Saar Muller
Petra Nederlof
Willem Nooijen
Frank Pameijer
Renée van Pel
Warner Prevoo
Marc Roef
Efraim Rosenberg
Marielle Ruijs
Michiel Sinaasappel
Ferida Sivro
Marcel Stokkel
Jelle Teertstra
Renato Valdes Olmos
Hester van Boven
Fijs van Leeuwen
Olaf van Tellingen
Loes van Velthuysen
Mark van de Vijver
Lizet van der Kolk
Senno Verhoef
Wouter Vogel
Jelle Wesseling
Bart van de Wiel
Christine Arkes (secretary)
Carla van Tiggelen (secretary)
Medical Oncology
John Haanen (head)
Joke Baars
Paul Baas
Jos Beijnen
André Bergman
Christian Blank
Jan Paul de Boer
Willem Boogerd
Henk Boot
Dieta Brandsma
Wieneke Buikhuisen
Sjaak Burgers
Annemieke Cats
Leo Gualtherie van Weezel
Michel van den Heuvel
Alwin Huitema
Martijn Kerst
Sabine Linn
Anne Lukas
Serena Marchetti
Sjoerd Rodenhuis
Jan Schellens
Gabe Sonke
Neeltje Steeghs
Babs Taal
Margot Tesselaar
Marchien van der Weide
Mariëlle de Kwant (secretary)
Karin van Leuveren (secretary)
Radiotherapy
Marcel Verheij (head)
Berthe Aleman
Harry Bartelink
José Belderbos
Monique Bloemers
Eugène Damen
Roel de Boer
Luc Dewit
Paula Elkhuizen
Rick Haas
Olga Hamming-Vrieze
Wilma Heemsbergen

Frank Hoebers
Edwin Jansen
Joost Knegjens
Han Krewinkel
Joos Lebesque
Ben Mijnheer
Luc Moonen
Arash Navran
Heike Peulen
Floris Pos
Coen Rasch
Babs Reichgelt
Peter Remeijer
Nicola Russell
Govert Salverda
Christoph Schneider
Jan-Jakob Sonke
Joep Stroom
Marcel van Herk
Baukelien van Triest
Karijn Verschueren
Corine van Vliet-Vroegindeweij
Marieke van Zwienen
Thelma Witteveen
Frits Wittkämper
Patricia Haye-Fewer
(section coordinator)
Surgical oncology
Theo Ruers (head)
Marc van Beurden
Michiel van den Brekel
Alfons Balm
Annemieke Ackerstaff
Arend Aalbers
Axel Bex
Biljana Zupan-Kajcovski
Charlotte Zuur
Dirk Buitelaar
Emiel Rutgers
Frans Hilgers
Frits van Coevorden
Gemma Kenter
Henk van de Poel
Hester Oldenburg
Houke Klomp
Bing Tan
Ingeborg Vergouwe
Inka Nieuweboer-Krobotova
Joris Hage
Johanna van Sandick
Jos van der Hage
Julia ten Cate
Katina Efthymiou
Leonie Woerdeman
Lotti Lubsen-Brandsma
Ludi Smeele
Marianne Piek-den Hartog
Marie-Jeanne Baas-Vrancken Peeters
Marieke van der Berg
Martine van Huizum
May Ronday
Michael Srámek
Michel Wouters
Omgo Nieweg
Peter Schutte
Peter Lohuis
Petra Biewenga
Simon Horenblas
Vic Verwaal
Wietze van der Veen
Willemien van Driel
Wim Meinhardt
Annemieke Hoogland
(offi ce manager)
Biometrics department
Otilia Dalesio
Financial Administration
Frieda Boekweit
General Facilities, ICT and Personnel
Department
Eric de Wilde
General Research Coordination
Jacques Neefjes, deputy scientifi c
director
Henri van Luenen, director of
operations
11
research divisions

12
introduction
INTRODUCTION
I am pleased to present our Scientifi c Annual Report 2010.
It provides an overview of the scientifi c activities at The
Netherlands Cancer Institute - Antoni van Leeuwenhoek
Hospital (NKI-AVL). Additional information can be found
at www.nki.nl. A lucid general overview of our activities
with illustrations can be found in our brochure (available
on our website).
The NKI-AVL is a Comprehensive Cancer Centre,
combining hospital and research laboratories under one
roof in a single independent organization. The hospital
comprises 180 beds, an outpatient clinic and a large
radiotherapy department. Facilities for clinical research
include a large patient database, clinical data management,
extensive diagnostic facilities, a pharmacy with a
production unit for experimental drugs, and active research
groups in medical, surgical and diagnostic oncology,
radiotherapy, pharmacy, epidemiology and psychosocial
oncology. The laboratory covers all major areas of cancer
research, with special emphasis on cell-based screens,
mouse tumour models, cell biology, structural biology, and
immunology. Translational research is an integral activity
of many research groups and is fostered by collaborations
between clinical and basic scientists.
Clinical activities showed again substantial growth in 2010
facilitated by a new temporary outpatient clinic that became
operational in January. In the course of 2011 we will decide
on the further expansion of the hospital so that a previously
planned 50% growth can be accommodated. We also try
to accommodate a national children oncology hospital
on the premises of the NKI-AVL. Earlier in the year an
independent committee installed by the Dutch organisation
of paediatric oncologists and parent organisation of
children with cancer selected the NKI-AVL as the preferred
location for such a specialised hospital. It will become
clear in the course of 2011 whether the building of this
hospital close to the NKI-AVL can be realised. Execution
of the plan to establish satellite radiotherapy treatment
centres near two other hospitals in the area will start with
constructions in the course of 2011 at one of the locations.
The activities to establish a proton therapy centre together
with the Erasmus University, the University Medical Centre
Leiden, and the Delft Technical University, have been
slowed down temporarily due to uncertainties with regard
to reimbursement guarantees and new developments with
regard to instrumentation. We secured together with a
number of other European cancer centres a 12 million EU
grant to implement robust high-throughput diagnostic
platforms for genomics/proteomics assays. We need such
platforms to identify suitable biomarkers that predict
response to therapy. We are also in the process to establish
with UMC-Utrecht, Hubrecht Laboratory, and ErasmusMC
a foundation that will provide deep sequencing services
of tumours and normal tissue of individual patients. This
is an integral part of our plans to advance personalised
medicine. Furthermore, we have succeeded in fi lling most
of the vacancies for nursing staff and pathologists, but are
still struggling to attract personnel in some other clinical
Director of Research Ton Berns
disciplines. Since the shortage of medical experts and
skilled nursing staff is expected to further increase in the
years to come we have to make the NKI-AVL stand out as
“the place to be” for employees. This is an important task
for the Institute.
We concluded 2010 with a healthy profi t for the hospital.
As a result no harsh measures with regard to personnel and
services will be needed to accommodate the substantially
reduced reimbursement that we expect for clinical services
in 2011. Our research expenditure also remained within
budget in 2010. However, this could only be achieved by not
fi lling the positions of staff members that left the Institute.
The renovation of the laboratories in the old research
building is in full swing and we expect to move into the
renovated labs in the course of 2011. Bids for building the
new animal facility will be evaluated in the beginning of
2011. We expect to select the contractors shortly after.
Discussions with the Ministry of Health, aimed to increase
our research budget as was strongly advised by the site visit
team that evaluated the Institute in 2009, have not been
successful. While it is evident that the government has to
cut costs, it is disappointing that in the current political
climate investment in research has a relatively low priority.
Many other countries clearly choose to increase the support
for research as they all share the view that high quality
research is critical for securing the future prosperity of a
country. Fortunately, the Dutch Cancer Society is more
receptive to our needs and we are negotiating a new 5-year
agreement for core funding which we hope to fi nalise early
2011.
We have installed an introductory program for basic
researchers in the clinic. In this way they can get a feel for
the daily work of their clinical colleagues. We expect that
this will also foster discussions and collaboration between
basic researchers and clinicians in the Institute. It also
can serve as a step-up to the twinning of clinicians with
investigators in the laboratory to become effective teams.
In 2010 two new spin-off companies were founded. UbiQ
focuses on reagents, both for research and diagnostic
purposes as well as to identify lead compounds for
targeting regulatory pathways controlled by ubiquitination
and deubiquitination reactions. This is an important new
fi eld with promising perspectives. The second company,
MODRA, has as general goal the commercialisation of

Table 1
Core research funding NKI-AVL from the Dutch Cancer Society and the Ministry of Health in the period 2002-2010
in million euro’s.
inventions jointly made by Slotervaart Hospital and NKI-
AVL. MODRA initially focuses on making existing drugs
orally available through co-administration of inhibitors
of drug transporters that prevent intestinal uptake under
normal conditions. The Technology Transfer Offi ce of the
NKI has played an important role in defi ning the conditions
and licenses for these new start-ups.
HIGHLIGHTS
The institute continues to have a strong scientifi c output;
2010 was again a productive year. It is always diffi cult to
estimate the impact of research when the results are still
fresh. The research highlights summarised here serve
primarily as a sampling of work currently on-going in the
Institute. A more complete and detailed account of specifi c
projects can be found in the reports of individual group
leaders in this SAR and on the website (www.nki.nl).
The Neefjes lab fi nished a data-based systems description
of an essential process in the immune system: antigen
presentation by MHC class II molecules. This has yielded
a series of novel pathways controlling transcription and
transport of these molecules.
Huib Ovaa and colleagues have established a novel
synthesis scheme allowing the production of unique
ubiquitin pairs. His group also succeeded in reversing the
digestion of peptides by the proteasome.
In a collaborative effort of the P Borst, Jonkers, Linn and
Wessels groups, led by Sven Rottenberg, the mouse model
for Brca1-associated breast cancer was used to identify a
new marker for chemotherapy response: low expression of
the Xist gene correlated with good response to cisplatin and
this marker was also found to predict long recurrence-free
survival of a group of breast cancer patients treated with
platinum-based chemotherapy.
The Piet Borst group found a function for base J, a DNA
base modifi cation they discovered in 1993 in the nuclear
DNA of some pathogenic protozoa. Deep sequencing
revealed base J to be present at transcription termination
sites. In the absence of J, RNA Polymerase II fails to
halt and reads on. The resulting RNA chaos in the cell
is probably responsible for the death of the parasite.
The biosynthetic pathway of base J remains therefore
an interesting pharmacological target to eradicate these
parasites.
In the Jonkers group, Peter Bouwman and Mark
Pieterse found that inactivation of 53BP1 suppressed the
proliferation arrest induced by BRCA1 loss and partially
restored homologous recombination. Aberrant 53BP1
expression was also frequently found in human BRCAmutated
and triple-negative breast cancers and associated
with decreased metastasis-free survival. Interestingly,
Janneke Jaspers and Sven Rottenberg found that Brca1mutated
mouse mammary tumours acquired resistance to
PARP inhibitors through inactivation of 53BP1.
13
introduction
Year 2002 2003 2004 2005 2006 2007 2008 2009 2010
Cancer Society 8.7 9.1 8.9 8.6 8.3 8.8 9.3 10.0 10.8
Ministry Health 9.9 10.1 9.2 9.3 9.4 9.5 9.6 10.2 10.3
Total * 18.6 19.2 18.1 17.9 17.7 18.3 18.9 20.2 21.1
In addition to the core funding the NKI acquired support through external grants, donations, and research agreements.
The contribution of these sources to the total budget has steadily increased over the years and represents roughly 65% of
the total budget in 2010.
Tanja van Harn and Floris Foijer (Te Riele group) found
that loss of the retinoblastoma (Rb) proteins can cause
genomic instability. Upon mitogen deprivation, Rbdefective
cells progressed through S phase, but then
arrested with high levels of DNA breaks and defective
sister-chromatid cohesion. When arrested cells were restimulated
to proliferate, the daughter cells were often
aneuploid. Thus, genomic instability can ensue from
defective cell cycle control in combination with (transiently)
inappropriate culture conditions.
Using a mouse model for small cell lung cancer, Joaquim
Calbo, Erwin van Montfort and Natalie Proost of my own
group found that the tumours are often composed of
phenotypically different cells with either a neuroendocrine
or a mesenchymal marker profi le, a feature also noted
in human SCLC cell lines. Crosstalk between these
mesenchymal and neuroendocrine endowed the
neuroendocrine cells with metastatic capacity, illustrating
the potential relevance of tumor cell heterogeneity in
dictating tumour properties.
Chromatin composition is still poorly characterized. The
Van Steensel group has generated genome-wide highresolution
binding maps of 53 chromatin components
in Drosophila cells. They demonstrated that the genome
consists of fi ve discernable chromatin types. One represents
a new repressive chromatin type that covers about half of

14
introduction
the genome and lacks classic heterochromatin markers
whereas two represent transcriptionally active euchromatin.
Their results provide an overarching view of chromatin
diversity and domain organisation in metazoan cells.
Oncogene-induced senescence is a p53-dependent
defence mechanism against uncontrolled proliferation.
Consequently, about 50% of human tumours harbour p53
mutations while others show a dysfunctional p53 pathway,
frequently by unknown mechanisms. The group of Agami
used functional genetic screens with RNA-interference
libraries and identifi ed the gene BRD7 as a tumour
suppressor that is part of the p53 pathway. In human breast
tumours harbouring wild-type p53, the BRD7 gene locus
is frequently deleted and low BRD7 expression caused by
promoter silencing was found in a subgroup of tumours.
This knowledge can be used to develop novel cancer drugs.
Autotaxin (ATX) or ecto-nucleotide pyrophosphatase/
phosphodiesterase-2 (ENPP2) is a secreted
lysophospholipase D that generates the lipid mediator
lysophosphatidic acid (LPA), a mitogen and chemoattractant
for many cell types. The role of autotaxin is
extensively studied by the group of Wouter Moolenaar.
ATX-LPA signalling has roles in various pathologies
including tumour progression and infl ammation. However,
the molecular basis of substrate recognition, catalysis,
and the mechanism of interaction with target cells, has
remained elusive. The group of Tassos Perrakis has
determined the crystal structure of ATX, alone and in
complex with a small-molecule inhibitor. They identifi ed a
hydrophobic lipid-binding pocket and mapped key residues
required for catalysis and selection between nucleotide and
phospholipid substrates. They showed that ATX interacts
with cell-surface integrins via its N-terminal somatomedin-
B-like domains, using an atypical mechanism. Their results
defi ne determinants of substrate discrimination by the
ENPP family, suggest how ATX promotes localised LPA
signalling, and enable new approaches to target ATX with
small-molecule therapeutics.
The Begg group and colleagues from the division of
Radiotherapy found that expression of the putative stem cell
marker CD44 was strongly associated with local recurrence
after radiotherapy for laryngeal cancer. The fi nding was
made from genome-wide expression profi ling (microarrays)
of pre-treatment biopsies, and then validated on a separate
clinical series using immunohistochemical staining
for CD44. The predictive potential of this marker was
independent of known clinical predictors. This fi nding is
likely to lead to a better understanding of treatment failure
and more individualised treatments for this type of head
and neck tumour.
The release of the 4D-CBCT guided radiotherapy software
application now permits high dose - high precision lung
cancer irradiation in other radiotherapy centres in the
Table 2
Short-term citations and impact of scientifi c articles published by the NKI research staff 1995 - 2007
Publication year Publications Citations* Citations/publication Impact**
1995 287 3648 12.7 1415
1996 260 4056 15.6 1520
1997 274 4174 14.1 1811
1998 276 3138 14.2 1392
1999 280 3474 12.4 1766
2000 301 4314 16.0 1699
2001 339 4944 16.2 1554
2002 407 7436 21.3 2324
2003 357 5084 15.4 1963
2004 347 5254 16.2 2018
2005 399 6261 17.3 2442
2006 432 6302 16.2 2584
2007 429 5515 13.8 2590
2008 445 5671 13.9 2553
2009 517 3122
2010 606 2945
As from 1997 publication year of articles is the criterion, instead of Scientifi c Report-year listing.
From 2008 the online publication data are used as criteria for the year of publication.
* Citations in the two years after publication, excluding self-citations. Starting 1989 citation analysis has been carried out on
line. This allows detection (and elimination) of all self-citations. Before 1989 this pruning was limited to fi rst authors.
** The impact factor is the average number of citations per year of an article in a given journal. The total impact is the sum of
the impact of all articles published that year.

world. The in-house developed 3D-EPID dosimetric
verifi cation of radiation treatment software received a 3 rd
ranking in the National Patient Safety Contest.
QUALITY OF RESEARCH
The quality of research can be monitored in several
ways. Our scientifi c productivity as assessed by objective
bibliometric parameters (citations and impact of scientifi c
articles published by the NKI staff) has shown a steady
increase over time (Table 2). It is satisfying to note that the
Institute retains an internationally prominent position in
cancer research.
While a high citation score is gratifying, it is only one
measure of scientifi c productivity. The quality of our
work should also be gauged by the invitations of our staff
to present at international scientifi c meetings, awards
obtained by our staff and signifi cant grants that were
obtained. We score high on all these accounts. Sjaak Neefjes
and Ton Schumacher both obtained a prestigious ERC
grant. Flora van Leeuwen obtained a Queen Wilhelmina
Research Award, Reuven Agami received a VICI grant,
and VIDI grants were awarded to Sven Rottenberg and Fijs
van Leeuwen. Lodewyk Wessels, Jos Jonkers and Bas van
Steensel were successful in securing a 4.5 million grant to
establish a Cancer System Biology Centre at the NKI.
The quality of research of the groups in each division is
evaluated approximately every 5 years by an external site
visit team. In November 2010 the Division of Radiotherapy
was evaluated. The reviewers were very positive about the
Division, which they rated to the top in the world.
HONOURS AND APPOINTMENTS
The NKI-AVL cannot award university degrees. However,
many of our staff members hold special part-time chairs at
Dutch Universities. This allows them to award PhD degrees
to graduate students receiving their training at The Netherlands
Cancer Institute. In 2010, 32 staff members had
professorships at one of the Dutch Universities. Theo Ruers
was appointed professor of Oncology at the University of
Twente, Bas van Steensel as Professor of Chromosome
Biology at the Erasmus Medical Centre in Rotterdam, and
Paul Baas as professor of Pulmonary Oncology at the
Academic Medical Centre in Amsterdam. Jos Beijnen was
elected as member of the Royal Netherlands Academy of
Sciences and Ton Schumacher as member of the European
Molecular Biology Organisation (EMBO).
There were substantial changes in our clinical staff mostly
due to retirements. Fortunately, we have been able to recruit
a number of excellent successors. Fred van Leeuwen was
appointed as tenured staff member in the Division of Gene
Regulation. Wim van Blitterswijk and Wouter Moolenaar
retired. However, Wouter Moolenaar will continue to run
his group as an extraordinary staff member. Laura van
‘t Veer left the Institute to take a prestigious position at the
Cancer Center at UCSF. Maarten Fornerod left the institute
to become staff member at the Erasmus MC, and Kenneth
Gilhuijs moved to the UMC Utrecht.
Staff of the NKI-AVL fulfi lled numerous functions in
national and international organisations, on boards of
scientifi c journals, as members of study sections, and
as organisers or co-organisers of scientifi c meetings,
workshops and congresses.
15
introduction
OUTLOOK AND ACKNOWLEDGEMENTS
I am confi dent that the NKI-AVL will continue to fl ourish,
even in the face of all the uncertainties that are associated
with the current economic crisis. However, it is imperative
that our core funding increases or, alternatively, that a
full-costs reimbursement model is installed for external
project grants. We are very successful in securing external
grants but these grants provide almost no overhead
thereby putting considerable strain on the core budget.
The arrangements we hope to fi nalise with the Dutch
Cancer Society will be critical for our capacity to stay in
the forefront of international cancer research. More than
ever breakthroughs will come from the close collaboration
of basic and clinical investigators. A continuous fl ow of
ideas in both directions is needed not only to bring new
concepts and drugs to the clinic, but also to understand at
the molecular level why new promising treatments often
do not work or quickly result in resistance. In this regard,
state of the art molecular typing of tumours using genomic
and proteomic techniques combined with robotic screens to
identify pathways responsible for drug resistance are critical
for unravelling mechanisms of resistance. I fi rmly believe
that breakthroughs in cancer diagnosis and treatment can
be realised with approaches that are now broadly applied
in the NKI. However, to make an impact in this area, we
do need to conduct these studies at a suffi ciently large
scale. This requires additional investments in molecular
pathology and therefore has high priority. With the drive
and enthusiasm that is so characteristic of the individuals
that shape our Institute, we should be able to convince
funding agencies as well as individuals to support the NKI-
AVL, an institution that is at the international forefront of
cancer research.
I want to end by thanking all our employees and those
who continue to support us: The Dutch Cancer Society,
the most reliable and signifi cant sponsor of our research;
the Ministry of Health, which provides a substantial core
grant to the Institute and has provided the funds for the
renovation of our research facilities; and, last but not least,
the many individuals who provide us with fi nancial, moral,
and practical support. Only with their help can we continue
to develop new ideas that can improve the perspectives of
cancer patients. I hope you will be inspired by this report.
Ton Berns
Director of Research

16
education in oncology
EDUCATION IN ONCOLOGY
The Netherlands Cancer Institute offers a variety of
opportunities for practical and theoretical training to
(trainee) technicians, University Master students, PhD
students and post-doctoral fellows. Research and clinical
staff and their group members are involved in theoretical
and practical training. Many staff members have joint
appointments as professors at Dutch universities and an
even larger number contribute to the regular curriculum at
various universities. The research divisions attract students
from universities throughout the country. The NKI has a
formal affi liation with the Science faculty of the University
of Amsterdam (UvA) and is committed to make a
contribution to Master student teaching. The institute
participates in the Oncology Graduate School Amsterdam,
together with the medical faculties of the UvA and the
Free University (VU), referred to as Academic Medical
Center (AMC) and VU medical center (VUmc), respectively.
All educational activities are supervised by the Teaching
Committee, which consists of Jannie Borst (chair and dean
Master students), Hein te Riele (general affairs), Roderick
Beijersbergen (Master course), John Hilkens, John Collard
(HLO students and publicity), Titia Sixma (dean PhD
students) and Fons Balm (clinical teaching). Peter Peters
functions as dean for the post-docs.
MASTER STUDENTS
The program in Experimental Oncology attracts Master
students of all national universities, see http://www.nki.nl/
Research/Career/Masters+students. Students generally
have a background in (Medical) Biology, Health Sciences,
Chemistry, Pharmacology, Medicine, or Psychology. The
program offers combined practical and theoretical training
in various aspects of experimental oncology. Practical
training includes participation in ongoing research projects
for a minimum of 4 months. In 2010, 41 Dutch university
Master students and 2 students from abroad completed
a placement of 5-9 months at the biomedical research
divisions. The students came primarily from the Free
University Amsterdam (VU) (19) and the University of
Amsterdam (UvA) (10), but also from Utrecht University (5),
Wageningen (4), Leiden (1), Nijmegen (1), and Groningen
(1). The institute also provides practical training
opportunities to trainee technicians, who stay for similar
periods of time as the university students and like these,
often make signifi cant contributions to research progress
of the PhD students and post-docs who supervise them.
There is an increasing demand from Universities for
placing Bachelor students for the three month internship
that concludes their program and we have accommodated
3 of them this year. The core element of theoretical training
is the course in Experimental Oncology, given twice yearly
(Table 1). This course is compulsory for Master students
who do an internship at the NKI, but in addition attracts
many students from throughout the country. We routinely
host about 40 students per course. It includes lectures and
tutorials given by our highest level clinical and research
professionals and is rated very highly in University
evaluations.
Table 1 - Course in Experimental Oncology
Epidemiology F van Leeuwen, M Rookus
Surgery E Rutgers
Radiodiagnostics W Vogel
Pathology D de Jong
Molecular diagnostics* R Kerkhoven, S Linn
Conventional pharmacotherapy S Rodenhuis
Radiotherapy** M Verheij
DNA damage response and
apoptosis* A Begg, J Borst
Tumor development* R Beijersbergen
Genomic instability* H te Riele
Signal transduction* W Moolenaar
Cell cycle R Bernards
Oncogene-tumor suppressor
gene interactions D Peeper
Cell division R Wolthuis
Cell adhesion* E Roos
Tumor microenvironment K de Visser
Mouse models of cancer* J Jonkers
Immunology and
immunotherapy* T Schumacher, J Haanen
Analysis of protein structure T Sixma
Rational drug development A Huitema
Drug delivery* A Schinkel
* including tutorial
** including tour
PHD STUDENTS
The PhD students at the NKI-AVL participate in the
Oncology graduate school Amsterdam (OOA) together
with the oncology departments of the VUmc and the AMC.
In 2010, the institute had ~170 PhD students registered
with the OOA and 24 students defended a PhD thesis
at a Dutch university.
Students participate in research of their group and in
interdepartmental work discussions. In addition, the
students follow the OOA training program, which consists
of courses (Table 2) and an annual retreat on the island
Texel. Apart from courses on different topics in cancer
research, the OOA offers a course on scientifi c English.
Students with no prior background in cancer research can
participate in the Experimental Oncology course.
Part of the training of the NKI students are discussions
with experts in the fi eld of oncology. The Friday morning
seminar speakers take their lunch with a delegation of the
students. Twice a year a list of speakers is sent round and
each graduate student participates several times per year in
these opportunities to exchange views with experts in the
fi eld.
The PhD student retreat focuses entirely on the research of
the graduate students themselves. At this retreat, students
are not only presenting their work in the form of a poster in
the fi rst year and presentations in subsequent years, but
they are also in charge of chairing sessions and discussions.
In recent years, they also participate in peer review, giving a
prize for the best poster and best presentation. In this

manner, the retreat provides training in presentation and
interaction skills, but it also provides an overview of the
research in the OOA at an early stage of the student’s
career. This provides a good opportunity for translational
interaction and bottom-up research, allowing the graduate
students themselves to contribute signifi cantly to interaction
between different research groups.
In addition, senior graduate students can participate in a
joint retreat with other cancer institutes in Europe. In 2010,
this event was hosted by IFOM in Milan, with participants
from among others British CrUK institutes, the Italian
IFOM and the Spanish CNIO contributing to a program
of scientifi c lectures and posters as well as an enthusiastic
social session. This retreat gives students the opportunity
to compare notes among excellent cancer institutes
throughout Europe.
The progress and work is monitored annually by a
supervisory committee. This committee has independent
members within and outside the division. The committee
discusses progress with the supervisor and student
separately and participates in a joint discussion of the
research. After two years of PhD research the student,
supervisor and committee evaluate the state of the project
in a midterm review. At this more elaborate meeting the
likelihood of achieving a PhD within a reasonable time
frame is discussed. This meeting can be used to redefi ne
goals if necessary.
The students are represented in the OIO-council that
meets with the Dean of graduate student affairs on a
regular basis, as well as upon special request. They also
mediate communication between the graduate students
and research manager or board of directors.
Table 2 - OOA graduate student courses 2010
9-12 March The Macroscopic and Pathologic Anatomy
of the mouse
WH Lamers, CJF van Noorden
April English Writing and Presenting
November Annual Graduate Student Retreat, Texel
T Sixma
In the Footsteps of Antoni van Leeuwenhoek,
basic course
P Peters, C van Noorden, K Jalink, E Reits et al.
November English Writing and Presenting
POST-DOCS
Post-docs in our Institute perform challenging research
in an internationally competitive environment. They
participate in work discussions both within the group
and among different divisions. Within the research group
there are opportunities to receive training in supervision,
manuscript writing and presentation. In many cases, our
postdocs have a preference for working in science and
indeed, many of our post-docs go on in academia.
There are however many alternative options available for
an interesting career after a successful post-doc period.
The Postdoc Career Development Initiative (PDCI) was
initiated based on the post-doc platform at the NKI-AVL.
The activities can be seen at www.pcdi.nl.
17
education in oncology
RETREAT 2010
12 parallel workshops, 9 keynote lectures of senior peers,
3 days away from the lab. These were the main ingredients
for the retreat which took place from 21 - 23 April in
Kapellerput, Heeze, where postdocs and fi nal year PhD
students came together to refl ect on their current position
and their career perspectives. There are more determinants
to success than a publication list alone. Each year, about
100 young scientists gather to refl ect on their current
position and to learn what opportunities there are. ‘Love
what you are doing, choose a position that suits your
interests and skills best and to know how to sell yourself’,
were among the things they heard at the retreat.
At the retreat, successful academics gave career advice:
know how to get and use feedback from colleagues, how to
establish collaborations, how to present yourself and to gain
independence. Being aware of your transferable skills is
part of the masterplan to success.
Participants were also put to work during three parallel
sessions of workshops, to start drafting their individual
masterplan. The sessions focused on what the postdocs
actually enjoy doing and on the way to achieve that goal:
it is important that they know how to present themselves
and convince potential employers, collaborators and peers
of their qualities. In the workshops they learned the
do’s and don’ts when writing an application letter, and to
improve public speaking and presentations. Finally,
participants improved a transferable skill of choice:
non-verbal behaviour, grant writing, negotiation, project
management or how to give/ask/receive feedback.
On the fi nal day, PCDI invited a few dozen ex-researchers to
talk about their career choices and their current positions.
Networking is another part of the masterplan to success,
because the world of Life Sciences is not limited to
academic research, it includes technology transfer, biotech
companies, government and media, with a common goal:
improved health.
Under the leadership of Peter Peters the retreats have been
attended by more then 1100 postdocs over the last 11 years.
Our institute has landed again in the Scientist’s list 2011 of
top 10 best international places to work for postdocs.

18
biochemistry
Division head, group leader Titia Sixma
Titia Sixma PhD Group leader
Marcello Clerici PhD Post-doc
Alex Fish PhD Post-doc
Rick Hibbert PhD Post-doc
Prakash Rucktooa PhD Post-doc
Mariano Stornaiuolo PhD Post-doc
Azusa Seto PhD Post-doc
Alex Faesen MSc PhD student
Mark Vargas MSc PhD student
Francesca Mattiroli MSc PhD student
Judith Smit MSc PhD student
Flora Groothuizen MSc PhD student
Danny Sahtoe MSc PhD student
Pim van Dijk Technical staff
Herrie Winterwerp Technical staff
Publications
Lebbink JH, Fish A, Reumer A,
Natrajan G, Winterwerp HH, Sixma
TK. Magnesium coordination controls
the molecular switch function of DNA
mismatch repair protein MutS. J Biol
Chem. 2010;285:13131-41
Shanmugham A, Fish A, Luna-Vargas
MP, Faesen AC, El Oualid F, Sixma TK,
Ovaa H. Nonhydrolyzable ubiquitinisopeptide
isosteres as deubiquitinating
enzyme probes. J Am Chem Soc.
2010;132:8834-5
El Oualid F, Merkx R, Ekkebus R,
Hameed DS, Smit JJ, de Jong A,
Hilkmann H, Sixma TK and Ovaa
H. Chemical Synthesis of Ubiquitin,
Ubiquitin-based Probes and Diubiquitin
Angewandte Chemie 2010;49:10149-53
DIVISION OF BIOCHEMISTRY
STRUCTURAL BIOLOGY
Development of cancer is generally due to errors that occur in cellular pathways.
Understanding mechanisms of the underlying processes will help to determine
where the errors occur and how they can be treated. We use X-ray crystallography as
a tool to provide three-dimensional structures and we interpret the structural data
using a variety of biochemical and biophysical techniques. These studies provide
more insight in the molecular processes and they can also provide targets for drug
design studies. In our group we focus mainly on proteins involved in ubiquitin
conjugation in chromatin regulation, on DNA mismatch repair and nicotinic
receptor signaling.
DNA mismatch repair DNA mismatch repair plays a crucial role in ensuring
genomic stability. Defects in the mismatch repair cascade in humans predispose
to hereditary non-polyposis colorectal cancer and are associated with a variety
of sporadic cancers. DNA mismatch repair is initiated by the protein MutS (in
Escherichia coli) or its MSH homologs. MutS recognizes and binds to mismatches
or unpaired bases that have escaped the proofreading capacity of the DNA replication
machinery or are present in the genome during recombinatorial events between
non-fully complementary DNA strands.
We make use of variants of MutS that specifi cally maintain the dimer or tetramer
state of these proteins in order to be able to provide quantitative data on the
mismatch recognition cycle. In collaboration with the groups of Terence Strick
(Paris), Peter Friedhoff (Giessen) and Joyce Lebbink (Rotterdam) this allows a
precise analysis of the steps involved in mismatch recognition and the initiation of
repair. These mutants allowed novel crystallographic analysis of the proteins with
and without DNA, leading to new insight into the state of the protein prior to DNA
binding.
Our detailed analyses of the steps ps
following mismatch recognition n are
building up to a model that reveals eals
the details of the complex ATPase ase
cycle of the mismatch repair proteins. oteins.
Figure 1: Novel crystal form of E.coli
MutS in complex with 16-mer DNA
containing an AA mismatch revealed
that crystal packing did not affect
protein-DNA interactions, since all
contacts were conserved between the
two crystal forms (Lebbink et al, 2010)

Ubiquitin and SUMO conjugation Ubiquitin conjugation is critical for signaling
in almost all cellular processes, including DNA repair, apoptosis, cell cycle,
chromatin regulation and endocytosis. Since these processes are important
for cellular stability, deregulation of ubiquitin-dependent processes often leads
to cancer. Structure analysis of the proteins involved in ubiquitin and SUMO
conjugation could help the development of novel drugs inhibiting critical pathways
in ubiquitin conjugation.
The process of conjugation by ubiquitin-(like) proteins involves covalent linking of
one or more 76-amino-acid ubiquitins to a target protein by an E1/E2/E3 cascade
of enzymes. Correct ubiquitination requires the complex spatial arrangement of
ubiquitin, E2, E3 proteins and the target simultaneously in a precise but fl exible
manner. Although the overall mechanism has been defi ned, the atomic details have
been lacking and the specifi city determining factors are unclear. We study several
E2/E3 complexes involved in conjugation of ubiquitin and the related modifi er
SUMO as well as proteins involved in de-ubiquitination.
Deubiquitinating enzymes are found in fi ve classes, and there are many less
than there are E2/E3 combinations. This indicates that regulation may well be
taking place at a different level. In order to understand this we have undertaken
a comparison of a number of ubiquitin specifi c proteases. In collaboration with
Huib Ovaa we are making use of novel tools to study these enzymes. Thus we can
incorporate the analysis of all diubiquitin pairs for this type of analysis.
In terms of regulation we are studying the role of the ubiquitin-like Ubl domains
in a number of DUB enzymes. Although these domains have a similar fold to
ubiquitin, their sequence properties are very different. Sofar our studies have
included the Ubl domain that is internal to the Usp4 catalytic domain and we also
analyzed the role of the C-terminal domain of Usp7 and its activating properties for
catalytic activity.
In collaboration with Rolf Boelens in Utrecht we studied the chain formation activity
of the Rad6 E2 enzyme and how this is modulated by the presence of Rad18. We
studied the PCNA modifi cation by Rad6/Rad18 and showed that Rad18 binding
inhibits the chain forming activity of Rad6 (Hibbert et al, submitted for publication.
Figure 2: Crystals of
catalytic domain of Usp4
Nicotinic Acetylcholine receptor homolog AChBP The acetylcholine binding
protein (AChBP) is a homolog of the extracellular domain of the nicotinic
acetylcholine receptor, and a representative of the cys-loop receptor family. It
remains the best model for atomic resolution structures of ligand binding to this
pharmaceutically important family of ion channels.
In collaboration with the group of Iwan de Esch (VU) novel interactors for AChBP
have been identifi ed, using a fragment growing approach, into a specifi c binding
pocket (Edink et al, submitted for publication). Using a combination of different
thermodynamic analyses and structural studies we were able to explain the behavior
of the different ligands that were studied as part of this procedure. Interestingly the
comparison of surface plasmon resonance and isothermal calorimetry indicated very
similar results and provided an explanation for the observed variations in binding
mode. Structural studies of a series of reference compounds including toxins and
anti-smoking compounds provide insight in the details of subunit specifi city of the
nicotinic receptor homologs.
19
biochemistry

20
biochemistry
Group leader Anastassis Perrakis
Anastassis Perrakis PhD Group leader
Christophe Caillat PhD Post-doc
Eleonora von Castelmur PhD Post-doc
Krista Joosten PhD Post-doc
Robbie Joosten PhD Post-doc
Jens Haussman MSc PhD student
Tatjana Heidebrecht Technical staff
Figure 3:
The HA155 inhibitor bound to ATX
STRUCTURAL BIOLOGY
This year was marked by two breakthroughs on our major long-term in-house
collaboration projects: determining the structure of Autotaxin (with Wouter
Moolenaar, Division of Cell Biology I, and Huib Ovaa, Division of Cell Biology II)
and of JBP1 (with Piet Borst, Division of Molecular Biology). Concurrently, our
work on DNA replication licensing and on mitotic progression advanced and our
adventures in X-ray crystallography model building and refi nement are undermarked
by a change of focus towards structural boinformatics.
Structural studies of Autotaxin The role of the signaling phospholipid
lysophosphatidic acid (LPA) and Autotaxin (ATX), the protein that produces LPA
from lysophosphatidylcholine (LPC), was established over the last three decades,
largely owing to the efforts of the group of Wouter Moolenaar at the NKI. LPA and
ATX have been shown by numerous studies to be involved in cancer metastasis and
other pathogenic situations, such as infl ammation and neuropathic pain. ATX is
the protein ecto-nucleotide phosphodiesterase 2 (ENPP2), a 100 kDa glycoprotein,
capable of both lysoPLD and nucleotide pyrophosphatase activities. Over the last
few years we have engineered a human cell line (HEK293 derivative) that is stably
expressing glycosylation-defi cient mutants of rat autotaxin, which allowed us to
crystallize ATX, and determine its structure this year.
Autotaxin hydrolyzes different species of LPC (with different alkyl chain lengths)
and some other lipids, as well as nucleotide substrates. Our structure shows that
both nucleotides and lipids partially share the same binding pocket, but the alkylchain
of lipid substrates form additional hydrophobic contacts with ATX. This model
implies that the LPA product likely has higher affi nity for ATX than any nucleotide
substrate, a hypothesis consistent with the previous fi nding that LPA acts as an
inhibitor of ATX activity against a variety of substrates, but not of LPC hydrolysis.
Thus, LPA formed by the enzyme may act as a “substrate specifying factor”
effectively inhibiting the hydrolysis of nucleotides because it can only be displaced
by lysophospholipid substrates such as LPC. This mechanism could dictate ATX
activity in vivo, because the spectrum of available LPA species could defi ne ATX
activity against specifi c lysophospholipid substrates. Our fi ndings also suggest that
further analysis of structural determinants of substrate discrimination could lead to
the identifi cation of molecules that inhibit the hydrolysis of specifi c substrates, e.g.
long-chain rather than short-chain LPC species.
The group of Huib Ovaa has developed a series of small-molecule boronic acid
inhibitors of ATX-mediated LPC hydrolysis, that lower plasma LPA levels in mice
following intravenous administration. We also determined a structure of ATX
in complex with one of these inhibitors, HA155, which enables us to correlate the
activity of this inhibitor with its binding mode. We show that the boron atom on
one end of the inhibitor forms a reversible covalent bond with the nucleophile
hydroxyl group of the active site threonine nucleophile. The other end of HA155, a
hydrophobic fl uoro-benzene, is pointing directly into the deep hydrophobic pocket,
oriented perpendicular to the protein surface.
Our results will allow us to provide feedback to inhibitor development studies, which
are purused in-house in collaboration with CR-UK, but also by our collaborators
in Pfi zer and by Merck-Darmstadt. Importantly we have now formed the basis for
understanding how autotaxin sequesters its lipid substrates and we formulated
testable hypotheses for how ATX can specifi cally present the hydrolysis products to
the cell-surface LPA receptors, to elicit a variety of important responses. This will
allow us how Autotaxin is related to processes such as infl ammation, neuropathic
pain, obesity, and most importantly in cancer metastasis.
Structural studies of JBP1 The JBP1 protein, discovered by Piet Borst and
colleagues, binds to DNA that contains base J ( -D-glucosyl-hydroxymethyluracil).
JBP1 has been shown to be essential for survival in many major protozoa human
pathogens such as T. brucei (sleeping sickness), T. cruzi (Chagas’ disease) and
Leishmania species (various types of Leishmaniasis). Last year, it has been shown
that there exists a partial mammalian homologue of JBP1, which is localized
in a region of the TET proteins that are involved in myeloid leukemia and

necessary for the synthesis and maintenance of the important epigenetic marker
hyroxymethylcytosine.
We showed that JBP1 recognizes J-containing DNA through a 160-residue domain,
DB-JBP1, with ten thousand-fold preference over normal DNA. The crystal structure
of DB-JBP1 revealed a novel helix-turn-helix variant fold, a “helical bouquet” with a
“ribbon” helix responsible for DNA binding. Mutation of a single residue (Asp525)
in that helix abrogates specifi city towards J-DNA, rendering mutated JBP1 unable
to rescue the targeted deletion of endogenous JBP1 genes in Leishmania. Based on
mutational analysis and H/D-exchange mass-spectrometry data, a model of JBP1
bound to J-DNA was constructed, and validated by small-angle X-ray scattering. Our
results open possibilities for utilizing JBP1 as a drug target and a tool for detecting
the important mammalian epigenetic marker hydroxymethylcytosine.
Geminin and its homologues in DNA replication licensing A new twist to
our research on the role of Geminin in replication licensing was provided by the
discovery of two homologue of Geminin: Idas and Lygeas (in Greek mythology,
´ and ´ are the cousins of the two Gemini). Lygeas (who unfortunately
made its fi rst appearance in scientifi c literature under the more mundane name
GemC1) directly mediates replication initiation through TopBP1- and Cdk2dependent
recruitment of Cdc45 onto replication origins. Idas, has a much more
tissue-specifi c localization (predominantly in the mouse forebrain), interacts with
Geminin, and inhibits the capacity of Geminin to sequester the pre-replication
origin component Cdt1. We have recently determined the structure of the
Geminin:Idas complex and have shown both in Xenopus and with biophysical
methods, how Idas binding reduces the inhibitory effect of Geminin to replication
initiation.
Structural studies of proteins involved in mitotic progression Our focus
this year has been on understanding the role of the regulatory domain of Mps1
kinase. The MPS (MonoPolar Spindle) family of kinases was shown to be be a
dual specifi city protein kinase in vitro, capable of autophosphorylation on serine,
threonine and tyrosine residues. Mps1 is essential for maintaining chromosomal
stability by allowing resolution of merotelic attachments and to ensure that single
kinetochores achieve the strength of checkpoint signaling suffi cient to prevent
premature anaphase onset and chromosomal instability. We have determined the
structure of the N-terminal domain of Mps1, which adopts the TPR fold, also found
in the Bub1 and BubR1 family of kinases.
In collaboration with the group of Geert Kops (UMC Utrecht), we have shown with
site directed mutagenesis and domain deletion and swapping studies, that the TPR
domain of Mps1 is important for localization of Mps1 to the kinetochores. Pulldown
experiments and peptide binding analysis, suggest that Mps1 is recruited to
kinetochores through the combined action of Aurora B and Ncd80, via interactions
with the TPR domain. We are currently analyzing how these interactions regulate
the biological roles of Mps1.
We are continuing our work on the FoxM1 transcription factor that is implicated
in a cell’s proliferation potential and in cancer. Our goal remains to extend
our work on the binding of the forkhead domain to DNA promoter sequences,
towards understanding the structure of full-length FoxM1 and deciphering how
phosphorylation regulates transcriptional activity.
Methods for X-ray crystallography The crystallographic models in the Protein
Data Bank (PDB) were deposited there over several decades, and were created using
the methods and software available at the time. We decided it is timely to consider
updating these models by using state of the art software, to improve their accuracy
and correct errors. This was addressed by PDB_REDO, a procedure to refi ne PDB
models for which experimental data are available. This year, we transfered some
functionality of our crystallographic model-building software ARP/wARP to
extend PDB_REDO to rebuild protein models. These new improved models are
freely available to a user community of tens of thousands of biologists, chemists,
bioinformaticians and drug designers.
Publications
21
cell biology i
biochemistry
Littler DR, Brown LJ, Breit SN, Perrakis
A, Curmi PM. Structure of human CLIC3
at 2 A resolution. Proteins. 2010;78:1594-
600
Littler DR, Alvarez-Fernandez M, Stein A,
Hibbert RG, Heidebrecht T, Aloy P, et al.
Structure of the FoxM1 DNA-recognition
domain bound to a promoter sequence.
Nucleic Acids Res. 2010;38:4527-38
Hausmann J, Christodoulou E, Kasiem
M, De Marco V, van Meeteren LA,
Moolenaar WH, et al. Mammalian cell
expression, purifi cation, crystallization
and microcrystal data collection of
autotaxin/ENPP2, a secreted mammalian
glycoprotein. Acta Crystallogr Sect F
Struct Biol Cryst Commun. 2010;66(Pt
9):1130-5
Brooks MA, Gewartowski K, Mitsiki E,
Letoquart J, Pache RA, Billier Y, et al.
Systematic bioinformatics and
experimental validation of yeast complexes
reduces the rate of attrition during
structural investigations. Structure.
2010;18:1075-82

22
cell biology I
Publications (continued)
Division head, group leader Arnoud Sonnenberg
Arnoud Sonnenberg PhD Group leader
Michael Ports PhD Post-doc
Ruben Postel PhD Post-doc
Pablo Secades PhD Post-doc
Severine Laval PhD Post-doc
Evelyne Frijns MSc PhD student
Dirk-Jan de Groot MSc PhD student
Mirjam Ketema MSc PhD student
Coert Margadant MSc PhD student
Norman Sachs MSc PhD student
Maaike Kreft Technical staff
Ingrid Kuikman Technical staff
Publications
van Laake LW, van Donselaar EG,
Monshouwer-Kloots J, Schreurs C,
Passier R, Humbel BM, Doevendans PA,
Sonnenberg A, Verkleij AJ, Mummery
CL. Extracellular matrix formation after
transplantation of human embryonic stem
cell-derived cardiomyocytes. Cell Mol Life
Sci 2010;i67:277-90
Steiner-Champliaud M-F, Schneider
Y, Favre B, Paulhe F, Pratzel-Wunder
I, Faulkner G, Wiche G, Konieczny
P, Adebola A, Liem RK, Langbein L,
Sonnenberg A, Fontao L, Borradori L.
BPAG1-b: complex distribution pattern in
striated and heart muscle and association
with plectin and -actinin. Exp Cell Res
2010;316:297-313
Margadant C, Sonnenberg A. Integrin-
TGFbeta crosstalk in fi brosis, cancer and
wound healing. EMBO Rep 2010;11:97-105
Raymond K, Richter A, Kreft M, Frijns
E, Janssen H, Slijper M, Praetzel-
Wunder S, Langbein L, Sonnenberg A.
Expression of the Orphan Protein Plet-1
during Trichilemmal Differentiation of
Anagen Hair Follicles. J Invest Dermatol
2010;130:1500-13
le Duc Q, Shi Q, Blonk I, Sonnenberg
A, Wang N, Leckband D, de Rooij
J. Vinculin potentiates E-cadherin
mechanosensing and is recruited to actinanchored
sites within adherens junctions
in a myosin II-dependent manner. J Cell
Biol 2010;189:1107-15
DIVISION OF CELL BIOLOGY I
RECEPTORS FOR MATRIX ADHESION
The main objective of our group is to study the mechanisms involved in cell
adhesion. We are specifi cally interested in understanding the signifi cance of and
characterizing the interactions that take place between cells and the extracellular
matrix component laminin in both the epidermis and the glomerulus of the kidney.
A second line of research involves the outer nuclear membrane protein nesprin-3,
which binds to the cytoskeletal linker protein plectin, thereby establishing a link
between the intermediate fi lament system and the nucleus. The physiological roles
of these proteins and their interaction are studied in model organisms, including
mice and zebrafi sh.
Integrins and tumor progression The integral components of cellular mediated
adhesion are the integrin heterodimers, which specify ligation to particular
extracellular matrix (ECM) proteins and focus signaling responses while connecting
cytoskeletal structures intracellularly. Outside-in activation of downstream signaling
events mediated by integrin ligation leads to activation of pathways similar to the
activated signals initiated by growth factor receptors. During the progression of
cancer, tumor cells must overcome many implemented points of regulation to
sustain uninhibited growth, invasion, and survival in new microenvironments in
tissues. This work aims to investigate if signaling initiated by integrin expression
leads to enhanced survival of tumor cells during metastasis.
We have identifi ed several breast cancer cell lines that express minimal amounts
of 1 integrin. Using these cells we have overexpressed 3 1, 5 1 and 6 1,
known to be expressed during carcinoma progression. Expression of 5 1 leads to
a morphologic change reminiscent of EMT and altered survival signaling pathways
via Akt. Using a zebrafi sh embryo metastasis model we were able to demonstrate
that 5 1 promotes survival of breast tumor cells in the circulation. This is a novel
model for understanding the rate limiting step of metastasis and should reveal new
mechanisms of integrin biology involved in metastatic dissemination of tumor cells.
Kindlins, talin, and integrins Loss-of-function mutations in kindlin-1 cause the
blistering disorder Kindler syndrome (KS). KS keratinocytes express 1-integrins
at lower levels than normal keratinocytes, resulting in poor cell adhesion and
spreading, low numbers of focal adhesions (FAs), and disturbed cytoskeletal
organization, all of which are restored by kindlin-1 re-expression. Treatment with
a 1-activating antibody partially rescues the defects, indicating that the integrins
are functional. Integrin activation depends on binding of an NPxY motif in the
1-tail by talin, connecting the integrin to the cytoskeleton, whereas the kindlins
bind a different NPxY motif. To address the regulation of 1 functions by talin and
the kindlins, we generated 1-mutants defective in talin-binding ( 1 Y783A ), kindlinbinding
( 1 Y795A ), or both ( 1 Y783/795A ), and stably expressed these in 1-null cells.
Wild-type 1 induces an EMT-like morphological change characterized by a 1oss of
cell-cell contacts, cell scattering, increased motility, high RhoA activity, fi bronectin
fi brillogenesis, and a contractile morphology with multiple protrusions and large
peripheral FAs. This phenotype is recapitulated by 1 Y795A but not by 1 Y783A or
1 Y783/795A , indicating that binding to talin but not to kindlin is critical for 1
functions. However, 1 Y795A accumulates in intracellular vesicles and is expressed at
low levels on the cell surface, which results in reduced numbers of FAs, much like in
the KS cells. In conclusion, whereas talin is a critical regulator of 1 activity, kindlin
regulates its cell surface expression.
Regulation of hemidesmosome disassembly by growth factors receptors
Hemidesmosomes (HDs) are specialized junctional complexes that mediate fi rm
adhesion of keratinocytes to the underlying basement membrane. Migration of
keratinocytes requires a regulated and dynamic turnover of HDs. We have previously
identifi ed three serine residues on the 4 cytoplasmic domain that play a critical

ole in the regulation of HD disassembly. However, only two of these residues (S1356
and S1364) are phosphorylated in keratinocytes after stimulation with either PMA or
EGF. The PMA- and EGF-stimulated phosphorylation of 4 is mediated by ERK1/2
and its downstream effector kinase p90RSK1/2. EGF-stimulated phosphorylation
of 4 increased keratinocyte migration, and reduced the number of stable HDs.
Mutation of the two serines in 4 to phospho-mimicking aspartic acid decreased
its interaction with the cytoskeletal linker protein plectin, as well as the strength of
6 4-mediated adhesion to laminin-332. During mitotic cell rounding, when the
overall cell substrate area is decreased and the number of HDs is reduced, 4 was
only phosphorylated on S1356 by a distinct, yet unidentifi ed, kinase.
Integrin 31/CD151 complex The tetraspanin CD151 forms a stable complex with
integrin 3 1. Using in situ proximity ligation assays we show that both molecules
are in close juxtaposition in adult glomeruli in vivo. Preliminary data suggests that
complex formation increases during the course of glomerulogenesis which leads to
increased podocyte adhesion during differentiation. Indeed we show in vitro that
wild-type glomerular epithelial cells (GECs) adhere more strongly to laminin-332
than CD151 knockout GECs or GECs expressing CD151 incapable of binding 3.
The 3 1/CD151 complex has been described not only to be present at sites of cell-
ECM adhesion, but also to be a component of the large cadherin-catenin complex
that regulates cell-cell adhesion. Since CD151 knockout mice develop renal failure,
we studied the role of sustained mechanical stress on GECs in vitro. Preliminary
data suggests that CD151-positive cells maintain cell-cell contacts better than cells
lacking CD151 when exposed to sustained low-level fl uid shear stress. Furthermore,
since the electrical resistance of a confl uent monolayer is a measure of the tightness
of its cell-cell contacts, experiments have been carried out in which the electrical
resistance of a growing monolayer of CD151 wild-type or knockout cells was
monitored. The higher resistance measured in CD151-positive monolayers again
suggests a role of CD151 in strengthening cell-cell adhesion. Because the infl uence
of CD151 on cell-cell and cell-matrix adhesions seems to be greatly infl uenced by
mechanical stress we decided to alter blood pressure and thereby transcapillary
fi ltration pressure in vivo. We fi rst decreased blood pressure in susceptible CD151
knock-out FVB mice by blocking the angiotensin converting enzyme, which leads to
a signifi cantly higher survival rate compared to untreated mice. We then increased
the blood pressure in resistant C57BL6 mice leading to proteinuria only in the CD151
knockout group. Using both mouse strains, we are now in the process of identifying
modifying gene(s) conferring susceptibility or protection to CD151 knockout induced
kidney failure.
Nesprin-3 in zebrafish The nuclear envelope (NE) protein nesprin-3 can establish
a link between the nucleus and the intermediate fi lament system via its interaction
with plectin. To defi ne the function of nesprin-3 in vivo, we made use of zebrafi sh as
a vertebrate model system. In situ hybridization and immunofl uorescence analyses
showed that nesprin-3 is primarily expressed at the NE of epidermal and skeletal
muscle cells during zebrafi sh development. Surprisingly, nesprin-3 defi cient
zebrafi sh do not display an overt abnormal phenotype; they are viable and fertile
with no defects in embryonic development. However, at the cellular level, these
zebrafi sh are characterized by a reduced association of keratin fi laments with the
NE in epidermal cells. Similar to mice, zebrafi sh express two nesprin-3 isoforms:
nesprin-3 and -3 . In contrast to nesprin-3 , nesprin-3 is only expressed during
the initial stages of development. This latter isoform lacks seven amino acids in
its fi rst spectrin repeat which are crucial for plectin binding and recruitment to
the NE. The seven amino acid motif is highly conserved between species and we
demonstrated by immunoprecipitation and co-localization studies that two residues
within this motif are essential for the interaction of nesprin-3 with the plectin
actin-binding domain (ABD). Furthermore, we show that the binding sites for
nesprin-3 and 4 on the plectin ABD partially overlap and that these proteins
might compete with each other for plectin binding.
Publications (continued)
23
cell biology i
Sakai T, Takagi J, Giudici C, Yamada Y,
Arikawa-Hirasawa E, Deutzmann R,
Timpl R, Sonnenberg A, Bächinger H,
Tonge DT. Laminin 121-recombinant
expression and interaction with integrins.
Matrix Biol 2010;29:484-93
Margadant C, Charafeddine RA,
Sonnenberg A. Unique and redundant
functions of integrins in the epidermis.
FASEB J 2010;24:4133-52
Frijns E, Sachs N, Kreft M, Wilhelmsen
K, Sonnenberg A. EGF-induced MAPK
signalling inhibits hemidesmosome
formation through phosphorylation of
the integrin 4 subunit. J Biol Chem
2010;285:37650-62
Postel R, Ketema M, Kuikman I, de
Pereda JM, Sonnenberg A. Nesprin-3
augments peripheral nuclear localization
of intermediate fi laments in zebrafi sh.
Identifi cation of amino acids essential for
nesprin-3 intereation with plectin. J. Cell
Sci., (in press)
Figure 1: Molecular composition of
hemidesmosomes and focal adhesions

24
cell biology i
Publications (continued)
Linn SC, Wesseling J. Molecular tests for
breast-cancer diagnosis? Lancet Onol
2009;10:314-5
Group leader Wouter Moolenaar
Wouter Moolenaar PhD Group leader
Maaike Alderliesten PhD Post-doc
Elisabetta Argenzio PhD Post-doc
Maikel Jongsma PhD Post-doc
Elisa Matas PhD Post-doc
Leonie Van Zeijl PhD Post-doc
Dalila Elouarrat MSc PhD student
Anna Houben MSc PhD student
LIPID GROWTH FACTOR SIGNALING
Our group has a longstanding interest in the lipid growth factor lysophosphatidic
acid (LPA), its signaling properties and role in health and disease. LPA signals
through at least six distinct G protein-coupled receptors and thereby stimulates the
proliferation, migration and survival of many cell types, both normal and malignant.
LPA is produced extracellulary from lysophosphatidylcholine by a secreted
lysophospholipase D (lysoPLD), named autotaxin (ATX), which was originally
identifi ed as an autocrine motility factor for melanoma cells. The ATX-LPA
signaling axis is essential for vascular development and is a signifi cant effector of
tumor progression in mice. As such, ATX qualifi es as an attractive target for therapy.
Our current research focuses on the regulation of ATX and its in vivo functions as
well as the characterization of novel LPA receptors and downstream effectors. Our
longstanding collaboration with the structural biology group of A. Perrakis (Division
of Biochemistry) has recently led to the elucidation of the crystal structure of ATX,
an important breakthrough that provides new insights into ATX structure-activity
relationships. Our studies should lead to novel ways of interfering with ATX-LPA
receptor signalling and with inappropriate LPA production in the tumor-stroma
microenvironment.
Autotaxin, a secreted lysophospholipase D implicated in tumor progression
We previously have established that ATX is essential for vascular development in
mice. Increasing evidence points to a signifi cant role of the ATX-LPA receptor axis
in cancer. In particular, enforced overexpression of ATX or individual LPA receptors
promotes tumor progression in experimental animals, while elevated expression
of certain LPA receptors is associated with poor clinical outcome in breast cancer
patients. Obviously, there is now an urgent need for pharmacological inhibitors of
ATX and, furthermore, a better understanding of how ATX activity is regulated and
how newly produced LPA is presented to its cognate receptors. We therefore set out
to discover potent inhibitors and to determine the crystal structure of ATX.
ATX inhibitors
To identify small-molecule inhibitors of ATX, we have performed high-throughput
screening of chemical compound libraries (collaboration Huib Ovaa). Among the
positive hits were several new chemical structures that have been optimized to reach
IC(50) values in the nanomolar range. Specifi city, selectivity and toxicity of these
pharmacological inhibitors are being further evaluated in cell-based and animal
studies. A boronic-based ATX inhibitor was found to rapidly lower plasma LPA levels
in mice, indicating that LPA turnover in the circulation is much more dynamic than
previously appreciated.
Crystal structure of ATX The LPA-generating activity of ATX has been well
characterized, but the molecular basis of substrate recognition and catalysis, and
how ATX may interact with target cells has been elusive. Through collaborations
with the groups of Anastassis Perrakis (Division of Biochemistry), Andrew Morris
(University of Kentucky, USA) and Huib Ovaa (Division of Cell Biology II), the
crystal structure of ATX has been determined, alone and in complex with a smallmolecule
inhibitor. The ATX structure reveals a unique hydrophobic lipid-binding
pocket and allowed us to map key residues required for catalysis and selection
between nucleotide and phospholipid substrates. ATX was found to interact with
cell-surface integrins via its N-terminal somatomedin-B-like domains, using an
atypical mechanism. Our results defi ne determinants of substrate discrimination by
ATX and its family members, suggest how ATX promotes localized LPA signaling,
and enable new approaches to target ATX by small-molecule inhibitors.
The melanoma-derived ATX isoform The prototypic and best studied isoform of
ATX is identical to plasma lysophospholipase D. The original “melanoma-derived”
isoform of ATX (ATXm) contains a 52-residue polybasic insert in the catalytic
domain. The function of this polybasic stretch remains enigmatic to date. We fi nd
that the ATXm insert is cleaved by the protease furin, yet cleavage does not affect the
catalytic activity of ATXm. Proteolytic cleavage of ATXm likely serves to modulate

the interaction of ATXm with negatively charged heparan sulphate proteoglycans at
the cell surface. We are currently exploring this scenario.
LPA receptor signaling
LPA-RhoA signaling: CLIC4 as a new player
LPA receptors couple to the G(13)-RhoA pathway to regulate the actin cytoskeleton.
We previously reported that LPA-induced RhoA activation is consistently
accompanied by rapid recruitment of “Chloride Intracellular Channel” protein 4
(CLIC4) to the plasma membrane. CLIC4 is a soluble protein structurally related to
omega-type glutathione-S-transferases (GSTs) and implicated in various biological
processes, ranging from chloride channel formation to epithelial and endothelial
morphogenesis. We found that Rho-dependent CLIC4 translocation depends on
conserved residues, whose equivalents are critical for the enzymatic function of
GSTs. This suggests that CLIC4 may function as a RhoA-regulated transferase that
catalyses a yet unknown chemical reaction. Through yeast two-hybrid screening
and pull-down assays, we have identifi ed and validated novel binding partners of
CLIC4 that shed new light on its possible function(s) in receptor signalling and
morphogenesis.
Figure 2: The ATX-LPA receptor signaling system. The lipid mediator LPA arises from the cleavage of
lysophosphatidycholine (LPC) by autotaxin (ATX). LPA acts on specifi c G protein-coupled receptors to
stimulate cell proliferation, migration and survival.
Novel LPA receptors: LPA5 and P2Y5
LPA is a potent chemoattractant for many cell types. In contrast, the novel LPA5
receptor was found to mediate inhibition of melanoma cell chemotaxis. LPA5
stimulation leads to a persistent rise in cAMP, which can account at least in part for
the inhibitory action of this receptor. Thus, selective agonists of LPA5 may prove
useful to counteract the migration of tumor cells in which LPA5 is the predominant
receptor.
Another novel LPA receptor, termed LPA6 (encoded by P2RY5), couples specifi cally
to RhoA activation and has been implicated in tumor suppression. We are in the
process of generating conditional P2ry5 knockout mice to establish the importance
of this unique LPA receptor in tumor development (collaboration P. Krimpenfort,
Division of Molecular Genetics).
Publications
25
cell biology i
Jalink K, Moolenaar WH. G proteincoupled
receptors: the inside story.
BioEssays. 2009;32:13-6
Albers HM, Dong A, van Meeteren LA,
Egan DA, Sunkara M, van Tilburg EW,
Schuurman K, van Tellingen O, Morris
AJ, Smyth SS, Moolenaar WH, Ovaa H.
Boronic acid-based inhibitor of autotaxin
reveals rapid turnover of LPA in the
circulation. Proc Natl Acad Sci USA
2010;107:7257-62
Albers HM, van Meeteren LA, Egan DA,
van Tilburg EW, Moolenaar WH, Ovaa
H. Discovery and optimization of boronic
acid based inhibitors of autotaxin. J Med
Chem 2010;53:4958-67
Kremer AE, Martens JJ, Kulik W,
Ruëff F, Kuiper EM, Buuren HR,
Erpecum KJ, Kondrackiene J, Prieto
J, Rust C, Geenes VL, Williamson
C, Moolenaar WH, Beuers U, Oude
Elferink RP. Lysophosphatidic acid is a
potential mediator of cholestatic pruritus.
Gastroenterology 2010;139:1008-18
Cavalli S, Houben A, Albers H, van
Tilburg E, de Ru A, Aoki J, van Veelen P,
Moolenaar WH, Ovaa H. Development
of an activity-based probe for autotaxin.
Chem Bio Chem 2010;11:2311-7
Hausmann J, Christodoulou E,
Kasiem M, De Marco V, van Meeteren
LA, Moolenaar WH, Axford D, Owen
R, Evans G, Perrakis A. Mammalian cell
expression, purifi cation, crystallization
and microcrystal data collection of
Autotaxin/ENPP2, a secreted mammalian
glycoprotein. Acta Crystallogr F
2010;66:1130-5
Chun J, Hla T, Lynch KR, Spiegel
S, Moolenaar WH. International
Union of Pharmacology (IUPHAR):
Lysophospholipid Receptor Nomenclature.
Pharmacol Rev 2010;62:579-87
Hausmann J, Kamtekar S, Christodoulou
E, Day J, Wu T, Fulkerson Z, Albers H,
van Meeteren L, Houben A, van Zeijl L,
Jansen S, Andries M, Hall T, Kasiem M,
Harlos K, Vander Kooi C, Smyth S, Ovaa
H, Bollen M, Morris A, Moolenaar W,
Perrakis A. Structural basis for substrate
discrimination and integrin binding by
autotaxin. Nature Mol Struct Biol 2010
(in press)

26
cell biology i
Publications (continued)
Linn SC, Wesseling J. Molecular tests for
breast-cancer diagnosis? Lancet Onol
2009;10:314-5
Group leader John Collard
John Collard PhD Group leader
Sandra Iden PhD Post-doc
Sander Mertens MSc PhD student
Saskia Ellenbroek MSc PhD student
Ronny Schaefer Technical staff
Helma van Riel Technical staff
Figure 3: Keratinocytes that lack either
the Par3 polarity protein, the Junctional
adhesion molecule JAM-A or the Rac
activator Tiam1, show impaired tight
junction maturation compared to control
keratinocytes (siCtrl). As Par3, Tiam1 and
JAM-A associate with each other these
data suggest that these proteins act in
concert to establish epithelial cell polarity.
GENETIC CONTROL OF INVASION AND METASTASIS
The aim of our research is to identify and characterize genes that play an essential
role in processes of tumor formation and in particular tumor progression. Insight
into the molecular mechanisms that underlie the progression of tumors may lead to
the development of novel diagnostic tools or improved therapeutic strategies.
Rho family proteins Using functional screens we have identifi ed different invasioninducing
genes. Of these, the invasion-inducing Tiam1 gene encodes an activator
(GEF) for the Rho-like GTPase Rac. Rho family proteins, which include Cdc42,
Rac1 and RhoA, control a wide range of biological processes such as cell adhesion,
cell migration and cell polarity. In particular, they act in signaling pathways
that regulate the reorganization of the actin cytoskeleton in response to receptor
stimulation.
Tiam1-Rac signaling and cell polarity Complex formation of GEFs with various
scaffold proteins is an important mechanism to determine signaling towards and
downstream of Rho GTPases. Tiam1 associates with Par3 of the Par polarity complex
- consisting of Par3, Par6 and PKCz - and thereby regulates polarity processes in
various cell types and in different contexts. Tiam1 promotes E-cadherin-based
cell-cell adhesions in epithelial cells whereas knock down of Tiam1 leads to loss
of cell-cell adhesions and epithelial-mesenchymal transition in MDCKII cells.
In keratinocytes, we found that Tiam1 controls tight junction (TJ) formation by
activation of the Par polarity complex, which is required for the establishment
of apical-basal epithelial cell polarity. Interestingly, both Par3 and Tiam1 bind
independently to the junctional adhesion molecule A (JAM-A), which functions as
a membrane localized scaffold to initiate epithelial cell polarity. JAM-A orchestrates
the link between the Par-complex and Tiam1-mediated Rac signaling to control cell
polarization.
In the absence of cell-cell adhesions, epithelial cells develop front-rear polarization
and migrate in a persistent fashion involving Tiam1-Par complex signaling. Upon
the induction of migratory signalling cascades in astrocytes, the MTOC and the
Golgi apparatus are reoriented in a position in front of the nucleus, a process
dependent on Cdc42. The MTOC faces the direction of migration, and Tiam1 is
required for the Rac-mediated protrusion outgrowth in astrocytes but not for the
Cdc42-mediated MTOC orientation. In T lymphocytes, Tiam1-Par signaling is
required for chemokine- and S1P-induced Rac activation and subsequent directional
cell migration. Tiam1-defi cient T-cells show reduced chemotaxis in vitro, and
impaired homing, egress and contact hypersensitivity in vivo. Par/PKCz/Tiam1/Rac
signaling is essential for the stabilization of polarization and effi cient crawling of
T-cells on endothelial cells.
Tumorigenicity in Tiam1-deficient mice Tiam1-defi cient (Tiam1 -/- ) mice develop,
grow, and reproduce normally. In mouse skin, Tiam1 is present in basal and
suprabasal keratinocytes of the epidermis as well as in hair follicles. Tiam1 -/- mice
display resistance to DMBA/TPA-induced (Ras-induced) skin tumor formation
but the frequency of malignant conversion of the tumors that do develop is
increased. We also studied the function of Tiam1 in tumorigenesis induced by other
oncogenic signaling pathways in different cell types. Tiam1 is a Wnt-responsive
gene and promotes b-catenin/TCF-induced intestinal tumor formation. In addition,
mammary tumor formation and progress ion induced by MMTV-c-neu - but not
MMTV-myc - are delayed and reduced in the absence of Tiam1. In general, during
tumor initiation Tiam1-mediated Rac activation promotes survival signals and
thereby prevents apoptosis leading to increased tumorigenicity. As Tiam1 often
functions in conjunction with the Par polarity complex and polarity proteins may
play a role in processes related to different aspects of cancer, we are currently
analyzing the function of polarity proteins in tumor formation. Our results indicate
that similarly to Tiam1-defi ciency, deletion of Par3 in mouse leads to a reduced
number of DMBA/TPA induced skin tumors. Par3-defi cient keratinocytes show also
lower survival signals and increased apoptosis in vitro, suggesting that Tiam1 and
Par polarity proteins affect tumorigenicity by acting in the same signaling pathway.

ACTIN DYNAMICS IN CANCER CELLS
The polymerization of actin monomers into fi laments produces mechanical force
that sculptures protrusions and invaginations on membranes. Actin dynamics
control the remodeling of the plasma membrane and are essential to support cell
migration. Not surprisingly, sophisticated mechanisms have evolved to harness the
activity of actin nucleators, enzymes required for actin to effi ciently form fi laments.
To date, the Arp2/3 complex and Formins are the best characterized actin nucleators
in mammalian cells.
Actin polymerization by the Arp2/3 complex results in the formation of new
actin fi laments arranged in a dendritic meshwork. The WASP/WAVE family of
Nucleation-Promoting Factors (NPFs) stimulates the weak basal activity of the
Arp2/3 complex. WAVE proteins are involved in the formation of lamellipodia,
veil-like protrusion of the plasma membrane. Lamellipodia/ruffl es are the primary
organelles of motility for cells adopting mesenchymal-type movement. WAVE
proteins confi ne Arp2/3-complex activity to the lamellipodium tip, which faces the
plasma membrane. In this way, localized actin polymerization allows the plasma
membrane enclosing lamellipodia/ruffl es to advance. WASP proteins are instead
implicated in endocytosis and traffi cking: they activate the Arp2/3 complex on
clathrin-coated pits and cytosolic vesicles.
Actin polymerization by the Diaphanous-related Formin mDia2 controls the
formation of fi lopodia, fi nger-like extensions of the plasma membrane. At variance
from lamellipodia, the role of fi lopodia in cell migration is still debated. mDia2 also
regulates vesicle traffi cking, which provides supplies to the leading edge of crawling
cells.
Figure 4: HeLa cells
transfected with a
constitutively active
mutant of mDia2.
Filamentous actin
(left) and mDia2
(right) are displayed.
Regulation of WAVE2- and N-WASP-based complexes WAVE- and N-WASPbased
core complexes have been shown to spatially and temporally restrict Arp2/3dependent
actin polymerization. However, they fail to provide a mechanistic
explanation for the high versatility of the WASP/WAVE proteins.
Our research is revealing that dedicated subunits are required to confer functional
specifi city on both WAVE- and N-WASP-based core complexes. Moreover, these
studies are uncovering new and NPF-independent roles for these proteins and
unexpected links between different classes of actin nucleators.
Regulation of mDia2 How mDia2 regulates actin dynamics is still matter of debate.
In order to fully understand the biological function(s) of mDia2, we have isolated its
interactome. This information is helping us dissect the mechanics controlling actin
nucleation by mDia2. In addition, it is suggesting new roles for mDia2.
Mechanisms of formation and roles of filopodia in cell migration Although
fi lopodia are likely to be fundamental in completing both developmental and
homeostatic programs, little is known about their formation and functions in cell
migration. Do fi lopodia originate from lamellipodia and steer crawling cells or are
they repressed by lamellipodia and counteracting cell locomotion? To answer these
questions, we have recently generated a genetically modifi ed cell line that allows us
to induce fi lopodium formation in vitro and to perform systematic studies. This tool
enables us to undertake pioneering loss-of-function genetic and proteomic screens
that will inventory fi lopodium-regulatory proteins and reveal the fi lopodium protein
signature, respectively.
Group leader Metello Innocenti
27
cell biology i
Metello Innocenti PhD Group leader
Zhen Liu PhD Post-doc
Magda Galovic MSc PhD student
Tadamoto Isogai MSc PhD student
Rob van der Kammen MSc Technical staff

28
cell biology i
Publications (continued)
Linn SC, Wesseling J. Molecular tests for
breast-cancer diagnosis? Lancet Onol
2009;10:314-5
Group leader Kees Jalink
Kees Jalink PhD Group leader
Linda Henneman MSc Post-doc
Daan Visser MSc PhD student
Kasia Kedziora MSc PhD student
Jeffrey Klarenbeek MSc Technical staff
Publications
Jalink K, van Rheenen J. Nano-imaging
of membrane topography affects
interpretations in cell biology. Nat
Methods 2010;7:486
Goedhart J, van Weeren L, Hink MA,
Vischer NO, Jalink K, Gadella TW, Jr.
Bright cyan fl uorescent protein variants
identifi ed by fl uorescence lifetime
screening. Nat Methods 2010;7:137-9
Jalink K, Moolenaar WH. G proteincoupled
receptors: the inside story.
Bioessays 2010;32:13-6
Borst JW, Willemse M, Slijkhuis R, van
der Krogt G, Laptenok SP, Jalink K,
Wieringa B, Fransen JA. ATP Changes the
Fluorescence Lifetime of Cyan Fluorescent
Protein via an Interaction with His148.
PLoS ONE 2010;5:e13862
Gloerich M, Ponsioen B, Vliem MJ,
Zhang Z, Zhao J, Kooistra MR, Price
LS, Ritsma L, Zwartkruis FJ, Rehmann
H, Jalink K, Bos JL. Spatial regulation
of cyclic AMP-Epac1 signaling in cell
adhesion by ERM proteins. Mol Cell Biol
2010;30:5421-31
BIOPHYSICS OF CELL SIGNALING
Employing advanced imaging and other biophysical techniques, we study cell
signaling events with high spatial and temporal resolution. Electrophysiological
(e.g. patch clamping) and advanced imaging (e.g. Fluorescence Resonance Energy
Transfer (FRET), Fluorescence Lifetime Imaging (FLIM), Fluorescence Cross
Correlation Spectroscopy (FCCS) and photorelease of caged compounds) are used in
research projects in our group as well as in a number of collaborations within and
outside our institute. We also contribute to the development of hardware, software
and FRET sensors for various intracellular messengers.
The cation channel TRPM7 in the control of invadosomes and invasive
migration. Podosomes and invadopodia are related cytoskeletal structures
implicated in (tumor) cell invasion. These “invadosomes” mediate cell-matrix
contact, sense mechanical forces and serve as focal secretion sites for proteases
that degrade the extracellular matrix. Invadosomes consist of an actin-dense core
surrounded by a characteristic juxtamembrane ring containing contractile and
regulatory proteins. Among these are force-generating myosins, actin-bundling and
-capping proteins, signaling proteins and proteins involved in secretion of proteases.
In an ongoing collaboration with the group of Dr. F.N. van Leeuwen (Nijmegen)
we identifi ed the atypical ‘channel-kinase’ TRPM7 as novel component of the
invadosome ring. TRPM7 is a membrane ion channel fused to a protein kinase
domain which functions as a mechanosensor and regulator of local Ca 2+ entry.
Strikingly, forced expres sion of TRPM7 in neuro blastoma cells is suffi cient to induce
invadosome formation. Phospholipase C (PLC) signaling triggers TRPM7-mediated
Ca 2+ infl ux and enhances invado some formation. Thus, TRPM7 may function as a
master regulator of invadosomes under the control of GPCR signals.
We found that TRPM7 confers a highly invasive phenotype on otherwise noninvasive
neuroblastoma cells, both in vitro (time-lapse imaging, Transwell
assays) and in vivo (tail-vein injection in nude mice). Conversely, RNAi-mediated
knockdown of TRPM7 strongly suppresses migration in MDA-MB-231 breast
carcinoma cells, a model for invasive breast cancer. Moreover, mRNA expression
profi ling of 246 human breast carcinoma specimens reveals that high expression
of TRPM7 at the time of diagnosis predicts a poor prognosis and is correlated with
distant metastasis. This result has now been confi rmed in large published databases
and, by QPCR, in an independent set of tumor biopsies in Nijmegen. These fi ndings
provide strong support for a role of TRPM7 in tumor cell dissemi nation.
Mass spectrometry analysis of TRPM7 immuno-complexes identifi ed some 40
proteins implicated in cytoskeletal regulation, cell adhesion and -migration. The
large majority of these proteins localizes to invadosomes. Proteins associated with
Ca 2+ /PLC signaling are amply represented in the set, suggesting an important
role for these intracellular messengers. Consistent with this notion we fi nd that
TRPM7 medi ates local Ca 2+ infl ux to specifi cally activate PLCd1 in invadosomes,
leading to PIP 2 hydrolysis and sustaining the TRPM7 open-channel state. Our
current investigations address, by combining biophysical readout techniques with
mutational analysis of the TRPM7 C terminus, the details of TRPM7 sensitivity to
Ca 2+ and phosphoinositides, and the exact role of PLCd1 which appears to mediate
a Ca 2+ -infl ux dependent feedback loop in the activation of TRPM7. We have also
started analyzing localization and role of novel invadosome components identifi ed
in the mass spec screen. Strikingly, a high percentage of the protein components
identifi ed in the mass spectrometry screen also correlate signifi cantly with poor
outcome.
Figure 5: TRPM7 overexpression signifi cantly enhances neuroblastoma metastasis to liver. Tail veininjected
N1E-115 neuroblastoma cells specifi cally metastasize to liver and bone. Neuroblasto mas that
overexpress TRPM7 just ~3-fold over endogenous levels are much more effective in invading those tissues.
Tumor load was visualized by luciferase imaging.

DIVISION OF CELL BIOLOGY II
ANTIGEN GENERATION AND PRESENTATION BY MHC CLASS
MOLECULES AND CONTROL OF THE IMMUNE RESPONSE
Our aim is to understand the molecular mechanism of how the immune system
detects infections and tumor cells. Understanding the control of the immune
system also allows manipulation to improve immune responses for cancer control
or to attenuate responses with autoimmunity. We also developed chemical biology
tools to identify small molecules allowing manipulation and to integrate that with
other screens. Ultimately, we aim at identifying target-lead combinations that can be
further developed into strategies for manipulating immune responses and tumors.
In this development program, the effects of compounds and siRNAs are considered
in a broad context. By studying the cell biology of antigen presentation, we can
understand the mechanism of many targets and some compounds in molecular
detail. We use a large variety of state-of-the-art technologies such as high contenthigh
resolution microscopy, lentiviral shRNA screens, chemistry and biophysical
techniques to achieve our goal: translating insights in relevant biological systems
into chemical tools for manipulation.
The turn-over of proteasomes The proteasome is a large abundant and very stable
protein complex involved in the destruction of most intracellular proteins. How
such stable protein complexes are destroyed is unclear but this provides interesting
options for manipulation. We have introduced a color-switch cassette (developed
with Dr F van Leeuwen, NKI) in one of the protein subunits of the proteasome,
which allow us to follow the half-life (ie the decay of the green signal). Since the
green signal also decays by dilution as the result of growth, we arrested yeast growth
by food deprivation. Under these conditions, the proteasome left the nucleus and
aggregated in the cytosol. Following food addition, the aggregates dissolved and the
proteasome entered the nucleus again. We have introduced a yeast knock-out library
in our yeast strain to identify the proteins involved in the various parameters. These
yeast mutants are screened in a timed fashion and analyzed by confocal microscopy.
We have established a semi-automatic microscopy system and automated image
analyses and are currently completing the analyses of hits involved in the various
systems. We aim to identify pathways and will transfer these to mammalian systems
when the mammalian homologues are identifi ed. This should yield new biology and
potentially targets for further manipulation.
Cell Biology of anti-cancer compounds We have compared the cell biological
activities of two topo-isomerase inhibitors; doxorubicin and etoposide. Using cell
biological tools, we showed that histones were transiently leaving chromatin of
non-mitotic cells only when cells were exposed to doxorubicin. This is due to the
anthracyclin structure rather than double strand break formation as result of topo-II
inhibition. Using FAIRE-Seq, we showed that histones are released from gene-
29
cell biology ii
Division head, group leader Jacques Neefjes
Jacques Neefjes PhD Division head
Gosia Garstka PhD Post-doc
Coen Kuijl PhD Post-doc
Victoria Menendez PhD Post-doc
Charlotte Sadaka PhD Post-doc
Tiziana Scanu PhD Post-doc
Jeroen Bakker MSc PhD student
Tineke van den Hoorn MSc PhD student
Marlieke Jongsma MSc PhD student
Baoxu Pang MSc PhD student
Petra Paul MSc PhD student
Izhar Salomon MSc PhD student
Sjoerd van Deventer MSc PhD student
Rik van der Kant MSc PhD student
Amy Wu MSc PhD student
Ruud Wijdeven MSc PhD student
Lennert Janssen BSc Technical staff
Figure 1: Genome-wide landscape of
histone evicton induced by doxorubicin
or aclarubicin treatment. Formaldehyde
Assisted Isolation of Regulatory Elements
followed by next generation sequencing
(FAIRE-seq) is used to explore the whole
human genome. Chromosome 11 is used
as an example. Unique nucleosome free
regions due to histone eviction are observed
when cells were treated with doxorubicin
or aclarubicin, compared to control cells.
Etoposide treatment is similar to control
situation because it could not evict
histones.

30
cell biology ii
Publications
Kok M, Zwart W, Holm C, Fles R,
Hauptmann M, Van ‘t Veer LJ, et al.
PKA-induced phosphorylation of ERalpha
at serine 305 and high PAK1 levels is
associated with sensitivity to tamoxifen in
ER-positive breast cancer. Breast Cancer
Res Treat 2010
Pang B, Neefjes J. Coupled for crosspresentation
in tumor immunotherapy.
Sci Transl Med 2010;2:44ps40
van den Hoorn T, Paul P, Jongsma M,
Neefjes J. Routes to manipulate MHC
Class II Antigen Presentation. Curr Opin
Immunol. 2010 (in press)
van Deventer S, Neefjes J. The
Immunoproteasome Cleans up after
Infl ammation. Cell 2010;142:517-8
Verzijlbergen KF, Menendez-Benito
V, van Welsem T, van Deventer
SJ, Lindstrom DL, Ovaa H, et al.
Recombination-induced tag exchange
to track old and new proteins. Proc Natl
Acad Sci U S A 2010;107:64-8
Zwart W, de Leeuw R, Rondaij M,
Neefjes J, Mancini MA, Michalides R.
The hinge region of the human estrogen
receptor determines functional synergy
between AF-1 and AF-2 in the quantitative
response to estradiol and tamoxifen. J Cell
Sci 2010;123:1253-61
Salomon I, Janssen H, Neefjes
J. Mechanical forces used for cell
fractionation can create hybrid membrane
vesicles. Int J Biol Sci 2010;6:649-54
Zwart W, Peperzak V, de Vries E, Keller
AM, van der Horst G, Veraar EA,
et al. The invariant chain transports TNF
family member CD70 to MHC class II
compartments in dendritic cells. J Cell Sci
2010;123:3817-27
de Wit J, Souwer Y, Jorritsma T, Klaasse
Bos H, ten Brinke A, Neefjes J, et al.
Antigen-specifi c B cells reactivate an
effective cytotoxic T cell response against
phagocytosed Salmonella through crosspresentation.
PLoS One 2010;5:e13016
rich areas. As also H2Ax is released, doxorubicin – but not etoposide – strongly
attenuates DNA repair of double strand breaks induced by the compounds. We show
that the result of these manipulations is massive alterations of the transcriptome
in tissue culture cells as well as in vivo. Especially the heart of mice is affected by
doxorubicin alone, which may correlate to the altered transcriptome.
Using novel technology on an ‘old’ anti-cancer compound allows identifi cation of
novel cell biological mechanisms occurring in response to doxorubicin.
A systems analysis of MHC class II antigen presentation MHC class II
molecules are expressed on so-called Antigen Presenting Cells, such as DC,
monocytes and B cells and control almost all immune responses. Although various
proteins have been defi ned as involved in MHC class II antigen presentation, a full
description of these is lacking. We have performed a fl ow cytometry-based siRNA
screen for factors controlling MHC class II expression and peptide loading.
To place the 290 hits in pathways, we broke down the MHC class II pathway in
two essential components: regulation by transcription and by cell biology. We
determined higher order regulation of a transcriptional network controlling MHC
class II expression by analysis of literature and of yeast two hybrid data that defi ned
a new molecule in the TGFb pathway in control of MHC class II expression.
All hits were also tested in a microscopy-based assay where automated image
analyses and computer-based machine learning were used to place cellular effects
in clusters. These clusters probably represent pathways and this is considered the
fi rst step toward pathway defi nition. To test this, we studied the mechanism of
MHC class II export in dendritic cells following activation signals. We selected
the corresponding cluster and silenced the hits in immature DC while scoring
for a maturated phenotype and identifi ed 8 hits that – when silenced – produced
immature DC with a maturated phenotype. We subsequently identifi ed a novel
pathway operational in human dendritic cells that couple a novel GTPase on MIIC
compartments with myosin Ie motor protein control.
We now aim to further improve our data set by introducing the results of other data
sets published in literature. In addition, a chemical library screen is performed and
now integrated in the siRNA screen to defi ne novel target-lead combinations. This
integrated chemical biology approach should yield biological understanding and
leads for manipulating MHC class II antigen presentation.
Bacterial infection, the PKB/Akt pathway and cancer We have studied the
biology of intracellular infections using a combination of chemical biology and
siRNA screening followed by a cell biological elucidation of pathways. We showed
that inhibiting Akt1 was suffi cient to activate phagosomal fusion with lysosomes and
elimination of the bacteria, by manipulating Rab14. We have identifi ed the effector
for Rab14 that brings together a series of other Rab proteins to control intracellular
transport. We have (in collaboration with Hermen Overkleeft, LIC, Leiden)
generated a kinase compound library to improve our Akt1 inhibitors. This yielded a
highly selective Flt-3 inhibitor that is active against a subset of ALL, and a reasonably
selective Akt1 inhibitor with poor activity on Akt2. We are further improving these
compounds and testing the Flt-3 inhibitors in xenotransplantation mouse models for
ALL.
We repeated the infection experiments with a phosphatase siRNA library and place
phosphatases in kinase networks. We identifi ed the fi ve subunits of PPP2 which
controls Akt. We also identifi ed different other phosphatases. As we performed a
simultaneous chemical library screen for esterases and phosphatases, we aim at
identifying new target-lead combinations with biological activities.
Activation of Akt is an important driver for cell transformation. We tested whether
intracellular bacteria can induce transformation and infected MEFs with one or
multiple pre-transforming mutations with Salmonella before culturing the cells
in soft agar. Soft agar growth was detected provided Akt activation in MEFs with
at least three pre-transforming mutations. This study is now expanded to other
systems and molecular pathology to test the role of bacterial infections in cancer
formation.

ESTROGEN RECEPTOR AND BREAST CANCER
Resistance to tamoxifen treatment is observed in about half of the recurrences in
breast cancer where the anti-estrogen tamoxifen acquires agonistic properties for
transactivation of ERa. Tamoxifen resistant breast cancer often remains sensitive to
other endocrine treatments and the tumors still express ERa. The clinical benefi ts
of identifying pathways underlying resistance can therefore be profound, as a way
to initiate personalized medicine where tamoxifen is only given to patients that
are going to respond. We aim for molecular understanding of the mechanism
of resistance to tamoxifen and other anti-estrogens and to develop tools to use as
clinical decision markers in anti-estrogen therapy of breast cancer patients.
Target(s) of ERa phosphorylated at Serine 305 by Protein kinase A
The target(s) for ERa-S305P were identifi ed from comparing the transcriptomes of
U2OS containing a stably integrated ERa wt or the S305A mutant that cannot be
phosphorylated by Protein Kinase A (PKA). The cells were treated with tamoxifen
and PKA was activated for up to 24 hours. This resulted in 709 hits specifi c for
ERa-S305P under tamoxifen conditions. Tumor relevant hits were further identifi ed
by comparison with expression profi les of 58 primary breast tumors, of which
the metastases were treated with tamoxifen, and of which concurrent ERa-S305P
staining was known. This resulted in 17 targets associated with presence of ERa-
S305P and 6 targets associated with early recurrence in this tumor set. These
numbers were reduced to respectively six and two by comparing these targets with
expression profi les of tamoxifen-insensitive breast tumors in a meta-analysis study.
The resulting eight hits that fulfi lled all criteria are subject of further studies on the
tumor relevant target(s) of ERa-S305P.
Properties of ERa phosphorylated at Serine 305 by Protein kinase A
Protein stability studies indicated that the phosphorylation of ERa at Serine 305 by
PKA is rapidly lost during tamoxifen exposure as the result of dephosphorylation
or degradation. We will study this ligand-specifi c and conformation-dependent loss
of ERa-Serine 305P in more detail. In collaboration with Pamgene (Den Bosch), we
observed that ERa phosphorylated at Serine 305 recruits cofactors more effi ciently in
the presence of tamoxifen than the non-phosphorylated form of ERa. This further
underlines the signifi cance of phosphorylation of ERa at Serine 305 as a mechanism
of tamoxifen resistance.
Clinical relevance of phosphorylation of ERa In collaboration with Marleen Kok
and Sabine Linn (Division of Molecular Biology) and Goran Landberg and colleagues
(University of Lund), we found that phosphorylation of the Estrogen Receptor at
Serine 305 predicted a poor response in a randomized trial that evaluated adjuvant
tamoxifen treatment as well as in metastatic breast cancer patients. A combined
measurement of two independent markers, phosphorylation of the Estrogen
Receptor at Serine 305 by Protein kinase A and expression of the kinase PAK-1,
identifi ed approximately 60% of all tamoxifen resistant cases of ER-positive breast
cancer. The opposite has been found for phosphorylation of Serine 118 in ERa,
which is associated with an activated ER and with a benefi t from treatment with
tamoxifen.
We now studied two activated kinases, i.e. phospho-ERK1/2 and phospho-PKA in
order to obtain a more detailed view of the in vivo association between ERK1/2,
PKA, PAK1 and ERaS118-P and ERaS305-P as well as response to tamoxifen.
Phospho-ERK1/2 (pERK1/2) was signifi cantly linked to increased phosphorylation
of ERaS118-Pas well as ERaS305-P. There was an association between cytoplasmic
pPKA and pERK1/2, suggesting a crosstalk between these two kinase pathways.
Surprisingly, PAK1 did not correlate to any of the ERa phosphorylation sites or
analysed kinases. In an analysis of subgroups, patients with high expression of
ERaS118-P still had a better recurrence-free survival compared to control patients.
Overall, our results indicate that resistance to tamoxifen is largely determined by
two opposite effects of phosphorylation of ERa.
Group leader Rob Michalides
31
cell biology ii
Rob Michalides PhD Group leader
Xanthippi Alexi PhD Post-doc
Renée de Leeuw MSc PhD student
Cristiane Bentin Toaldo Technical staff
Desiree Verwoerd Technical staff
Publications
Zwart W, de Leeuw R, Rondaij M,
Neefjes J, Mancini M and Michalides R.
The hinge region of the human Estrogen
Receptor determines functional synergy
between AF-1 and AF-2 in the quantitative
response to estradiol and tamoxifen. J.Cell
Science 2010;123:1253-1261
Kok M, Zwart W, Holm C, Renske
Fles R, Hauptmann M, Van ’t Veer
LJ, Wessels LFA, Neefjes JJ, Stål O,
Linn SC, Landberg G and Michalides
R. Predictive algorithm for tamoxifen
resistance in breast cancer patients based
on PKA-induced phosphorylation of ER
at serine 305 and PAK1. Breast Cancer Res
Treatment 2011;125:1-12
Wigerup C, Kok M, Svensson-Mansson
S, Hauptmann M, Michalides R, Stål O,
Linn S, Landberg G. In vivo association
between kinase activation and ER
phosphorylation in premenopausal breast
cancer and links to tamoxifen response.
Breast Cancer Res Treatment 2011
(in press)
de Vries-van Leeuwen IJ, da Costa
Perreira D, Yalcin Z, Michalides R,
Feenstra KA, de Boer AH. 14-3-3 binding
to the Estrogen Receptor Alpha F-domain
is enhanced by the small molecule
ligand Fusicoccin and reduces receptor
dimerization and activation. Proc Natl
Acad Sci USA, 2011

32
cell biology ii
Group leader Huib Ovaa
Huib Ovaa PhD Group leader
Boris Rodenko PhD Senior post-doc
Alessia Amore PhD Post-doc
Farid El Oualid PhD Post-doc
Remco Merkx PhD Post-doc
Sander van Kasteren PhD Post-doc
Paul Geurink PhD Post-doc
Celia Berkers PhD Post-doc
Harald Albers MSc PhD student
Annemieke de Jong MSc PhD student
Reggy Ekkebus MSc PhD student
Rieuwert Hoppes MSc PhD student
Henk Hilkmann Ing Technical staff
Dris El Atmioui Ing Research assistant
Kim Wals Ing Research assistant
Yves Leestemaker MSc Research assistant
Dharjath Hameed MSc Research assistant
Publications
Albers HM, Dong A, van Meeteren LA,
Egan DA, Sunkara M, van Tilburg EW,
et al. Boronic acid-based inhibitor of
autotaxin reveals rapid turnover of LPA in
the circulation. Proc Natl Acad Sci U S A.
2010;107:7257-62
Albers HM, van Meeteren LA, Egan DA,
van Tilburg EW, Moolenaar WH, Ovaa
H. Discovery and optimization of boronic
acid based inhibitors of autotaxin. J Med
Chem. 2010;53:4958-67
Berkers CR, de Jong A, Ovaa H, Rodenko
B. Transpeptidation and reverse proteolysis
and their consequences for immunity.
Int J Biochem Cell Biol. 2009;41:66-71
Berkers CR, Ovaa H. Drug discovery
and assay development in the ubiquitinproteasome
system. Biochem Soc Trans.
2010;38:14-20
Cavalli S, Houben AJ, Albers HM,
van Tilburg EW, de Ru A, Aoki J, et al.
Development of an activity-based probe for
autotaxin. ChemBioChem. 2010;11:2311-7
Hoppes R, Ekkebus R, Schumacher
TN, Ovaa H. Technologies for MHC
class I immunoproteomics. J Proteomics.
2010;73:1945-53
CHEMISTRY AND BIOLOGY OF UBIQUITIN-MEDIATED
PROTEOLYSIS AND ANTIGEN PRESENTATION
The proteasome is a multi-catalytic proteolytic machine that is abundant and
responsible for the turnover of many critical regulatory proteins including
tumor suppressor proteins and cell cycle regulators. The destructive force of
the proteasome as an important determinant of protein half-life is regulated by
ubiquitination. Substrates are tagged with multiple ubiquitin (Ub) molecules for
destruction by the proteasome. Ubiquitin is a 76 amino acid protein that can be
conjugated onto substrates to guide protein destruction. The majority of proteins are
targeted for proteasomal proteolysis by Ub polymers. Despite a wealth of literature
on ubiquitination of proteasome substrates, little is known about the degradation
process at a more detailed molecular level; ubiquitination status and protein stability
currently cannot be predicted. It is clear however, that a ubiquitin code exists and
that protein turnover by the proteasome is a tightly regulated and complex process
that includes not only the complexity of the proteasome but also Ub polymer
formation and remodeling and Ub recycling. Because of this complexity, tools that
allow detailed studies of the effects of ubiquitination status on protein turnover are
urgently needed.
Our lab aims to develop tools to profi le cellular enzymatic activities associated with
post-translational modifi cation of proteins with ubiquitin and we study proteasome
activity, antigen production and antigen presentation by designing tools that
interfere with individual components of these systems. We search for inhibitors of
enzymatic activities and ligands of receptors both by high throughput screening
of small molecule compound collections using in vitro biochemical screens and
cell-based assays and by rational chemical design followed by chemical optimization
and all biochemistry further required. Ligands and inhibitors form the basis for the
development of research probes that we use to achieve our goals.
Research is centered around one central theme: chemistry and biology of ubiquitinmediated
proteolysis and MHC class I antigen presentation, and divided into three
main topics:
(1) ubiquitin chain chemistry, biochemistry and proteomics
(2) proteasome inhibition and activation
(3) MHC class I antigen presentation
Chemical Synthesis of Ubiquitin conjugates By the concerted action of E1, E2
and E3 enzymes, Ub is linked to target proteins via an isopeptide bond between the
Ub C-terminal carboxylate and the -amine of a lysine residue or N-terminus of the
target protein. The ability to generate well-defi ned Ub conjugates biochemically is
limited by the requirement of prior identifi cation and production of specifi c E2/
E3 enzymes. In addition, the generation of Ub mutants by biochemical methods is
limited to the natural amino acid repertoire. To have full control over Ub conjugation
and structure, we have recently developed chemical approaches that allow us to
synthesize virtually any well-defi ned Ub conjugate and mutant (fi gure 2). Using
Figure 2:
A) Amino acid sequence
of Ubiquitin. B) LC profi le
of a commercial Ubiquitin
sample and C) our crude
synthetic Ubiquitin.
D) Mass spectrometric
analysis of crude synthetic
Ub, calc 8565 Da, found
8565 Da (deconvoluted
spectrum right).

these chemistries, we prepared fl uorogenic Ub conjugates and showed that these
are fully functional (fi gure 3). In another synthetic effort we focused on the ability
of Ub to be self-conjugated onto any of its 7 lysine residues (i.e. K6, K11, K27, K29,
K33, K48 and K63). Although all linkages have been identifi ed in cells, only K11,
K48- and K63-linkages have been studied so far as these are the only linkages that
can currently be obtained biochemically. To overcome this limitation, we have
developed a very effective synthetic approach towards all seven isopeptide linked
diUb conjugates. Figure 4 shows analysis of the synthesized K6, K11, K27, K29 and
K33 linked diUb conjugates. Overall, routine strategies for the chemical construction
of Ub mutants, Ub chains or specifi c C-terminal modifi cations, now bring virtually
any Ub derivative within practical reach.
Chemical reporters of UPS activity Pharmacological interference with UPSmediated
protein degradation holds much promise. However, the only example
of pharmacological modulation of the UPS approved for use in the clinic so far is
the proteasome inhibitor bortezomib and tools to study proteasome action are in
demand. A chemical approach using irreversible covalent inhibitors equipped with
reporter groups offers several advantages over traditional approaches, including their
applicability to any cell line or tissue. We recently developed such probes which have
been shown to provide information that correlates directly with the functional state
of enzyme active sites: active forms only and not latent or (auto)inhibited activities
are reported. Using fl uorescent proteasome activity reporters we have analyzed
proteasome activity in mice and we were able to monitor pharmacological inhibition
in vivo and to visualize active proteasomes in cells both by confocal microscopy and
fl ow cytometry.
Conditional MHC class I ligands for epitope mapping MHC-bound peptides
are essential for the stability of the MHC class I complex. Hence, standard strategies
for the production of recombinant MHC complexes are based on in vitro refolding
reactions with specifi c peptides and this severely limits the ability to produce
large collections of peptide-MHC complexes. To address this issue, we developed
in collaboration with the Schumacher lab conditional MHC ligands that can be
cleaved in the MHC bound state under conditions that do not affect the integrity
of the MHC structure. MHC class I molecules can effi ciently be produced with
these conditionally cleavable peptide ligands. These UV-labile ligands have been
shown to disintegrate in the MHC-bound state upon exposure to UV light under
mild conditions (neutral pH, 4-37 o C). Importantly, when UV-mediated cleavage is
performed in the presence of another MHC binding peptide, an effi cient ligand
exchange reaction results in a class I molecule complexed with the epitope of
choice. Ligands that disintegrate on command have now been identifi ed for various
different human MHC alleles indicating that it is straightforward to identify
conditional ligands for other MHC class I alleles. Furthermore, this MHC exchange
technology has been adapted to high-throughput applications in automated ELISA
and fl uorescence polarization assays.
Improvements and development of new technologies to profi le enzymatic UPSrelated
activities and MHC loading will allow new approaches to understanding
the ubiquitin proteasome system and antigen presentation, unraveling novel
phenomena. Despite major achievements in chemical probe development and in
chemical investigations into the ubiquitin proteasome system and MHC class I
antigen presentation, many challenges remain.
Publications (continued)
33
cell biology ii
Shanmugham A, Fish A, Luna-Vargas
MP, Faesen AC, El Oualid F, Sixma TK,
et al. Nonhydrolyzable ubiquitinisopeptide
isosteres as deubiquitinating
enzyme probes. J Am Chem Soc.
2010;132:8834-5
Verzijlbergen KF, Menendez-Benito
V, van Welsem T, van Deventer
SJ, Lindstrom DL, Ovaa H, et al.
Recombination-induced tag exchange
to track old and new proteins. Proc Natl
Acad Sci U S A 2010;107: 64-8
Ovaa H, Kuijl C, Neefjes, J. Recent
and new targets for small molecule anticancer
agents. Drug Discovery Today:
Technologies. e3-e11
Frederiks F, Stulemeijer IJE, Ovaa H,
van Leeuwen F. A modifi ed epigenetics
tool box to study histone modifi cations
on the nucleosome core. ChemBiochem.
2010 2010;12:308-313
El Oualid F, Merkx R, Ekkebus R,
Hameed DS, Smith J, de Jong A,
et al. Chemical Synthesis of Ubiquitin,
Ubiquitin-Based Probes, and Diubiquitin.
Angew Chem Int Ed Engl. 2010
2010;49:10149-10153
Figure 3: Turnover of commercial and
synthetic UbAMC by the deubiquitinating
enzyme USP7 shows identical Michaelis-
Menten kinetics.
Figure 4: A) SDS-PAGE analysis of
synthetically prepared K6, K11, K27, K29
and K33 linked diUb conjugates.
B) LC-MS analysis of purifi ed K33 linked diUb.
C) Deconvulated mass spectrum of K33 linked
diUb (calc 17112 Da, found 17113 Da)

34
cell biology ii
Group leader Peter Peters
Peter Peters PhD Group leader
Nicole Van der Wel PhD Associate staff scientist
Sue Godsave PhD Post-doc/EU project manager
Pekka Kujala PhD Post-doc
Musa Sani PhD Post-doc
Matthijn Vos PhD Post-doc
Abdallah Abdallah PhD Post-doc
Diane Houben MSc PhD student
Jason Pierson MSc PhD student
Hans Jansen BSc Technical staff
Maaike van Zon BSc Technical staff
Karin de Punder BSc Technical staff
Nico Ong Research assistant
Publications
Pierson J, Ziese U, Sani M, Peters PJ.
Exploring vitreous cryo-section-induced
compression at the macromolecular
level using electron cryo-tomography;
80S yeast ribosomes appear unaffected.
J Struct Biol. 2010
Sani M, Houben EN, Geurtsen J, Pierson J,
de Punder K, van Zon M, Wever B,
Piersma SR, Jiménez CR, Daffé M,
Appelmelk BJ, Bitter W, van der Wel N,
Peters PJ. Direct visualization by cryo-EM
of the mycobacterial capsular layer: a
labile structure containing ESX-1-secreted
proteins. PLoS Pathog. 20105;6:e1000794
Barker N, Huch M, Kujala P, van de
Wetering M, Snippert HJ, van Es JH, Sato
T, Stange DE, Begthel H, van den Born M,
Danenberg E, van den Brink S, Korving
J, Abo A, Peters PJ, Wright N, Poulsom
R, Clevers H. Lgr5(+ve) stem cells drive
self-renewal in the stomach and build longlived
gastric units in vitro. Cell Stem Cell.
2010;6:25-36
Weerdenburg EM, Peters PJ, van der
Wel NN. How do mycobacteria activate
CD8+ T cells? Trends Microbiol.
2010;18:1-10
NANOBIOLOGY
The clue of the treatment of various diseases is inside biological nanomachines.
Investigating these machines becomes more attractive and serious when there is a
possibility to realize these experiments without isolation. Obtaining high resolution
images at 3nm from cryo-electron microscopy tomograms from native cell will
give immense information. Besides our work on mycobacteria we have therefore
made a main focus in our research line on visualization of nanomachines in their
native cellular environment and strive to integrate this in cancer and mycobacteria
research.
The cellular nanocosm is made up of numerous types of macromolecular complexes
or biological nanomachines. These form functional modules that are organized
into complex subcellular networks. Information on the ultra-structure of these
nanomachines has mainly been obtained by analyzing isolated structures, using
imaging techniques such as X-ray crystallography, NMR, or single particle electron
microscopy (EM). Yet there is a strong need to image biological complexes in a native
state and within a cellular environment, in order to gain a better understanding
of their functions. Emerging methods in EM are now making this goal reachable.
Cryo-electron tomography bypasses the need for conventional fi xatives, dehydration
and stains, so that a close-to-native environment is retained. As this technique is
approaching macromolecular resolution, it is possible to create maps of individual
macromolecular complexes. Atomic structures made by X-ray and NMR can
be ‘docked’ or fi tted into the lower resolution particle density maps to create a
macromolecular atlas of the cell under normal and pathological conditions. The
majority of cells, however, are too thick to be imaged in an intact state and therefore
methods such as ‘high pressure freezing’ and ‘cryo-sectioning of unperturbed
vitreous fully hydrated samples’ have been introduced for electron tomography.
This year we continued on improving methods for visualizing nanomachines in a
close-to-physiological, cellular context. EM is in a renaissance and with a Dutch team
of colleagues we obtained grants to create a national centre of large infrastructure
where two Titan Krios cryo-TEMs will be located. For more information see www.
necen.nl.
Cryo-electron tomography of vitreous cryo-sections is the most suitable method
for exploring the 3D organization of biological samples that are too large to be
imaged in an intact state. Producing good quality vitreous cryo-sections, however,
is challenging. We focused on the major obstacles to success: contamination in
and around the microtome, and attachment of the ribbon of sections to an electron
microscopic grid support fi lm. The conventional method for attaching sections to
the grid has involved mechanical force generated by a crude stamping or pressing
device, but this disrupts the integrity of vitreous cryo-sections. Furthermore,
attachment is poor, and parts of the ribbon of sections are often far from the support
fi lm. This results in specimen instability during image acquisition and subsequent
diffi culty with aligning projection images. We have implemented a protective glove
box surrounding the cryo-ultramicrotome that reduces the humidity around and
within the microtome during sectioning. We also introduced a novel way to attach
vitreous cryo-sections to an EM grid support fi lm using electrostatic charging with
a major effort of the Dutch company ‘Simco’ as a gesture of charity. The ribbon of
vitreous cryo-sections remains in place during transfer and storage and is devoid
of stamping related artifacts. We illustrated these improvements by exploring the
structure of putative cellular 80S ribosomes within 50nm, vitreous cryo-sections of
Saccharomyces cerevisiae.
We then explored vitreous cryo-section-induced compression at the macromolecular
level using electron cryo-tomography and focused on the 80S ribosomes. Vitreous
cryo-section-induced compression infl uences the interpretation and the reliability
of electron microscopy images and tomographic reconstructions. We showed this
year that electron cryo-tomographic reconstructions of vitreous cryo-sections show
that 80S ribosomes, both intracellular and in an isolated state, appear able to resist
section-induced compression.

Figure 5: Proposed
cross-presentation
pathways from
literature.
Mycobacteria Infl ammation is a localized protective reaction to injury or infection,
but it also has a pathogenic role in many diseases, including cancer. Whereas acute
infl ammation is critical for host defense, chronic infl ammation can contribute
to tumorigenesis and metastatic progression. The latter, including macrophages
and lymphocytes, are important elements of the tumour microenvironment.
Mycobacterium tuberculosis is a devastating pathogen that kills >1.5 million people
each year, but controlled use of its cell wall activates macrophages in ways that can
be harnessed for therapy. For example, M. bovis Bacille Calmette-Guérin (BCG) is
one of the most widely used antitumor adjuvant therapies in humans. Injection
of BCG into the bladder mediates regression of transitional cell carcinomas by
stimulating a vigorous local immune response, bathing tumors in cytokines and
activated immune cells. That is why we focused earlier this year on writing a review
called “How do mycobacteria activate T cells?”
CD8(+) T cells are activated upon presentation of antigens from the cytosol.
Therefore, it was unclear how pathogenic mycobacteria could prime this type of
lymphocyte, given that these microbes were thought to remain in phagosomes
and, hence, be shielded from the host cytosol. Recently, we showed that pathogenic
mycobacteria can enter the cytosol through translocation from phagolysosomes
(Cell 2008), providing a direct mechanism for CD8(+) T cell priming. However,
this mechanism does not apply to other mycobacteria such as BCG, which do not
enter the cytosol. We discussed the different hypotheses to explain the induction of
CD8(+) T cell responses in mycobacterial infections.
A lot of effort went into the direct visualization by cryo-EM of the mycobacterial
capsular layer. The cell envelope of mycobacteria, a group of Gram positive bacteria,
is composed of a plasma membrane and a Gram-negative-like outer membrane
containing mycolic acids. In addition, the surface of the mycobacteria is coated
with an ill-characterized layer of extractable, non-covalently linked glycans, lipids
and proteins, collectively known as the capsule, whose occurrence is a matter of
debate. By using plunge freezing cryo-electron microscopy technique, we were able
to show that pathogenic mycobacteria produce a thick capsule, only present when
the cells were grown under unperturbed conditions and easily removed by mild
detergents. This detergent-labile capsule layer contains arabinomannan, alphaglucan
and oligomannosyl-capped glycolipids. Further immunogenic and proteomic
analyses revealed that Mycobacterium marinum capsule contains high amounts of
proteins that are secreted via the ESX-1 pathway. Finally, cell infection experiments
demonstrated the importance of the capsule for binding to cells and dampening
of pro-infl ammatory cytokine response. Together, this study showed a direct
visualization of the mycobacterial capsular layer as a labile structure that contains
ESX-1-secreted proteins.
We continued our successful collaboration with the laboratory of Hans Clevers
(Hubrecht Institute, Utrecht).
Publications (continued)
35
cell biology ii
Pierson J, Fernández JJ, Bos E, Amini S,
Gnaegi H, Vos M, Bel B, Adolfsen F,
Carrascosa JL, Peters PJ. Improving the
technique of vitreous cryo-sectioning for
cryo-electron tomography: electrostatic
charging for section attachment and
implementation of an anti-contamination
glove box. J Struct Biol. 2010;169:219-25
Figure 6: The spatial organization of the
mycobacterial cell envelope exhibiting the
capsule. This scheme represents the relative
size and organization of the different
layers of the envelope including the plasma
membrane, mycomembrane, periplasmic
space and capsular layer. The positions
of some of the constituents analyzed are
depicted.

36
experimental therapy
Division head, group leader Adrian Begg
Adrian Begg PhD Group leader
Conchita Vens PhD Research associate
Caroline Verhagen MD PhD student
Monique De Jong MD PhD student
Ingrid Hofl and Technical staff
Manon Verwijs Technical staff
DIVISION OF EXPERIMENTAL THERAPY
MECHANISMS AND MODULATION OF RADIOSENSITIVITY
Fanconi Anemia (FANC) mutations in sporadic head and neck cancer Patients
with defects in the FANC pathway have an increased incidence of head and neck
cancer (HNSCC) indicating an involvement of the Fanconi DNA repair pathway in
the development of sporadic HNSCC. This repair pathway is essential to cell survival
upon exposure to crosslinking agents such as mitomycin C (MMC) and cisplatin.
Since combinations of cisplatin with radiation are the standard therapy option for
patients with advanced HNSCC, this might have implications for the clinic.
A panel of 30 primary HNSCC tumour cell lines derived from patients was therefore
studied as basis for the evaluation of the functional integrity of the FANC pathway
in sporadic HNSCC. These tumour cell lines were tested for MMC-induced G2 cell
cycle blocks, as seen in FANC pathway defective cells. Approximately 20% of the cell
lines showed blocks similar to confi rmed FANC A or G defi cient human fi broblasts.
We further analysed survival after MMC in these cells and found a considerable
proportion of the cell lines showed a sensitive phenotype, comparable to the FANCdefi
cient controls. G2 block data correlated well with the survival data. The cell lines
are being further characterized by mutation and expression analyses in order to
identify the FANC defects, which will be confi rmed by further functional assays.
In parallel, we are carrying out high throughput DNA sequencing on a cohort of
HNSCC tumours for aberrations and mutations in FANC genes and several other
DNA repair genes. For this purpose a capture array has been designed that enriches
for over 500 selected genes involved in DNA repair and shown to play a role in
HNSCC. First tests show good enrichment and specifi city, also indicating high
coverage during sequencing, thereby optimizing output and sensitivity.
Figure 1: CD44 mRNA
expression is predictive
for local control after
radiotherapy for early
stage larynx cancer.
Prediction of outcome In a recent study of early stage larynx tumors from patients
treated with radiotherapy alone, we found that expression of the putative stem
cell marker CD44 was the most signifi cant predictor of local control. This was
validated in a separate series of larynx tumors in which CD44 staining intensity
and frequency on a tissue microarray made from pretreatment biopsies correlated
with outcome after radiotherapy. We have therefore pursued the relationship of
CD44 expression with factors known to infl uence radiotherapeutic response. We
found no relationship of CD44 expression with intrinsic radiosensitivity in a series
of 9 larynx cancer cell lines. Further, we knocked down CD44 expression in two
squamous cell lines using an inducible shRNA vector and again found no effect on
radiosensitivity. In a study to test for a possible relationship with hypoxia, we found
no relationship between CD44 expression and either CA9 or HIF-1 alpha expression

y immunohistochemistry on the larynx tissue microarray. Further, in a series of
patients given the hypoxia marker drug pimonidazole, no relationship was found
between pimonidazole staining and CD44 staining. Finally, a signifi cant correlation
was found between CD44 expression and plating effi ciency in the 9 larynx cell lines.
Figure 2: Example of the
concept of tumor targeted
radiosensistization:
tumor cells defi cient in base
excision repair (PolBDN)
show greater radiosensitization
(black arrow) by an ATM
inhibitor (ATMi) than repair
profi cient cells (Vec: grey
arrow).
These data together support the idea that stem cell content is the dominant factor
determining outcome of early laryngeal cancer treated with radiotherapy.
In an expression profi ling study on 33 head and neck carcinoma cell lines, we
found a “classifi er” of 288 genes showing a signifi cant correlation with intrinsic
radiosensitivity. Of interest was the presence of epithelial-mesenchymal transition
(EMT) genes in this gene set. To study the role of EMT genes more directly, we
expressed a mutant HIF1alpha containing a deletion in the oxygen dependent
degradation domain (HIF1-deltaODD). This forced EMT and these cells were
found to be more radioresistant. In a second cell line pair, EMT forced by SNAIL
expression was also accompanied by increased radioresistance. The relationship of
radioresistance with EMT has to our knowledge not been previously reported.
Publications
37
experimental therapy
De Jong MC, Pramana J, Knegjens
JL, Balm AJ, Van den Brekel MW,
Hauptmann M, Begg AC, Rasch CR.
HPV and high risk gene expression profi les
predict response to chemoradiotherapy
in head and neck cancer, independent
of clinical factors. Radiother Oncol.
2010;95:365-70
Neijenhuis S, Verwijs-Janssen M, Van
den Broek L, Begg AC, Vens C. Targeted
radiosensitization of cells expressing
truncated DNA polymerase beta. Cancer
Res. 2010;70:8706-14
De Jong MC, Pramana J, Van der Wal JE,
Lacko M, Peutz-Kootstra CJ, De Jong JM,
Takes RP, Kaanders JH, Van der Laan BF,
Wachters J, Jansen JC, Rasch CR, Van
Velthuysen MF, Grénman R, Hoebers FJ,
Schuuring E, Van den Brekel MW, Begg
AC. CD44 Expression Predicts Local
Recurrence After Radiotherapy In Larynx
Cancer. Clin Cancer Res. 2010;16:5329-38
Vens C, Begg AC.Targeting base excision
repair as a sensitization strategy in
radiotherapy. Semin Radiat Oncol.
2010;20:241-9
Begg AC. Radiobiology: state of the present
art. A conference report. Int J Radiat Biol.
2010;86:71-8
Verheij M, Vens C, Van Triest B. Novel
therapeutics in combination with
radiotherapy to improve cancer treatment:
rationale, mechanisms of action and
clinical perspective. Drug Resist Update.
2010;13:29-43
West C, Rosenstein BS, Alsner J,
Azria D, Barnett G, Begg A, Bentzen S,
Burnet N, Chang-Claude J, Chuang E,
Coles C, De Ruyck K, De Ruysscher D,
Dunning A, Elliott R, Fachal L, Hall J,
Haustermans K, Herskind C, Hoelscher
T, Imai T, Iwakawa M, Jones D, Kulich
C; EQUAL-ESTRO, Langendijk JH,
O’Neils P, Ozsahin M, Parliament M,
Polanski A, Rosenstein B, Seminara D,
Symonds P, Talbot C, Thierens H, Vega
A, West C, Yarnold J. Establishment of a
Radiogenomics Consortium. Int J Radiat
Oncol Biol Phys. 2010;76:1295-6

38
experimental therapy
Publications (continued)
Linn SC, Wesseling J. Molecular tests for
breast-cancer diagnosis? Lancet Onol
2009;10:314-5
Group leader Fiona Stewart
Fiona Stewart PhD Group leader
Nicola Russell MD PhD Academic staff
Paul Baas MD PhD Academic staff
Fijs Van Leeuwen PhD Academic staff
Saske Hoving PhD Post-doc
Marion Scharpfenecker PhD Post-doc
Ingar Seemann MSc PhD student
Ben Floot Technical staff
Johannes Te Poele Technical staff
Nils Visser Technical staff
Publications
Hoving S, Heeneman S, Gijbels MJJ, Te
Poele JAM, Bolla M, Pol JFC, Simons MY,
Russell NS, Daemen MJ, Stewart FA.
NO-donating aspirin and aspirin partially
inhibit age-related atherosclerosis but not
radiation-induced atherosclerosis in ApoE
null mice. PLoS one 2010;5:e12874
Stewart FA, Hoving S, Russell NS.
Vascular damage as an underlying
mechanism of cardiac and cerebral toxicity
in irradiated cancer patients. Radiat Res.
2010
Rosenfelder N, Stewart F, Brada M.
Vascular effects of radiation in the central
nervous system. In: Shrieve DC, Loeffl er
JS (eds). Human Radiation Injury.
Philadelphia: Lippincott Williams &
Wilkins, 2010 (in press)
RADIATION-INDUCED VASCULAR DAMAGE
Vascular damage in normal tissues is a serious late complication of cancer
patients after radiotherapy. This manifests as atherosclerosis in large vessels and
telangiectasia (dilated, thin-walled blood vessels prone to bleeding) and perfusion
defects in capillaries. In our studies we focus on the mechanisms of development of
radiation-induced vascular damage, with the ultimate goal of designing appropriate
intervention strategies to inhibit or prevent this.
Radiation-induced microvascular damage and fibrosis development
(collaboration with Peter ten Dijke, LUMC, Leiden)
The mechanisms whereby telangiectasia develop are largely unclear. The vascular
phenotype suggests that aberrant repair responses, including TGF- signalling, are
involved. In addition to vascular injury, irradiation causes excessive connective tissue
formation (fi brosis). Fibrosis develops mainly through the actions of TGF- and its
downstream targets.
We used irradiated mouse kidneys (a microvascular rich organ) as a model to study
the molecular mechanisms and cellular alterations leading to telangiectasia and
fi brosis. These studies identifi ed the TGF- co-receptor endoglin as being critically
involved in both processes. Mice expressing reduced levels of endoglin (Eng +/- mice)
developed less radiation-induced vascular damage and less fi brosis. Eng +/- mice
showed reduced TGF- levels and decreased expression of downstream target genes
related to fi brosis (Serpine1, Ctgf, and Col3a1). However, this was not suffi cient to
explain the vascular phenotype in Eng +/- mice as vascular repair genes (Vegfa, Id1)
were unaffected. We also did not measure any differences in proliferation, which
might have explained improved vessel repair in irradiated Eng +/- mice.
Further studies showed that irradiation triggered leukocytes infi ltration, which was
reduced in Eng +/- mice. As leukoctyes are a rich source of cytokines, chemokines
and growth factors, we investigated whether these cells might infl uence vascular
repair after irradiation. Immunohistochemical studies showed that the infi ltrate
mainly consisted of macrophages. Comparison of the mRNA expression profi le
in irradiated in wild type and Eng +/- kidneys showed that Eng +/- mice expressed
reduced levels of the pro-infl ammatory/angiogenic/fi brotic cytokines Il6 and Il1 .
Moreover, when we studied the ability of isolated bone marrow-derived cells from
wild type and Eng +/- mice to respond to LPS stimulation, we detected impaired
upregulation of Il6 and Il1 in cells derived from Eng +/- mice. Staining of irradiated
kidneys confi rmed that these molecules are indeed produced by macrophages.
These results suggest that pro-infl ammatory cytokine production by macrophages
contributes to the vascular and fi brotic phenotype after irradiation.
In a new project we will investigate the role of endoglin on bone-marrow-derived
cell infi ltration and cytokine production, versus its role in endothelial cells.
Furthermore, we will interfere with infl ammatory cell infi ltration and cytokine
production in order to prevent erroneous repair and fi brosis development. In
addition, we are aiming at stimulating proper vascular repair by promoting the
recruitment of endothelial progenitor cells. We will also investigate the effects of
combining anti-vascular treatments used in the clinic with radiotherapy on the
development of late normal tissue damage.
Radiation induced atherosclerosis in large vessels (collaboration with Mat
Daemen, Cardiovascular Research Institute, Maastricht)
We have previously shown that irradiation (1 x 8-14 Gy or 20 x 2 Gy in 4 weeks)
accelerated the development of atherosclerotic plaque in carotid arteries of ApoE -/-
mice (which have elevated cholesterol levels and are prone to development of agerelated
atherosclerosis). Irradiation also predisposed to the formation of a vulnerable,
infl ammatory, thrombotic plaque phenotype with a thin fi brous cap.
Based on these results, the adhesive and thrombogenic properties of carotid
endothelium were evaluated at 1-4 weeks after irradiation. High doses (14 Gy)
led to an early reduction (1 week) in infl ammatory markers ICAM1 and VCAM1,
whereas these markers have been associated with the initiation of age-related
atherosclerosis. There was no change in levels of eNOS and MCP1 after irradiation.
High dose irradiation did cause an increase in prothrombotic tissue factor at 4

weeks, but this was counterbalanced by an increase in expression of anti-thrombotic
thrombomodulin. Anti-infl ammatory and anti-thrombotic drugs (aspirin and nitric
oxide releasing aspirin) did not inhibit initiation or progression of radiation-induced
atherosclerosis, although they did inhibit development of age-related atherosclerosis.
These results would suggest that either there are other infl ammatory/thrombotic
mediators of radiation-induced atherosclerosis, or that other mechanisms are
involved. We have now broadened our search for candidate molecules involved in
triggering the radiation-induced atherosclerosis by running microarray analysis of
RNA from irradiated and control arteries.
Radiation induced cardiac damage (collaboration with Mat Daemen, Cardiovascular
Research Institute, Maastricht)
The goal of this study is to understand the functional and structural alterations of
radiation-induced heart diseases, with the aim of identifying suitable intervention
strategies. Male C57BL/6J mice received local heart irradiation with single doses of
2-16 Gy. Changes in cardiac function, determined by SPECT/CT and ultrasound
imaging, were correlated with histo-pathological and microvascular changes at 20 to
40 weeks after irradiation. The epicardium showed infl ammatory thickening, with
iron-containing macrophages, from 20 weeks after ≥ 8 Gy. At 40 weeks, myocardial
thickness was reduced at all radiation doses, while interstitial collagen increased
after ≥ 8 Gy. Diffuse amyloid deposits, probably indicative of vascular leakage,
were present in the myocardium at 40 weeks after 16 Gy. Microvascular density
was decreased at 40 weeks after 16 Gy (26%). Remaining microvessels were well
perfused, but there was a 50% decrease in expression of alkaline phosphatase and,
after 16 Gy, a signifi cant increase in pro-thrombotic von Willebrand Factor. Cardiac
function tests showed decreased end diastolic and systolic volumes at 20 weeks after
all doses (maximum 26% and 39% decrease, respectively) and increased ejection
fraction (maximum 28%) after 16 Gy. There was no progression of functional
damage at 40 weeks, despite progressive changes in cardiac structure. This is
indicative of compensatory mechanisms to maintain cardiac function, until the
damage becomes so severe that heart failure occurs.
Cardiac damage in irradiated wild type C57BL/6J mice is also being compared
with atherosclerosis prone ApoE -/- mice, and Eng +/– mice (defi cient in TGF
signaling and microvascular repair). Preliminary results do not indicate more severe
functional damage at 20 weeks after irradiation. However, Eng+/- mice had more
severe, early loss of alkaline phosphatase expression (indicative of microvascular
damage) than wild type litter mates.
Angiogenic tissue res ponse in patients with malignant mesothelioma after
treatment with cisplatin, pemetrexed and Axitinib (collaboration with Arjan
Griffi oen, VUMC, Amsterdam )
Malignant pleural mesothelioma (MPM) have a high microvessel density and
express increased levels of vascular endothelial growth factor receptors VEGFR1-3,
and other angiogenic factors like platelet-derived growth factor (PDGF). Recent
studies reported a negative correlation between the microvessel density and
VEGF levels in MPM biopsies, and survival. Axitinib is a potent kinase inhibitor
of VEGFR1-3 and PDGFR-B. A prospective, randomized, phase I/II trial has been
initiated in the NKI (coordinator P Baas) to determine safety and effi cacy of the
addition of Axitinib to standard chemotherapy for patients with previously untreated
MPM. A major limitation in evaluation of ‘targeted agents’, is the lack of noninvasive
methods to determine effi cacy in an early stage. We therefore initiated
a feasibility study to investigate the effects of Axitinib on tumor vascularization.
Thoracoscopic biopsies (obtained before treatment and after three treatment courses)
were collected and angiogenic parameters, e.g. microvessel density, proliferating
endothelial cells and expression and activation of VEGFR2, were assessed. Moreover,
plasma samples were taken during the treatment course and VEGF protein levels
were determined by ELISA. Preliminary results demonstrate a reduction in vessel
density and endothelial cell proliferation in the Axitinib-treated tumours. Moreover,
VEGFR2 protein expression and activity and VEGF protein levels correlated with
treatment response. We are now including more patients in this study and results
will be correlated with clinical chemistry data and CT or X-ray scans.
39
experimental therapy

40
experimental therapy
Group leader Marcel Verheij
Marcel Verheij MD PhD Group leader
Albert Van Hell PhD Post-doc
Baukelien Van Triest MD PhD Academic staff
Conchita Vens PhD Research Associate
Shuraila Zerp MSc Technical staff
Gerben Koning Erasmus MC, Rotterdam
Publications
Van Lummel M, Van Blitterswijk WJ,
Vink SR, Veldman RJ, Van der Valk MA,
Schipper D, Dicheva BM, Eggermont
AMM, ten Hagen TLM, Verheij M,
Koning GA. Enriching lipid nanovesicles
with short-chain glucosylceramide
improves doxorubicin delivery and effi cacy
in solid tumors. The FASEB Journal 2010
(in press)
Verheij M, Vens C, Van Triest B. Novel
therapeutics in combination with
radiotherapy to improve cancer treatment:
Rationale, mechanisms of action and
clinical perspective. Drug Resist Update
2010;13:29-43
STRATEGIES TO ENHANCE RADIOSENSITIVITY AND
CHEMOSENSITIVITY
Within the Division of Experimental Therapy, our group conducts two projects.
The fi rst focuses on inhibitors of DNA repair as radiosensitizers (collaboration
with Conchita Vens). The NAD+-depleting agent APO866 was found to enhance
radiation-induced cell death and act as a radiosensitizer in various models, including
two prostate cancer cell lines. Restoration of cellular NAD levels abolished the
cytotoxic effect. In vivo MTD of APO866 was defi ned. PARP inhibitors are also good
candidates for combined use with DNA damaging agents. The main mechanism by
which both radiation and cisplatin kill tumor cells is by an accumulation of un- or
misrepaired DNA damage. PARP inhibitors increase radiation and chemotherapy
(Cisplatin) response in preclinical studies. PARP inhibitors have been shown to
specifi cally kill homologous recombination defi cient tumor cells as single agents. We
have designed 3 phase I-II studies evaluating the safety and tolerability of Olaparib
in combination with (cisplatin-based chemo-) radiotherapy in locally advanced breast
cancer, NSCLC and head and neck cancer. Olaparib exhibits low systemic toxicity
profi les when given as monotherapy. When combined with chemotherapeutic agents
more toxicity is seen.
In a second project we investigate the patented concept of improved drug delivery
by short chain sphingolipid-enriched liposomes in vitro and in vivo. We examined
this novel concept of GC-directed membrane permeabilization for doxorubicin in a
spontaneous mouse tumor model that mimics human Invasive Lobular Carcinoma
(ILC) breast cancer. We found that GC-enriched liposomal doxorubicin enhanced
the uptake of doxorubicin in ILC cell cultures at least fi ve-fold, and inhibited tumor
outgrowth in vivo much more effectively than either of the two clinically applied
formulations of doxorubicin, (free, non-liposomal) Adriamycin ® or (standard/
conventional liposomal) Caelyx ® . Mice treated with GC-liposomal doxorubicin
showed an extended overall survival (up to 2-fold) compared with conventional
liposomes. No increase in normal tissue toxicity was observed upon the addition of
GC. These data hold promise for widening the therapeutic window of amphiphilic
anti-cancer agents, including doxorubicin, using this plasma membrane modulation
strategy. We are currently continuing the GC-enriched liposomal doxorubicin
towards clinical investigations.

MOLECULAR PATHOLOGY AND MOLECULAR EPIDEMIOLOGY
OF BREAST AND COLORECTAL CANCER
Genome-wide genomic profi ling in hereditary breast cancer (PI Petra Nederlof)
Breast tumors from BRCA1 and BRCA2 mutation carriers show specifi c
chromosomal changes, and these genomic profi les can be used to classify individual
tumors. We use a genome-wide array-CGH analysis containing ~3500 1 Mb spaced
BAC clones with routinely formalin-fi xed paraffi n embedded (FFPE) tumor material.
We showed that basal like sporadic tumors frequently show a BRCA1-like aCGH
pattern, in combination with methylation of the BRCA1 promoter and a TP53
mutation spectrum similar to that found in BRCA1 tumors. In collaboration with
Dr. P Devilee (Leiden) we have analyzed tumors from BRCA1/2 negative HBC
families (BRCAx). The tumors show heterogeneous aCGH profi les. Interestingly, a
subgroup of tumors show a similar pattern characterized by among others gain of
chromosome #22 and loss of chromosome #13. Several families showed this pattern,
which now allows linkage analysis in this subpopulation. Furthermore, sporadic
basal-like breast tumors resemble BRCA1 tumors in their aCGH profi le, IHC profi le
and p53 mutations, and frequently show inactivation of BRCA1 by methylation.
Genetic determinants of breast cancer risk and breast cancer outcome
A large part of breast cancer susceptibility is as yet unexplained. Large Genome
Wide Association Studies have identifi ed new loci, which have been validated in
large consortia. We validated within the Breast Cancer Association Consortium
(BCAC) 18 breast cancer susceptibility loci (common single nucleotide
polymorphisms) explaining ~8% of familial breast cancer. We showed that specifi c
breast cancer risk loci (e.g. in FGFR2) predispose to tumors of a certain breast tumor
subtype defi ned by immunohistochemical markers (ER, PR, HER2, CK5/6, EGFR).
Furthermore, these breast tumor subtypes predispose to different breast cancer
survival patterns. In our own Dutch breast cancer studies we found evidence that
two functional polymorphisms TP53 R72P (215G>C) and MDM2 SNP309 (-410T>G)
play a role in breast cancer survival within the group of patients with specifi c
tumors. Using the Tissue Micro Arrays constructed of tumors collected in our breast
cancer series diagnosed

42
experimental therapy
Publications (continued)
De Graauw M, Van Miltenburg
MHAM, Schmidt MK, Pont C, Lalai
R, Kartopawiro J, Le Dévédec S, Smit
VTBHM, Van der Wal A, Van ’t Veer LJ,
Cleton-Jansen AM, ten Dijke P, Van de
Water B. Annexin A1 Expression in Basal-
Like Breast Cancer Mediates Metastatic
Behavior Through TGF signaling. Proc
Natl Acad Sci USA. 2010;107:6340-6345
Enciso-Mora V, Broderick P, Van
Leeuwen FE, Broeks A, et al A genomewide
association study of Hodgkin’s
lymphoma identifi es new susceptibility
loci at 2p16.1 (REL), 8q24.21 and 10p14
(GATA3). Nat Genet. 2010;42:1126-30
Fles R, Hoogendoorn WE, Platteel I,
Scheerman CE, De Leeuw-Mantel G,
Mourits MJE, Hollema H, Van Leeuwen
FE, Van Boven HH, Nederlof PM.
Genomic profi le of endometrial tumors
depends on morphological subtype, not on
tamoxifen exposure. Genes Chromosomes
Cancer 2010;49:699-710
He Y, Van ’t Veer LJ, Lopez-Yurda M, Van
de Velde CJH, Marijnen CAM. Do rectal
cancer patients with PIK3CA mutations
benefi t from preoperative radiotherapy
with regard to local recurrences? Clin
Cancer Res. (in press)
Joosse SA, Brandwijk KI, Devilee P,
Wesseling J, Hogervorst FB, Verhoef
S, Nederlof PM. Prediction of BRCA2association
in hereditary breast
carcinomas using array-CGH. Breast
Cancer Res Treat. 2010
Joosse SA, Brandwijk KI, Mulder L,
Wesseling J, Hannemann J, Nederlof
PM. The genomic signature of BRCA1
defi ciency in sporadic basal-like breast
tumors. Genes Chrom Cancer (in press)
Mook S, Knauer M, Bueno-de-Mesquita
JM, Retel VP, Wesseling J, Linn SC,
Van ‘t Veer LJ, Rutgers EJ. Metastatic
potential of T1 breast cancer can be
predicted by the 70-gene MammaPrint
signature. Ann Surg Oncol. 2010;17:1406-
1413
Avoidance of overtreatment with radiotherapy in rectal cancer patients
The development of local recurrences (LR) is a major problem in the treatment of
rectal cancer and there is a high clinical need to identify those patients who are at
increased risk. In this study (collaboration C. Marijnen, LUMC), 1. we aim to develop
a gene expression profi le for the risk of LR in rectal cancer patients, allowing for
selection of patients for pre-radiotherapy (PRT). Non-irradiated fresh frozen tumor
samples from 240 stage I-III rectal cancer patients were collected from the Dutch
total mesorectal excision (TME) trial, in which patients were randomized for shortterm
pre-RT followed by TME or TME alone. 215 samples with > 50% tumor content
and high RNA quality were subjected to Illumina bead-array and gene expression
analysis is in progress. 2. By using parallel extracted DNA, we have shown that
PIK3CA mutations can be used as a biomarker in identifying rectal cancer patients
with an increased risk for LR. Comparison with irradiated patients within the
TME trial revealed a benefi cial effect of pre-RT on PIK3CA mutant patients in
preventing LR. Our fi ndings suggest that PIK3CA mutations may not only be a
prognostic factor, but may also be predictive with regard to PRT benefi t. If this is
confi rmed in further studies, it could potentially allow the selection of patients with
increased benefi t from PRT versus surgery only and spare others the side effects of
radiotherapy.
Quantitative Expression Profiling of RNA from FFPE and from Fresh Frozen
Tissues using DASL Illumina platform Tissues samples collected during surgery
and biopsies are often fi xed in formalin, followed by embedding in paraffi n. Many
of these samples represent clinical material with the potential to provide critical
insight into expression profi les associated with complex disease as the breast
cancer. This project aims to evaluate if the DASL-based expression profi ling assay,
specifi cally designed for degraded RNAs (Illumina Inc 2004) is a sensitive and
reliable method to apply on RNA from FFPE. We analyzed 70 samples, 50 breast
cancer samples (44 FFPE tissues and 6 FF tissues) and 20 liposarcoma samples (12
FFPE tissues and 8 FF tissues) on the 502 cancer-related genes DASL platform. The
results showed that DASL platform works well with paraffi n tissues, as we could see
in the correlation analysis with frozen tissues (Pearson correlation Breast tissues
0.74 ± 0.05, Liposarcoma 0.77 ± 0.04). Subsequently we enlarged the cohort of
breast samples pairs and analyze them with the whole genome DASL platform. We
profi led, in duplicate, 20 FFPE breast tissues and 20 matched FF breast tissues and
evaluated the concordance of the DASL results from FFPE and matched FF material.
We show that after proper normalization, all FFPE and FF pairs exhibit a high level
of similarity (Pearson correlation > 0.7), signifi cantly larger than the similarity
between non-paired samples. Predictions of gene expression signatures developed
on frozen material (Intrinsic subtype, Genomic Grade Index, 70 gene signature)
showed a high level of concordance between FFPE and FF matched pairs.
Target identification and evaluation for breast molecular imaging
By visualizing molecular changes accompanying breast carcinogenesis, molecular
imaging holds the promise to outperform conventional imaging modalities, possibly
improving screening, diagnosis, and therapy direction. In preclinical research,
many imaging probes targeting cancer-associated molecules have been successfully
developed and tested. One of the hurdles for human breast molecular imaging
however, is the reality that no single cancer-specifi c molecule is over-expressed in
all breast cancers. The Breast Imaging Target Evaluation (BrITE) project aims to
identify an optimal panel of imaging targets for breast molecular imaging, which
would greatly expedite the development and translation of molecular imaging
probes.

PHARMACODYNAMICS OF ANTI CANCER DRUGS
Effect of genetic polymorphism within DPYD on treatment outcome of
capecitabine-based chemotherapy In 568 patients with advanced colorectal
cancer treated with capecitabine-based chemotherapy, the effect of genetic
polymorphism within dihydropyrimidine dehydrogenase (the primary detoxifying
enzyme of capecitabine) was explored. The polymorphisms IVS14+1G>A
(DPYD*2A), 2846A>T, 1236G>A and 496A>G were signifi cantly associated with
grade 3-4 diarrhea. Due to toxicity, a mean capecitabine dose reduction of 50% was
applied in IVS14+1G>A and 25% in 2846A>T variant allele carriers.
Upfront genotyping of DPYD*2A to improve patient safety of fluoropyrimidine
therapy DPYD*2A has previously been associated with severe drug-induced toxicity
of fl uoropyrimidines. In this study, a total of 1600 patients were prospectively
genotyped for DPYD*2A prior to start of fl uoropyrimidine therapy. Patients with
DPYD*2A were treated with an initially reduced dose followed by dose titration
upon clinical tolerability. The risk of grade ≥ 3 toxicity thereby reduced from 68% in
historical controls to 15% by genotype-guided dosing. Drug-induced death reduced
from 10% to 0%. The cost-effectiveness analysis showed that upfront genotyping is
cost-effective.
Phase I/II study of docetaxel, oxaliplatin and capecitabine for advanced
gastric cancer A total of 33 patients with advanced gastric cancer were treated in
three-weekly cycles with the combination of docetaxel 50 mg/m 2 (day 1), oxaliplatin
100 mg/m 2 (day 1) and capecitabine 850 mg/m 2 bid (days 1-14) in 3-weekly cycles.
Grade ≥ 3 toxicity included neutropenia (36%), leukocytopenia (23%), febrile
neutropenia (14%) and diarrhea (5%). The overall response rate was 46%. Median
progression-free and overall survival were 6.9 months (95% CI: 5.6 – 8.2) and 11.6
months (95% CI: 8.7 – 14.5), respectively.
Simultaneous integrated boost – intensity modulated radiotherapy (SIB-
IMRT) with concomitant capecitabine and mitomycin-C for locally advanced
anal cancer A total of 18 patients with locally advanced anal carcinoma were treated
with SIB-IMRT in 33 daily fractions of 1.8/1.5 Gy plus capecitabine on radiation days
and mitomycin-C on day 1. Most predominant acute grade ≥ 3 toxicities included
dermatitis within the radiation area (61%), fatigue (22%) and pain (6%). 72%
(95%-CI: 51-94%) of the patients achieved a complete response, 28% had a partial
response.
Phase I study of everolimus plus capecitabine in advanced solid malignancies
A total of 18 patients with advanced solid malignancies were treated with everolimus
10 mg/day continuously, plus capecitabine bid (days 1-14) in three-weekly cycles.
Capecitabine was dose-escalated (500 – 1000 mg/m 2 bid): 1000 mg/m 2 bid was
declared the maximum tolerated dose. Toxicities were mostly grade ≤ 2 and included
fatigue (56%), stomatitis (50%), and hand-foot syndrome (33%). In three patients a
partial response was observed.
Effect of gastric surgery on the systemic exposure to oral capecitabine
In 86 gastrectomized patients with gastric cancer were treated with postoperative
capecitabine-based chemoradiotherapy. The pharmacokinetics (PK) of capecitabine
were determined and compared with the PK in 18 non-gastrectomized patients. A
total or partial resection resulted in a signifi cantly faster absorption, higher peak
plasma concentration, and higher systemic exposure of capecitabine compared to
non-gastrectomized patients.
Validation of a multi-parameter flow cytometry method for the determination
of DNA and phosphorylated extracellular-signal-regulated kinase (pERK) in
circulating tumor cells (CTCs)
Density gradient centrifugation of peripheral blood, followed by magnetic anti
epithelial cell adhesion molecule Micro Beads were used for the selective isolation of
CTCs. Multiparameter fl ow cytometry was used for identifi cation and enumeration
43
experimental therapy
Group leader Jan Schellens
Jan Schellens MD PhD Group leader
Jos Beijnen PhD Academic staff
Irma Meijerman PhD External staff
Serena Marchetti MD PhD Academic staff
Neeltje Steeghs MD PhD Academic staff
Maarten Deenen MSc PhD student
Lot Devriese MD MSc PhD student
Suzanne Leijen MD PhD student
Geert Frederix MSc PhD student
Dick Pluim Technical staff
Artur Burylo Technical staff
Structural collaborations:
Group Laura van ’t Veer
Daphne de Jong MD PhD,
Department of Pathology
Publications
Appels NM, Bolijn MJ, Van Eijndhoven
MA, Stephens TC, Beijnen JH, Schellens
JH. Characterization of the in vitro
activity of AZD3409, a novel prenyl
transferase inhibitor. Cancer Chemother
Pharmacol 2010 (in press)
Beijnen JH, Schellens JH. Personalized
Medicine in Oncology: A Personal
View with Myths and Facts. Curr Clin
Pharmacol. 2010;5:141-47
Fong PC, Yap TA, Boss DS, Carden CP,
Mergui-Roelvink M, Gourley C, De Greve
J, Lubinski J, Shanley S, Messiou C,
A’Hern R, Tutt A, Ashworth A, Stone J,
Carmichael J, Schellens JH, de Bono JS,
Kaye SB. Poly(ADP)-ribose polymerase
inhibition: frequent durable responses in
BRCA carrier ovarian cancer correlating
with platinum-free interval. J Clin Oncol.
2010;28:2512-19
Gourley C, Michie CO, Roxburgh P,
Yap TA, Harden S, Paul J, Ragupathy K,
Todd R, Petty R, Reed N, Hayward RL,
Mitchell P, Rye T, Schellens JH, Lubinski
J, Carmichael J, Kaye SB, Mackean M,
Ferguson M. Increased incidence of
visceral metastases in scottish patients
with BRCA1/2-defective ovarian cancer:
an extension of the ovarian BRCAness
phenotype. J Clin Oncol. 2010;28:2505-11

44
experimental therapy
Publications (continued)
Keizer RJ, Gupta A, Mac Gillavry MR,
Jansen M, Wanders J, Beijnen JH,
Schellens JH, Karlsson MO, Huitema AD.
A model of hypertension and proteinuria
in cancer patients treated with the antiangiogenic
drug E7080. J Pharmacokinet
Pharmacodyn. 2010;37:347-63
Keizer RJ, Van Benten M, Beijnen JH,
Schellens JHM, Huitema ADR. Piraña
and Pcluster: A modeling environment
and cluster infrastructure for NONMEM.
Comput Methods Programs Biomed. 2010
(in press)
Koolen SL, Oostendorp RL, Beijnen JH,
Schellens JH, Huitema AD. Population
pharmaco-kinetics of intravenously and
orally administered docetaxel with or
without co-administration of ritonavir in
patients with advanced cancer. Br J Clin
Pharmacol. 2010;69 465-74
Langenberg MH, van Herpen CM, De
Bono J, Schellens JH, Unger C, Hoekman
K, Blum HE, Fiedler W, Drevs J, Le Maulf
F, Fielding A, Robertson J, Voest EE.
Effective strategies for management of
hypertension after vascular endothelial
growth factor signaling inhibition therapy:
results from a phase II randomized,
factorial, double-blind study of Cediranib
in patients with advanced solid tumors.
J Clin Oncol 2010;27: 6152-59
Leijen S, Beijnen JH, Schellens JH.
Abrogation of the G(2) Checkpoint by
Inhibition of Wee-1 Kinase Results in
Sensitization of p53-Defi cient Tumor Cells
to DNA-Damaging Agents. Curr Clin
Pharmacol. 2010;5:186-91
Oostendorp RL, Buckle T, Lambert G,
Garrigue JS, Beijnen JH, Schellens JH,
Van Tellingen O. Paclitaxel in selfmicro
emulsifying formulations: oral
bioavailability study in mice. Invest New
Drugs 2010 (in press)
Vermaat JS, Van der Tweel I, Mehra N,
Sleijfer S, Haanen JB, Roodhart JM,
Engwegen JY, Korse CM, Langenberg
MH, Kruit W, Groenewegen G, Giles
RH, Schellens JH, Beijnen JH, Voest
EE. Two-protein signature of novel
serological markers apolipoprotein-A2 and
serum amyloid alpha predicts prognosis
in patients with metastatic renal cell
cancer and improves the currently used
prognostic survival models. Ann Oncol.
2009;21:1472-81
of CTCs and determination of their pERK and DNA content. The validation
parameters included specifi city, recovery, linearity, accuracy, precision, sensitivity
and stability (see fi gure 3 and 4). The applicability of the method was demonstrated
by successful enumeration of CTCs, and the determination of DNA, and pERK in
patients with metastatic cancer.
Circulating tumor cell (CTC) detection in advanced non-small cell lung cancer
(NSCLC): validation of qRT-PCR-based detection A CTC-detection assay in
peripheral blood of NSCLC patients Objective was optimized. Immunomagnetic
bead selection for EpCAM and RT-PCR for selected marker genes was used
and combined this in statistical analysis. In total 35 healthy controls and 45
NSCLC patients were assayed and discrimination between the groups achieved.
Discrimination between patients and controls was achieved.
Eribulin Mesylate pharmacokinetics in patients with solid tumors receiving
repeated oral Ketoconazole (ket) The effects of ketaconazole (KET), a CYP3A4
inhibitor, on eribulin pharmacokinetics were investigated in a phase I, randomized,
open-label, 2-way crossover study with pharmacokinetic analysis. Co-administration
of KET had no statistically signifi cant effect on single dose exposure to eribulin.
Eribulin dosing in patients with advanced solid tumors and hepatic
impairment The effect of hepatic impairment on eribulin exposure following a
single intravenous administration was studied in a phase 1 study design. In patients
with mild and moderate hepatic impairment, mean dose-normalized AUC(0-∞) was
1.75-fold and 2.79-fold, respectively, compared to normal hepatic function. Thus,
hepatic impairment increased exposure to eribulin in patients with advanced solid
tumors.
Eribulin Mesylate pharmacokinetics in patients with solid tumors receiving
Rifampin (RIF) The effects of rifampin (RIF), a CYP3A4 inducer, on eribulin
pharmacokinetics were explore in a phase I, open-label, 2-way crossover study with
pharmacokinetic analysis.In total 14 subjects were included and PK sampling was
performed. The study is ongoing for PK analysis and safety evaluation.
Phase I study of two schedules of oral topotecan in combination with
Pazopanib in patients with advanced solid tumors Safety, tolerability and
pharmacokinetics of oral topotecan in combination with pazopanib were studied in a
phase 1 study with bioavailability phase and 2 schedules of dose-escalation.
The bioavailability part was completed and showed a signifi cant effect of pazopanib
on the bioavailability of topotecan (1.6-fold increase). In total 18 patients were
included in ongoing dose-escalation.
Phase I dose escalation study of MEK inhibitor RO4987655, administered
orally as monotherapy in patients with advanced tumors The RAS/RAF/MEK/
ERK signaling pathway is dysregulated in many human cancers. RO4987655 is a
potent, highly selective ATP non-competitive MEK inhibitor. Preliminary results in
patients show that RO4987655 is well tolerated. Skin rash is frequently reported as
adverse effect. A substantial number of patients show stable disease.
Phase I dose escalation study of Wee1 inhibitor MK1775 in both monotherapy
and in combination with gemcitabine, cisplatin or carboplatin in adult
patients with advanced solid tumors MK-1775 is an inhibitor of the Wee1 kinase
regulating the G2 checkpoint leading to chemo-sensitization in p53 defi cient tumor
cells. Downregulation of pCDC2 upon treatment with MK and chemotherapy are
used as biomarker. Preliminary results show that MK-1775 with chemotherapy
is well tolerated and a substantial number of patients show stable disease/partial
remission.

MOLECULAR MARKERS TO PREDICT CHEMOSENSITIVITY IN
BREAST CANCER
Development of clinically useful and feasible tests using molecular
markers to predict the response of primary breast cancers to specific
chemotherapeutic agents or combinations Preoperative chemotherapy for locally
advanced breast cancer may lead to excellent responses in some tumors and to no or
partial responses in others. Therefore there is a strong need for tests that can predict
chemotherapy response. In the CTMM BreastCare project we aim to develop such
tests based on molecular markers.
One idea for such a test is based on the concept of BRCAness. We previously showed
that sporadic tumors with a BRCA-like genomic profi le showed a higher response
rate to chemotherapy with alkylating agents. We have now validated these fi ndings
in 249 stage-III breast cancer patients who were part of a randomized controlled trial
studying adjuvant high-dose platinum-based chemotherapy (HD-PB-chemotherapy)
versus conventional anthracycline-based chemotherapy. Using aCGH we were
able to select hormone-receptor positive and TN breast cancer patients for selective
benefi t of intensifi ed double-strand-break-inducing chemotherapy. This test should
now be investigated in studies where other alkylating agents or PARP-inhibitors are
employed.
A second idea was prediction based on basic pathology and molecular data in
ER+HER2- breast cancer. We analyzed all basic pathology data (ER, PR expression,
histological subtype, proliferation) and molecular subtype data from 211 ER+HER2-
tumors to identify a subset of these characteristics that could distinguish between
poor and good responders. PR negative and luminal B subtype tumors responded
remarkably better to neoadjuvant chemotherapy, but predictive power was poor. We
concluded that based on the tested variables it was not possible to discern good and
poor responders. The decision to refrain patients from chemotherapy in ER+HER2-
breast tumors could therefore not be based on present predictive markers.
A third idea for a predictive test is based upon results of gene expression microarray
analyses. To date we have performed 284 microarrays from tumors of patients
treated with neoadjuvant chemotherapy. The dataset now contains information
about 48,000 different transcripts. We plan to perform unsupervised clustering
and statistical analysis of microarrays to fi nd genes differently expressed between
responders and non-responders to neoadjuvant chemotherapy. Signatures from
literature will also be examined. From these data we will search for markers
predictive for neoadjuvant chemotherapy response.
Development of a human in mouse model of breast carcinoma to optimize
alkylating or targeted treatment Triple negative breast cancer (TNBC: negative for
ER, PR and HER2) tends to rapidly develop resistance to conventional therapy and
targeted therapy is not available. In the development of new therapies, the availability
of in vivo models is essential. Ideally, these in vivo models would recapitulate the
heterogeneity in characteristics and response to therapy found in human breast
cancers. Xenograft models in which human tumor tissue is engrafted directly into
recipient mice may be very useful for this purpose (Marangoni et al., Clin Can Res
2007;13:3989). We have therefore set up a panel of xenografts for human TNBC in
immunocompromised mice. After outgrowth, tumors are propagated by serially
transplantation and are characterized based on histology, immunohistochemistry,
gene expression profi le, aCGH, BRCA1 promoter methylation, mRNA and protein.
Figure 5: Cisplatin treatment of BRCA1 defi cient TNBC xenografts leads to complete remission (A),
recurrence of tumor (B) or tumor resistance (C). Arrows indicate times of treatment.
Western blot for BRCA1 in untreated and cisplatin resistant TNBC xenograft (D).
Publications
45
experimental therapy
Group leader Jelle Wesseling
Jelle Wesseling MD PhD Group leader
Jos Jonkers PhD Academic staff
Petra Nederlof PhD Academic staff
Sjoerd Rodenhuis MD PhD Academic staff
Lodewijk Wessels PhD Academic staff
Petra ter Brugge PhD Post-doc
Esther Lips PhD Post-doc
Jorma De Ronde PhD student
Petra Kristel Technical staff
Lennart Mulder Technical staff
Eline Van der Burg Technical staff
Blows FM, Driver KE, Schmidt MK,
Broeks A, Van Leeuwen FE, Wesseling J,
Cheang MC, Gelmon K, Nielsen TO,
Blomqvist C, Heikkila P, Heikkinen T,
Nevanlinna H, Akslen LA, Begin LR,
Foulkes WD, Couch FJ, Wang X,
Cafourek V, Olson JE, Baglietto L,
Giles GG, Severi G, McLean CA,
Southey MC, Rakha E, Green AR, Ellis
IO, Sherman ME, Lissowska J, Anderson
WF, Cox A, Cross SS, Reed MW,
Provenzano E, Dawson SJ, Dunning AM,
Humphreys M, Easton DF, Garcia-Closas
M, Caldas C, Pharoah PD, Huntsman
D. Subtyping of breast cancer by
immunohistochemistry to investigate a
relationship between subtype and short
and long term survival: a collaborative
analysis of data for 10,159 cases from
12 studies. PLoS Med 2010;7:e1000279
Joosse SA, Brandwijk KI, Devilee P,
Wesseling J, Hogervorst FB, Verhoef
S, Nederlof PM. Prediction of BRCA2association
in hereditary breast
carcinomas using array-CGH. Breast
Cancer Res Treat. 2010
Joosse SA, Brandwijk KI, Mulder L,
Wesseling J, Hannemann J, Nederlof
PM. The genomic signature of BRCA1
defi ciency in sporadic basal-like breast
tumors. Genes Chrom Cancer (in press)
Mook S, Knauer M, Bueno-de-Mesquita
JM, Retel VP, Wesseling J, Linn SC, Van ‘t
Veer LJ, Rutgers EJ. Metastatic potential
of T1 breast cancer can be predicted by the
70-gene MammaPrint signature.
Ann Surg Oncol. 2010;17:1406-13

46
experimental therapy
Publications (continued)
Hartog H, Horlings HM, Van der Vegt B,
Kreike B, Ajouaou A, Van de Vijver MJ,
Marike Boezen H, De Bock GH, Van der
Graaf WT, Wesseling J. Divergent effects
of insulin-like growth factor-1 receptor
expression on prognosis of estrogen receptor
positive versus triple negative invasive
ductal breast carcinoma. Breast Cancer
Res Treat. 2010
Knauer M, Cardoso F, Wesseling J,
Bedard PL, Linn SC, Rutgers EJ, Van
‘t Veer LJ. Identifi cation of a low-risk
subgroup of HER-2-positive breast cancer
by the 70-gene prognosis signature. Br J
Cancer. 2010
Straver ME, Glas AM, Hannemann J,
Wesseling J, Van de Vijver MJ, Rutgers EJ,
Vrancken Peeters MJ, Van Tinteren H,
Van ‘t Veer LJ, Rodenhuis S. The 70gene
signature as a response predictor
for neoadjuvant chemotherapy in
breast cancer. Breast Cancer Res Treat.
2010;119:551-8
De Ronde J, Wessels L, Wesseling J.
Molecular subtyping of breast cancer:
ready to use? Lancet Oncol. 2010;11:306-7
De Ronde JJ, Hannemann J, Halfwerk
H, Mulder L, Straver ME, Vrancken
Peeters MJ, Wesseling J, Van de Vijver M,
Wessels LF, Rodenhuis S. Concordance
of clinical and molecular breast cancer
subtyping in the context of preoperative
chemotherapy response. Breast Cancer Res
Treat. 2010;119:119-26
Vollebergh MA, Lips E, Nederlof PM,
Wessels LFA, Schmidt MK, Van Beers
EH, Cornelissen S, Holtkamp M,
Froklage FE, De Vries EGE, Schrama
JG, Wesseling J, Van de Vijver MJ, Van
Tinteren H, De Bruin M, Hauptmann
M, Rodenhuis S, Linn SC. A comparative
genomic hybridization classifi er derived
from BRCA1-mutated breast cancer and
benefi t of high-dose, platinum-containing,
chemotherapy in breast cancer patients.
Annals of Oncol. (in press)
Lips EH, Mulder L, Hannemann J,
Laddach N, Vrancken Peeters MT, Van
de Vijver MJ, Wesseling J, Nederlof PM,
Rodenhuis S. Indicators of homologous
recombination defi ciency in breast
cancer and association with response to
neoadjuvant chemotherapy. Ann Oncol.
(in press)
We have identifi ed a number of TNBC xenografts defi cient for BRCA1, through
mutations in the gene, leading to a truncated, non-functional protein, or through
epigenetic mechanisms, leading to down-regulation of BRCA1 expression. Since
BRCA1 plays an important role in the repair of double strand breaks, BRCA1
defi cient tumors are thought to be especially sensitive to alkylating agents that
induce DNA crosslinks and subsequent DNA breaks, as was recently shown for
cisplatin (Silver et al, J Clin Oncol 2010;28:1145, Byrski et al, J Clin Oncol 2010;28:375).
We have started therapeutic intervention studies on the xenografts using the
alkylating agents cisplatin, nimustin and melphalan. Treatment was discontinued
when tumors reached a size of 50% or less of the starting size. In case of relapse,
treatment was started again after relapse to the starting size. BRCA1 defi cient
models responded well to treatment with alkylating agents, with complete
disappearance of palpable lesion in most cases after 21 days of treatment (fi gure 5A).
However, some tumors relapsed and had to be retreated. After relapse, most tumors
remained sensitive to cisplatin (4/5 relapsed tumors; fi gure 5B) but resistance did
occur (1/5 relapsed tumors; fi gure 5C). Tumors that relapsed after melphalan or
nimustin treatment quickly developed resistance.
We have started investigating cisplatin resistance in BRCA1 defi cient xenograft
models. In a model that is characterized by BRCA1 defi ciency due to promoter
methylation (fi gure 5D, untreated), cisplatin resistance was accompanied by reexpression
of BRCA1 (fi gure 5D, resistant). We will continue by studying the
mechanism of BRCA1 re-expression, and also examining the mechanisms behind
resistance to other alkylating agents.
In conclusion, TNBC xenograft models are very suitable for studying response and
resistance to cytotoxic drugs, showing that BRCA1 defi cient tumors are sensitive to
alkylating agents and that re-expression of BRCA1 is a mechanism of resistance to
cisplatin.

DIVISION OF GENE REGULATION
MICRORNAS AND RNA BINDING PROTEINS IN CANCER
Our main research objective is to understand the cancerous process in humans
and identify essential cancerous genes. The knowledge we gather will allow us to
design novel therapeutic approaches. Most human tumors harbor multiple genetic
alterations that activate oncogenes, inhibit tumor suppressors and induce genomic
instability. As each tumor contains many genetic alterations, the study of the
contribution of each alteration to the cancerous phenotype was obscured. In the
past, we developed and successfully used an RNA interference (RNAi) approach to
inactivate genes in mammalian cells. We used this RNAi system to characterize
tumor suppressors, such as the bromodomain containing protein 7 (BRD7)
described below.
In the past we initiated studies to identify cancerous microRNAs (miRNAs), a newly
emerging gene family encoding for endogenous small RNAs. We developed novel
and unique genetic approaches to screen for cancer-causing and cancer-preventing
miRNAs. With these tools we discovered and characterized the role of the miR-372
family in tumor growth and metastasis as well as the oncogenic role of miR-221 in
glioblastoma.
Interestingly, we noticed that the regions surrounding many functional miRNA
targets (identifi ed by our genetic screens, for example) are highly conserved
throughout evolution. We postulated that these regions recruit RNA-binding
proteins (RBPs) that regulate miRNA function. We performed genetic screens
and identifi ed RBPs that can inhibit or potentiate the activity of miRNAs to their
target mRNAs. We propose that the genetic interaction between miRNAs and RBPs
determines developmental processes and cellular proliferation. How this affects
cancer initiation and development is under investigation now.
Functional genetic approaches identify novel tumor suppressor genes
Oncogene-induced senescence is a p53-dependent defense mechanism against
uncontrolled proliferation. Consequently, many human tumors harbor p53
mutations and others show a dysfunctional p53 pathway, frequently by unknown
mechanisms. Using functional genetic screens with RNAi libraries we identifi ed
BRD7 (bromodomain-containing 7) as a protein whose inhibition allows full
neoplastic transformation in the presence of wild-type p53 (fi gure 1). In human
breast tumors harboring wild-type, but not mutant, p53 the BRD7 gene locus was
frequently deleted and low BRD7 expression was found in a subgroup of tumors
(1A&B). Functionally, BRD7 is required for effi cient p53-mediated transcription of
a subset of target genes (1C). BRD7 interacts with p53 and p300 and is recruited to
target gene promoters, affecting histone acetylation, p53 acetylation and promoter
activity. Thus, BRD7 suppresses tumorigenicity by serving as a p53 cofactor required
for the effi cient induction of p53-dependent oncogene-induced senescence.
Figure 1:
A. BRD7 gene is deleted ed
in many wild type p53
breast cancer tumors.
B. Expression of BRD7 7
is signifi cantly low in
wild type p53 breast cancer ancer tumors.
C. BRD7 interacts with th p53 and
acetylated histones, and nd is required for
effi cient p53 transcriptional tional activity.
47
gene regulation
Division head, group leader Reuven Agami
Reuven Agami PhD Group leader
Eitan Zlotorynski PhD Post-doc
Rani Elkon PhD Post-doc
Nicolas Léveillé PhD Post-doc
Gijs van Haaften PhD Post-doc
Maria Gomez Benito PhD Post-doc
Henning Schaefer MD Guest
Carlos le Sage PhD Post-doc
Martijn Kedde PhD Post-doc
Mathias Jenal PhD Post-doc
Jarno Drost MSc PhD student
Marieke van Kouwenhove MSc PhD student
Arnold Bos MSc PhD student
Carlos Melo MSc PhD student
Mariette Schrier Technical staff
Joachim Oude Vrielink Technical staff
Publications
Agami R. MicroRNAs, RNA binding
proteins and cancer. Eur J Clin Invest
2010;40: 370-374
Drost J, Mantovani F, Tocco F, Elkon
R, Comel A, Holstege H, Kerkhoven R,
Jonkers J, Voorhoeve PM, Agami R, et al.
BRD7 is a candidate tumour suppressor
gene required for p53 function. 2010. Nat
Cell Biol 2010;12:380-389
Elkon R, Zlotorynski E, Zeller KI and
Agami R. Major role for mRNA stability
in shaping the kinetics of gene induction.
2010. BMC Genomics 2010;11:259
Kedde M, van Kouwenhove M, Zwart W,
Oude Vrielink JA, Elkon R, and Agami
R. A Pumilio-induced RNA structure
switch in p27-3’ UTR controls miR-221
and miR-222 accessibility. Nat Cell Biol
2010;12:1014-1020
Mann M, Barad O, Agami R, Geiger
B, and Hornstein E. miRNA-based
mechanism for the commitment of
multipotent progenitors to a single
cellular fate. Proc Natl Acad Sci U S A.
2010;107:15804-15809

48
gene regulation
Publications (continued)
Mantovani F, Drost J, Voorhoeve PM, Del
Sal G, and Agami R. Gene regulation and
tumor suppression by the bromodomaincontaining
protein BRD7. Cell Cycle
2010;9:2777-2781
Schaap-Oziemlak AM, Raymakers RA,
Bergevoet SM, Gilissen C, Jansen BJ,
Adema GJ, Kogler G, le Sage C, Agami
R, van der Reijden BA, et al. MicroRNA
hsa-miR-135b regulates mineralization
in osteogenic differentiation of human
unrestricted somatic stem cells. Stem Cells
Dev 2010;19:877-885
van Haaften G, and Agami R.
Tumorigenicity of the miR-17-92 cluster
distilled. 2010. Genes Dev 2010;24:1-4
Wilting SM, van Boerdonk RA, Henken
FE, Meijer CJ, Diosdado B, Meijer GA,
le Sage C, Agami R, Snijders PJ, and
Steenbergen RD. Methylation-mediated
silencing and tumour suppressive function
of hsa-miR-124 in cervical cancer. Mol
Cancer 2010;9:167
A
B
Figure 3: A. Kinetic induction created by
sequential transcription activation. B.
Kinetic induction created by differential
stability of mRNA.
Interplay between RNA-binding proteins and miRNAs regulates gene
expression microRNAs (miRNAs) interact with 3’-Untranslated regions (3’UTRs)
of messenger RNAs (mRNAs) to control the expression of a large proportion of
the protein coding genome during normal development and cancer. RNA-binding
proteins (RBPs) potentially control the biogenesis, stability, and activity of miRNAs
(fi gure 2A). In recent years we have demonstrated that miRNA accessibility to target
mRNAs can be controlled by RBPs. We provided fi rst proof of principle to this
phenomenon with an RNA binding protein called DND1, a gene required for proper
differentiation and survival of primordial germ cells. In addition, we generated
genome-wide tools for large-scale screen analysis to identify RBPs whose expression
controls miRNA accessibility to target mRNAs. This has led us to the identifi cation
of RBPs required cell cycle control, as well as for optimal p53 function (fi gure 2B).
Figure 2: A. Interplay between RNA-binding proteins (RBP) and miRNA biogenesis and activity.
B. Binding of RNA-binding proteins (RBP) to target mRNAs infl uences miRNA activity. This affects
cellular processes such as differentiation, cell cycle and stress response.
A Pumilio-induced RNA structure switch in p27-3´ UTR controls miR-221 and
miR-222 accessibility
Key regulators of 3´ untranslated regions (3´ UTRs) are microRNAs and RNAbinding
proteins (RBPs). The p27 tumor suppressor is highly expressed in quiescent
cells, and its downregulation is required for cell cycle entry after growth factor
stimulation. Intriguingly, p27 accumulates in quiescent cells despite high levels
of its inhibitors miR-221 and miR-222. Here we show that miR-221 and miR-222
are underactive towards p27-3´ UTR in quiescent cells, as a result of target site
hindrance. Pumilio-1 (PUM1) is a ubiquitously expressed RBP that was shown
to interact with p27-3´ UTR. In response to growth factor stimulation, PUM1 is
upregulated and phosphorylated for optimal induction of its RNA-binding activity
towards the p27-3´ UTR. PUM1 binding induces a local change in RNA structure
that favours association with miR-221 and miR-222, effi cient suppression of p27
expression, and rapid entry to the cell cycle. We have therefore uncovered a novel
RBP-induced structural switch modulating microRNA-mediated gene expression
regulation (fi gure 2).
Major role for mRNA stability in shaping the kinetics of gene induction
mRNA levels in cells are determined by the relative rates of RNA production and
degradation. Yet, to date, most analyses of gene expression profi les were focused
on mechanisms which regulate transcription, while the role of mRNA stability in
modulating transcriptional networks was to a large extent overlooked (fi gure 3A).
In particular, kinetic waves in transcriptional responses are usually interpreted as
resulting from sequential activation of transcription factors. We therefore examined
on a global scale the role of mRNA stability in shaping the kinetics of gene response
(fi gure 3B). Analyzing numerous expression datasets we revealed a striking global
anti-correlation between rapidity of induction and mRNA stability, fi tting the
prediction of a kinetic mathematical model. In contrast, the relationship between
kinetics and stability was less signifi cant when gene suppression was analyzed.
Frequently, mRNAs that are stable under standard conditions were very rapidly
down-regulated following stimulation. Such effect cannot be explained even by
a complete shut-off of transcription, and therefore indicates intense modulation
of RNA stability. Taken together, our results demonstrate the key role of mRNA
stability in determining induction kinetics in mammalian transcriptional networks.

CHROMATIN GENOMICS
In every eukaryotic cell, hundreds of chromatin proteins work together to control the
expression of thousands of genes. Each chromatin protein interacts with many other
proteins and regulates specifi c parts of the genome. This network of interactions
is enormously complex. To gain insight into this network and the roles in gene
regulation, we take a broad integrative genomics approach, using both fruit fl y and
mammalian cells as model systems. We conduct our studies in the living cell, in
the context of the entire genome. Thereby, we aim to identify general principles
that govern gene regulation by chromatin. We develop and apply new wholegenome
approaches to study the structure and composition of chromatin and the
mechanisms of gene regulation.
One of our major tools is our DamID technology, which enables us to generate
detailed in vivo genomic binding maps of a large variety of chromatin proteins and
transcription factors. These binding maps provide a wealth of new insights into the
roles of each protein in determining chromatin structure and gene regulation, and
form the basis for systematic functional analysis of gene regulation by chromatin
components. We have established a DamID “pipeline” that effi ciently generates fullgenome
binding maps (each consisting of ~385,000 data points) for a wide range of
proteins in a Drosophila cell line. Furthermore, a unique application of DamID is
the mapping of contacts of the genome with the nuclear lamina. This enables us to
study the folding of chromosomes inside nuclei of Drosophila and mammalian cells
in detail.
Principal chromatin types in Drosophila cells The diversity of chromatin
composition and the distribution along chromosomes are still poorly characterized.
We have generated genome-wide high-resolution binding maps of 53 broadly selected
chromatin components in Drosophila cells. By integrative computational analysis we
demonstrated that the genome is segmented into fi ve principal chromatin types that
are defi ned by unique yet overlapping combinations of proteins and form domains
that can extend over > 100 kb. We identifi ed a repressive chromatin type that covers
about half of the genome and lacks classic heterochromatin markers. Furthermore,
transcriptionally active euchromatin consists of two types that differ in molecular
organization and H3K36 methylation and regulate distinct classes of genes. Finally,
we provided evidence that the different chromatin types help to target DNA-binding
factors to specifi c genomic regions. These results provide a global view of chromatin
diversity and domain organization in a metazoan cell.
Group leader Bas van Steensel
49
gene regulation
Bas van Steensel PhD Group leader
Mario Amendola PhD Post-doc
Guillaume Filion PhD Post-doc
Jop Kind PhD Post-doc
Katy Kolodziej PhD Post-doc
Alexey Pindyurin PhD Post-doc
Joke van Bemmel MSc PhD student
Dominika Bijo´s MSc PhD student
Ulrich Braunschweig MSc PhD student
Wouter Meuleman MSc PhD student
Daniel Peric-Hupkes MSc PhD student
Ludo Pagie PhD Bioinformatician
Wendy Talhout BAS Technical staff
Arantxa Rosado BSc Technical staff
Figure 4: Binding profi les of 53 different
chromatin components in Drosophila cells.
A region of 1Mb from chromosome 2L is
shown. Each horizontal track represents
the binding pattern of a single protein.
Positions of genes are indicated at the
bottom.

50
gene regulation
Publications
Filion GJ, van Bemmel JG, Braunschweig
U, Talhout W, Kind J, Ward LD,
Brugman W, de Castro IJ, Kerkhoven
RM, Bussemaker HJ, van Steensel B.
Systematic protein location mapping
reveals fi ve principal chromatin types in
Drosophila cells. Cell. 201;143:212-224
van Steensel B, Braunschweig U, Filion
GJ, Chen M, van Bemmel JG, Ideker T.
Bayesian network analysis of targeting
interactions in chromatin. Genome Res.
2010;20:190-200
Peric-Hupkes D, Meuleman W, Pagie
L, Bruggeman SW, Solovei I, Brugman
W, Gräf S, Flicek P, Kerkhoven RM, van
Lohuizen M, Reinders M, Wessels L,
van Steensel B. Molecular maps of the
reorganization of genome-nuclear lamina
interactions during differentiation. Mol
Cell. 2010;38:603-613
van Bemmel JG, Pagie L, Braunschweig
U, Brugman W, Meuleman W, Kerkhoven
RM, van Steensel B. The insulator protein
SU(HW) fi ne-tunes nuclear lamina
interactions of the Drosophila genome.
PLoS ONE 2010
Pauli A, van Bemmel JG, Oliveira RA,
Itoh T, Shirahige K, van Steensel B,
Nasmyth K. A direct role for cohesin in
gene regulation and ecdysone response in
Drosophila salivary glands. Curr Biol.
2010;20:1787-1798
van Steensel B, Dekker J. Genomics tools
for unraveling chromosome architecture.
Nat Biotechnol. 2010;28:1089-1095
Kind J, van Steensel B. Genome-nuclear
lamina interactions and gene regulation.
Curr Opin Cell Biol. 2010;22:320-325
Filion GJ, van Steensel B. Reassessing
the abundance of H3K9me2 chromatin
domains in embryonic stem cells. Nat
Genet. 2010;42:4
Protein targeting interactions in chromatin Chromatin proteins often direct the
genomic binding pattern of other chromatin proteins, for example by recruitment or
competition mechanisms. The network of such targeting interactions in chromatin
is complex and still poorly understood. Together with Dr. Trey Ideker (University of
California, San Diego) we implemented Bayesian Network Inference, a probabilistic
computational method, to predict the targeting interactions among a broad set of
chromatin components in Drosophila cells, based on the DamID profi les of these
proteins. Experimental and computational validation confi rm the overall reliability
of these predictions. For example, we found that the homologous proteins HP1
and HP1c each target the heterochromatin protein HP3 to distinct sets of genes in
a competitive manner. We also discovered a central role for the remodeling factor
Brahma in the targeting of several DNA-binding factors, including GAGA factor,
JRA and SU(VAR)3-7. Our network model provides a global view of the targeting
interplay among dozens of chromatin components and provides a framework to
build an increasingly refi ned view of chromatin.
Chromatin Protein Discovery Project In 2008 we have started the Chromatin
Protein Discovery Project, which aims to generate genome-wide binding maps
for a large set of candidate novel chromatin proteins in Drosophila. The candidate
proteins are selected by computational predictions that take into account protein
domain structure, interactions with known chromatin proteins, and likelihood
of nuclear localization. For each of these we are generating full-genome, highresolution
DamID maps, which are expected to reveal many new molecular
interactions and functions of the hitherto uncharacterized proteins. We have now
generated informative binding maps of 40 novel proteins. Extensive analysis of
these binding maps is ongoing to predict the functions and molecular interactions
for each protein. This project will broaden our view of chromatin by identifying and
annotating dozens of novel components.
Genome – nuclear lamina interactions during differentiation The threedimensional
organization of chromosomes within the nucleus and its dynamics
during differentiation are largely unknown. To visualize this process in molecular
detail, we generated high-resolution maps of genome-nuclear lamina interactions
during subsequent differentiation of mouse embryonic stem cells via lineagecommitted
neural precursor cells into terminally differentiated astrocytes. This
reveals that a basal chromosome architecture present in embryonic stem cells
is cumulatively altered at hundreds of sites during lineage commitment and
subsequent terminal differentiation. This remodeling involves both individual
transcription units and multigene regions and affects many genes that determine
cellular identity. Often, genes that move away from the lamina are concomitantly
activated; many others, however, remain inactive yet become unlocked for activation
in a next differentiation step. These results suggest that lamina-genome interactions
are widely involved in the control of gene expression programs during lineage
commitment and terminal differentiation.
Nuclear lamina interactions in Drosophila By high-resolution DamID we
found that the Drosophila genome is also organized in discrete lamina-associated
domains (LADs), which are about fi ve times smaller than mammalian LADs but
contain on average a similar number of genes. We conducted a systematic analysis
of the positions of LADs relative to the binding of several insulator proteins,
which are thought to act as boundaries of chromatin domains. We found that only
SU(HW) binds preferentially at LAD borders and at specifi c positions inside LADs,
while GAF, CTCF, BEAF-32 and DWG are mostly absent from these regions. By
knockdown and overexpression studies we demonstrated that SU(HW) weakens
genome – NL interactions through a local antagonistic effect, but we did not obtain
evidence that it is essential for border formation. These results provide insights into
the evolution of LAD organization and identify SU(HW) as a fi ne-tuner of genome –
NL interactions.

EPIGENETIC REGULATION OF GENE EXPRESSION
In eukaryotic cells the DNA is packaged into chromatin by histone proteins. Posttranslational
modifi cations of the histone proteins can affect chromatin structure
and function and are involved in regulation of gene expression and DNA damage
response. Changes in chromatin modifi cation can also result in heritable changes
in gene expression without changes in the actual genetic code and these epigenetic
changes can lead to cancer. The mechanisms by which epigenetic imprints are
established or prevented are still poorly understood. Many chromatin modifi ers are
conserved from yeast to humans. Our group uses the budding yeast Saccharomyces
cerevisiae as a powerful model system to identify new epigenetic regulators and to
unravel the molecular mechanisms by which chromatin-modifying enzymes affect
chromatin structure, gene expression, and DNA damage response.
Function and regulation of histone H3 lysine 79 (H3K79) methylation by Dot1
We previously discovered a novel histone methyltransferase Dot1, which can add
one, two, or three methyl groups to lysine 79 of histone H3 on the surface of the
nucleosome core. Dot1, which is conserved from yeast to humans, infl uences
heterochromatin structure and the DNA damage response, and has been implicated
in oncogenic transformation in mammals. A major goal of our research is to
understand how H3K79 methylation affects chromatin structure and function and
how Dot1 is regulated. In contrast to other known protein methyltransferases,
Dot1 acts by a non-processive mechanism. This unusual mechanism of synthesis
affects the function of the methylated lysine and determines how it can be
regulated. In general, different forms of lysine methylation have specifi c functions
in chromatin. In contrast, the mono-, di-, and trimethylated forms of histone
H3K79 showed functional overlap in promoting silencing, suggesting that the
complex methylation pattern of H3K79 is read as a binary code of methylation or no
methylation. In addition, whereas histone methylation typically acts as a binding
signal, H3K79 methylation seems to act as an anti-binding signal. Since human
and yeast Dot1 are structurally very similar, we expect that similar rules apply to
human cells. Cell-cycle changes in H3K79 methylation in African trypanosomes are
controlled by two Dot1 proteins that have distinct enzymatic activities.
Chromatin dynamics One of the main goals of our group is to understand
how chromatin modifi cations can have long-term effects on gene expression.
Post-translational modifi cations of histones have been proposed to be involved
in epigenetic memory. When a cell divides, parental histones (containing the
epigenetic marks) and newly synthesized histones (unmodifi ed or in a ground
state) are somehow assembled onto the daughter DNA strands in a manner that
faithfully reproduces the transcriptional states of chromatin that existed prior to
chromosome duplication. The exact mode of histone inheritance is still unclear and
recent studies have shown that chromatin can be dynamic. We developed a novel
assay in yeast to determine protein turnover in vivo. This universally applicable assay,
called Recombination-Induced Tag Exchange (RITE), involves differential labeling
of existing and newly synthesized proteins by a genetic epitope-tag switch system
(see fi gure 5). Using RITE we found that histones
throughout the genome are subject to extensive
replication-independent exchange, suggesting
that histones and their post-translational marks
are not permanent residents in chromatin. We
have recently developed high-throughput methods
to identify proteins responsible for this mode of
histone deposition and eviction. We expect that our
studies will provide novel insights into the role of
histones in maintaining and erasing established
epigenetic patterns and gene expression programs.
51
gene regulation
Group leader Fred van Leeuwen
Fred van Leeuwen PhD Group leader
Iris Stulemeijer PhD Post-doc
Kitty Verzijlbergen MSc PhD student
Marit Terweij MSc PhD student
Hanneke Vlaming MSc PhD student
Tibor van Welsem Technical staff
Publications
Verzijlbergen KF, Menendez-Benito V,
van Welsem T, van Deventer SJ,
Lindstrom DL, Ovaa H, Neefjes J,
Gottschling DE, van Leeuwen F.
Recombination-induced tag exchange to
track old and new proteins. Proc Natl
Acad Sci U S A 2010;107:64-8
Frederiks F, van Welsem T, Oudgenoeg
G, Heck AJ, Janzen CJ, van Leeuwen F.
Heterologous expression reveals distinct
enzymatic activities of two DOT1 histone
methyltransferases of Trypanosoma brucei.
J Cell Sci 2010;123:4019-23
Frederiks F, Stulemeijer IJE, Ovaa H, and
van Leeuwen F. A modifi ed epigenetics
tool box to study histone modifi cations on
the nucleosome core. ChemBioChem (in
press)
Stulemeijer IJE, Pike BL, Faber AW,
Verzijlbergen KF, van Welsem T,
Frederiks F, Lenstra TL, Holstege FCP,
Gasser SM and van Leeuwen F. Dot1
binding induces chromatin rearrangements
by histone methylation-dependent and
-independent mechanisms. Epigenetics &
Chromatin (in press)
Figure 5: A genetic pulse-chase assay
to determine protein turnover in vivo.
The epitope tag on histone H3 can be
switched at the genomic level from an old
to a new tag in arrested yeast cells.
Upon re-entry into the cell cycle old
histone proteins disappear and new histone
proteins accumulate.

52
gene regulation
Group leader Jan-Hermen Dannenberg
Jan-Hermen Dannenberg PhD Group leader
Richard Heideman MSc Post-doc
Roel Wilting MSc PhD student
Eva Yanover Technical staff
Publications
Nogueira C, Kim KH, Sung H, Paraiso K,
Dannenberg JH, Bosenberg M, Chin L,
Kim M. Cooperative interactions of PTEN
defi ciency and RAS activation in melanoma
metastasis. Oncogene 2010;29:6222-32
Payne CJ, Gallagher SJ, Foreman O,
Dannenberg JH, DePinho RA, Braun, RE.
Sin3a is required by Sertoli cells to establish
a niche for undifferentiated spermatogonia,
germ cell tumors and spermatid elongation.
Stem Cells 2010;8:1424-34
Wilting RH, Yanover Ea , Heideman RMa ,
Jacobs H, Horner J, Van der Torre J,
DePinho RA, Dannenberg, JH. Overlapping
functions of Hdac1 and Hdac2 in cell cycle
regulation and hematopoiesis. EMBO J.
2010;29:2586-97 ( aauthors contributed
equally)
Dannenberg JH, Berns A. Drugging Drug
Resistance. Cell 2010;141:18-20
THE ROLE OF CLASS I HDACS IN TRANSCRIPTION,
DEVELOPMENT AND TUMORIGENESIS
P roteins are regulated by a vast number of modifying enzymes that balance posttranslational
modifi cations such as phosphorylation, methylation and acetylation.
Acetylation of lysine residues is controlled by histone acetyl transferases (HATs)
and histone deacetylases (HDACs). Both classes of proteins have been linked to
tumorigenesis as cancer-specifi c mutations are found in HATs (e.g. CBP) while
many tumor types display elevated levels of HDAC1, HDAC2 and HDAC3. Owing
to their anti-tumor activity, HDAC-inhibitors (HDACi) have entered the clinic in the
treatment of cutaneous T-cell lymphoma and are tested in various clinical trials for
combination therapy. Despite their clinical effi cacy little is known about the actual
mode of action of HDACi and which HDACs they are targeting to elicit anti-tumor
activity.
Our group focuses on dissecting the function of HDACs in development and
tumorigenesis by using mice and cells carrying Hdac1 and Hdac2 conditional
knockout alleles. Studies aimed at unraveling the transcriptome of these
transcriptional co-repressors in combination with the identifi cation of new
components of the HDAC1/2 mediated pathways may reveal new drug targets for
anti-cancer therapy.
Class I HDACs in development and cell cycle regulation Previously, we have
shown that Hdac2-defi cient mice are viable albeit that that they are born with a twofold
lower Mendelian frequency and display a 25% reduction in total bodyweight.
Furthermore, it was shown that Hdac2 functions in modulating synaptic plasticity
and long-lasting changes of neural circuits, which in turn negatively regulates
learning and memory (Guan et al., 2009). In order to study the role of Hdac1 and
Hdac2 in cell cycle regulation we generated a set of isogenic mouse embryonic
fi broblasts (MEFs) harboring combinations of Hdac1 and Hdac2 conditional
knockout alleles. Cre-recombinase mediated deletion of either Hdac1 or Hdac2
did not result in a cell cycle related phenotype due to functional compensatory
up-regulation of Hdac2 or Hdac1 protein levels, respectively. Indeed, deletion of
both Hdac1 and Hdac2 (double knock-out; DKO) resulted in a senescence-like G 1cell
cycle arrest. In contrast to expression of wild-type versions, catalytic inactive
Hdac1 or Hdac2 did not rescue the cell cycle arrest, suggesting that Hdac1 or
Hdac2 deacetylase activity is required for cell cycle progression. Despite strong upregulation
of the cell cycle inhibitor Cdkn1a (p21 Cip ) in DKO MEFs, simultaneous
genetic inactivation of either Cdkn1a or Trp53 did not result in a bypass of cell cycle
arrest and senescence. Moreover, inactivation of Hdac1 and Hdac2 in transformed
cells that have bypassed oncogene-induced senescence still resulted in a cell cycle
arrest, providing a rational for the anti-tumorigenic activity of HDACi.
Class I HDACs in hematopoiesis Treatment of cancer patients with HDACi
results often in hematological side-effects. In order to determine whether this is
due to off-target effects or due to inhibition of HDACs we inactivated Hdac1 and
Hdac2 in bone marrow using MxCre transgenic mice. While ablation of Hdac1
or Hdac2 does not affect hematological differentiation, loss of Hdac1 and Hdac2
results in severe anemia and thrombocytopenia as consequence of bone marrow
cytopenia. Deletion of Hdac1 and Hdac2 resulted in a dramatic decrease in early
progenitors and hematopoietic stem cells (HSCs) and consequently in a reduction
of all differentiated hematopoietic lineages. Detailed analysis of Hdac1 and Hdac2
knockout mice suggested an intrinsic role for Hdac1 and Hdac2 in hematopoietic
stem cell survival. Therefore, hematological related side-effects observed in HDACi
treated patients are very likely related to on-target Hdac1 and Hdac2 inhibition.
Figure 6: Hdac1 and Hdac2 regulate hematopoietic stem cell survival in a cell-autonomous
fashion. A. Deletion of Hdac1 and Hdac2 in a competitive bone marrow transplantation experiment
results in a progressive decrease of peripheral Ly5.2+ (Hdac1/2 defi cient; DKO) cells. B. Total numbers
of control and DKO hematopoietic stem cells (LSK) at the end of the competitive bone marrow
transplantation experiment as analyzed by FACS analysis

DIVISION OF IMMUNOLOGY
LYMPHOCYTE ACTIVATION AND SURVIVAL
Our interest is to determine how cells decide between living and dying. We focus
on the mechanism of action of TNF receptor family members, since these govern
such decisions. Lymphocytes are our main cell type of interest, since throughout
their existence, they mostly live “on the edge” between life and death. Our work is
inspired by the desire to improve cancer immunotherapy. The second aim of our
work is to contribute to the design of novel therapies aimed at killing cancer cells by
activating apoptotic pathways.
TNF receptor family members and control of the immune response From our
work, TNF receptor family member CD27 and its ligand CD70 have emerged as
interesting targets to improve anti-tumor immunity. This costimulatory receptor/
ligand pair promotes the generation and maintenance of effector CD8 T cells, the
formation of memory CD8 T cells and their secondary expansion. CD27 rescues
primed T cells from apoptosis, which partially explains its impact on the magnitude
of the CD8 T cell response.
T cell survival To determine by which molecular mechanisms CD27 directs the T
cell response, we have used genome-wide expression profi ling. In this way, we have
identifi ed IL-2 as a key CD27-directed gene product in primed CD8 T cells. In vivo
experiments with reconstituted T cells proved that CD27 promotes the survival of
effector CD8 T cells at the tissue site via an autocrine IL-2/IL-2 receptor pathway.
However, CD27 promotes survival of clonally expanding CD8 T cells at the priming
site via different mechanisms. One of these involves engaging the pro-metabolic
and anti-apoptotic Pim-1 serine/threonine kinase that appears to be a direct target of
CD27 signaling.
CD4 T cell function We have also determined the array of CD27 target genes in
CD4 T cells. CD27 supports the survival of CD4 T cells, but also has qualitative
effects on these cells. In the fi rst place, CD4 T cells require CD27 to deliver help for
the CD8 memory response. This involves a programming of CD8 cells during the
primary response, enabling them to express certain effector genes upon secondary
stimulation. One of these is a membrane molecule that we hypothesize to be
essential for memory T cell function. Secondly, CD27 target genes in CD4 T cells are
diagnostic for a T helper-1 (Th1) effector function, in agreement with data from other
researchers that identify CD70 on dendritic cells as a mediator of IL-12-independent
Th1 differentiation. Thirdly, we have recently found that CD27 signaling has a
dramatic effect on CD4 T cells that are on their way to develop into T helper-17
(Th17) cells. This pro-infl ammatory CD4 T cell subset has raised much attention in
recent years, because of its causative role in certain auto-immune diseases.
Deliberate CD27 stimulation of developing murine Th17 cells by means of an
agonistic recombinant CD70 that we produce in our laboratory disables Th17
effector function. This was also apparent in vivo, in a model of Th17-dependent
auto-immunity, experimental allergic encephalomyelitis (EAE), which is a disease
reminiscent of human multiple sclerosis. Absence of CD27-CD70 signaling in
CD27 -/- and our recently developed CD70 -/- mice led to increased severity of disease,
concomitant with increased numbers of Th17 cells. Conversely, in our CD70
transgenic mice that constitutively express CD70 on dendritic cells, the disease was
less severe than in control mice and fewer Th17 cells developed. CD27 signaling
does not affect the master regulators of transcription that determine commitment of
CD4 T cells towards the Th17 lineage, but selectively silences expression of the IL-17
gene by histone modifi cation. We are currently looking into the molecular details of
this important process.
Hematopoiesis By analysis of CD70 transgenic (tg) mice, we have discovered a
novel link between CD27-CD70 and bone homeostasis. CD70tg mice that express
CD70 under control of the CD11c promoter develop increased trabecular bone
mass, accompanied by decreased cellularity of bone marrow, progressive anemia
and extramedullary hematopoiesis. Osteoclast numbers, but not osteoblast
Publications
53
immunology
Division head, group leader Jannie Borst
Jannie Borst PhD Group leader
Yanling Xiao MD PhD Senior Post-doc
Jonathan Coquet PhD Post-doc
Ulf Geumann PhD Post-doc
Bert van de Kooij MSc PhD student
Chiel Maas MSc PhD student
Victor Peperzak MSc PhD student
Rogier Rooswinkel MSc PhD student
Elise Veraar MSc PhD student
Evert de Vries Technical staff
Gerda van der Horst Technical staff
Verbrugge I, Maas C, Heijkoop
M, Verheij M, Borst, J. Radiation
and anti-cancer drugs can facilitate
mitochondrial bypass by CD95/Fas via
c-FLIP downregulation. Cell Death Differ.
2010;17:551-61
Maas Ch, Verbrugge I, de Vries E, Savych
G, van de Kooij LW, Tait SWG, Borst J.
Smac/DIABLO release from mitochondria
and XIAP inhibition are essential to limit
clonogenicity of Type I tumor cells after
TRAIL receptor stimulation. Cell Death
Differ. 2010;17:1613-23
Maas Ch. Molecular regulation of
death receptor- and DNA damageinduced
apoptosis. Thesis, University of
Amsterdam, 2010
Xiao Y, Peperzak V, Van Rijn L, Borst J,
de Bruijn JD. Dexamethasone treatment
during the expansion phase maintains
stemness of bone marrow mesenchymal
stem cells. J Tissue Eng Regener Med.
2010;4:374-86
Peperzak V, Xiao Y, Veraar EAM, Borst J.
CD27 sustains survival of CTLs in virusinfected
non-lymphoid tissue in mice by
inducing autocrine IL-2 production. J Clin
Invest. 2010;120:168-78
Zwart W, Peperzak V, de Vries E, Keller
AM, van der Horst G, Veraar EA, Janssen
L, Janssen H, Naik SH, Neefjes J, Borst
J. The invariant chain transports TNF
family member CD70 to MHC class II
compartments in dendritic cells. J Cell
Sci. 2010;123:3817-27

54
immunology
Publications (continued)
Peperzak V, Veraar EAM, Keller AM,
Xiao Y, Borst J. The Pim kinase pathway
contributes to survival signaling in primed
CD8+ T cells upon CD27 costimulation.
J Immunol. 2010;185:667-78
Peperzak V. Molecular Mechanisms
underlying CD27-CD70 costimulation.
Thesis, University of Amsterdam, 2010
Yamaura K, Boenisch O, Watanabe
T, Ueno T, Vanguri V, Yang J, Tanaka
K, Guleria I, Borst J, Zhai Y, Kupiec-
Weglinski JW, Najafi an N. Differential
requirement of CD27 costimulatory
signaling for naïve versus alloantigenprimed
effector/memory CD8 + T cells.
Am J Transplant. 2010;10:1210-20
Salek-Ardakani S, Flynn R, Arens R,
Yagita H, Smith G, Borst J, Schoenberger
S, Croft M. TNFR family members
OX40 and CD27 link viral virulence to
protective T cell vaccines. J Clin Invest
2011; 121:296-307
Figure 1: CD27 mediates effector CD8+
T cell survival at the tissue site via an
autocrine IL-2 pathway.
A complementation assay, in which wildtype
or CD27-/- T cells were retrovirally
transduced to express the il-2 gene and
followed in vivo for their responsiveness
to infl uenza virus infection, proved that
CD27 signaling allows effector CD8 T cells
to survive in non-lymphoid (lung) tissue
via autocrine IL-2/IL-2 receptor signaling
(Peperzak V. et al., J Clin Invest 2010).
numbers were reduced in CD70tg mice. Strikingly, CD27 was found on a certain
cell population in normal bone marrow that showed a strong commitment toward
osteoclast formation. We conclude that CD27 hallmarks a newly defi ned osteoclast
progenitor and that sustained engagement of CD27 on these cells inhibits osteoclast
development, leading to an increased trabecular bone mass and perturbation of
the bone marrow niche. This negative feedback may be provided by CD70-bearing
activated immune cells and may underlie bone remodeling observed under such
pathological conditions.
Ligand traffi cking Deliberate constitutive expression of CD70 at the cell surface has
dramatic effects on naïve lymphocyte homeostasis, as demonstrated in CD70tg
mice. This can convert immunological tolerance or ignorance into immunity. We
have discovered that cell surface expression of CD70 is intricately regulated by its
intracellular storage and directed transport, which is governed, surprisingly, by the
MHC class II chaperone Invariant chain (CD74).
Conclusion Our novel fi ndings indicate that targeting the CD27-CD70 costimulatory
axis may be of interest not only in the context of anti-tumor immunity, but also in
auto-immune or infl ammatory disease. Expression of CD27 and CD70 in the mature
immune system are largely conserved between human and mouse, and CD70
expression hallmarks patients with constitutive immune activation. However, it
remains to be shown to which extent the mechanistic details of CD27-CD70 action
are conserved. In collaboration with former Organon (now MSD), we are exploring
the production of functionally active reagents that target the human molecules
and may prove clinically interesting. We have fi led a European patent on one such
reagent.
Apoptosis signaling and cancer therapy Pro-apoptotic agents are of great interest
for cancer therapy, provided that they can act in a tumor-specifi c fashion. The TRAIL
death receptors conform to this condition; they are not toxic on normal tissue and
induce apoptotic cell death in many different tumor types. Although the exact
mechanisms underlying this tumor-specifi city are not known, agonist reagents that
target the two TRAIL receptors in human have moved rapidly through preclinical
testing and are now in Phase I and II clinical trials. Interestingly, TRAIL receptor
agonists act synergistically with conventional and novel therapeutics in many
cases, by a variety of mechanisms. One is the p53-dependent upregulation of the
TRAIL receptors. We have revealed other sensitization mechanisms, including p53independent
downregulation of c-FLIP molecules that block death receptor-induced
inducer caspase activation.
Factors that modulate tumor cell sensitivity to TRAIL receptor agonists are of
interest, given the effi cacy of this novel type of anti-cancer therapeutic. We have
identifi ed two molecules that control the cell surface expression of TRAIL receptor-1,
but not TRAIL receptor-2. One of these impacts on intracellular transport of
TRAIL receptor-1 in the biosynthetic route, while the other is a ubiquitin ligase
that determines receptor turnover by endocytosis at the plasma membrane. These
mechanisms attenuate apoptosis sensitivity of the tumor cells and are therefore of
clinical relevance.
Apoptosis signaling relies on Bcl-2 family members that act either pro- or antiapoptotically.
BH3 domain-only proteins within this family are essential to convey
the apoptotic signal to mitochondria, from where caspase activation is initiated.
BH3 domain mimetic drugs are very promising anti-cancer therapeutics that act
tumor-specifi cally according to the principle of “oncogene” addiction. Tumor cells
may be more reliant on inhibitory Bcl-2 family members than normal cells, due
to their dangerous life style. Inactivating inhibitory Bcl-2 family members by BH3
domain mimetic drugs therefore allows for selective tumor cell killing. We study the
mechanism of action of certain BH3 domain-only proteins and their antagonists,
with focus on the role of ubiquitination in determining their activity and half life.

DISSECTING AND MANIPULATING ANTIGEN-SPECIFIC T CELL
IMMUNITY
The aim of our research is straightforward 1) to design novel technologies that can
be used to examine and modify antigen-specifi c T cell immunity 2) to use these
tools to unravel and manipulate the molecular processes underlying immune
recognition by T lymphocytes. Within these projects, a main focus is on the design
and testing of novel concepts for adoptive immunotherapy.
Dissecting antigen-specific T cell immunity There is now substantial evidence
that therapeutic manipulation of immune reactivity can result in clinically
meaningful effects on human cancer. For example, T cell responses induced by
either anti-CTLA4 treatment or infusion of ex vivo expanded tumor-infi ltrating
T cells (TIL therapy) have shown substantial activity in metastatic melanoma.
Importantly, at present we do not know which cytotoxic T cell reactivities mediate
cancer regression. Furthermore, as the number of potential melanoma-associated
antigens to which these responses can be directed is very high, classical strategies to
map cytotoxic T cell reactivity do not suffi ce. Knowledge of such reactivities would
be of obvious use, both to monitor current therapies and to design more targeted
strategies that selectively aim to induce immune reactivity against these antigens.
In the past years, we have set out to develop a broadly applicable platform that
allows one to dissect disease- and therapy-induced T cell reactivity. First, in earlier
work in collaboration with the group of Huib Ovaa (Division of Cell Biology) we
have designed MHC class I molecules occupied with UV-sensitive ‘conditional’
peptide ligands, providing a robust platform for the creation of very large collections
of pMHC complexes for T cell detection. Second, in more recent work we have
developed a ‘combinatorial coding’ strategy that allows the parallel detection of
dozens of different T cell populations within a single sample. Having established
this technological framework, we have used it in collaboration with the Haanen
group to assess whether TIL therapy induces a detectable alteration in the
melanoma-reactive T cell repertoire. Using TIL cell products generated either at the
NKI or by collaborators at NIH and in Israel, we have demonstrated that individual
TIL products contain unique combinations of antigen reactivities, and that the
combined magnitude of these responses is surprisingly low. Importantly, TIL
therapy of melanoma patients leads to a signifi cant increase in the tumor-reactive T
cell compartment, and T cell reactivity post-infusion can almost in full be explained
by the patterns of reactivity observed within the matched cell product. Collectively,
these results suggest that the clinical effi cacy of TIL therapy may be enhanced by the
preparation of more defi ned tumor-reactive cell products, and establish the value of
high-throughput monitoring for the analysis of immuno-active therapeutics.
Dissection of T cell immunity through genetic tagging and intravital
imaging The ability to visualize antigen-specifi c T cell responses and to determine
the differentiation pathways of different subsets of T cells is essential for our
understanding of pathogen- and vaccine-induced immunity. While MHC tetramer
technology makes it possible to follow the development of immunity at the T cell
population level, it doesn’t allow the analysis of cell fate and cellular differentiation
pathways.
To allow for such lineage tracking, we have invested in the development of
technologies with which individual T cells can be tagged with genetic barcodes.
This tagging technology relies on the use of oncoretroviral and lentiviral libraries
containing some 5,000 different barcodes. Infection of cell populations of interest
by these libraries of viral vectors and subsequent analysis of the barcodes present
within these cell populations can then be utilized to reveal lineage relationships.
Using a strategy for the retroviral barcode labeling of naïve T lymphocytes, we have
previously analyzed to what extent short-lived ‘effector’ T cells and memory T cells
are derived from the same naïve T cell clones. Contrary to models that assume that
T cell fate is to a large extent determined during the initial priming event, these
data show that effector and memory T cell subsets are more or less completely
derived from the same set of naïve T cell precursors. These data however did not
address whether memory and short-lived effector fates could occur as a consequence
Group leader Ton Schumacher
Publications
55
immunology
Ton Schumacher PhD Group leader
Gavin Bendle PhD Senior post-doc
Andreas Hollenstein PhD Post-doc
Shalin Naik PhD Post-doc
Jan Rohr MD Post-doc
Remko Schotte PhD Post-doc
Jenny Shu PhD Post-doc
Marit van Buuren MSc PhD student
Carmen Gerlach MSc PhD student
Jeroen van Heijst MSc PhD student
Carsten Linnemann MSc PhD student
Riccardo Mezzadra MSc PhD student
Laura Bies Technical staff
Nienke van Rooij Technical staff
Mireille Toebes Technical staff
Jos Urbanus Technical staff
Abrahamsen IW, Stronen E, Wälchli S,
Johansen JN, Kjellevoll S, Kumari S,
Komada M, Gaudernack G, Tjonnfjord G,
Toebes M, Schumacher TN, Lund-
Johansen F, Olweus J. Targeting B cell
leukemia with highly specifi c allogeneic T
cells with a public recognition motif.
Leukemia 2010;24:1901-9
Bendle GM, Linnemann C, Bies L,
Hooijkaas AI, de Witte MA, Jorritsma A,
Pouw N, Kaiser AD, Debets R, Kieback E,
Uckert W, Song J-Y, Haanen JB,
Schumacher TN. Lethal graft-versus-host
disease in mouse models of T cell receptor
gene therapy. Nat Med 2010;16:565-70
Borkner L, Kaiser A, Van de Kasteele AW,
Andreesen R, Mackensen A, Haanen JB,
Schumacher TN, Blank C. RNA
interference targeting programmed death
receptor-1 improves immune functions of
tumor-specifi c T cells. Cancer Immunol
Immunother. 2010;59:1173-83
Gerlach C, Van Heijst JW, Swart E, Sie D,
Armstrong N, Kerkhoven RM, Schepers K,
Schumacher TN.. One naive T cell - multiple
fates in CD8+ T cell differentiation. J Exp
Med 2010;207:1235-46
Hadrup SR, Schumacher TN. MHCbased
detection of antigen-specifi c CD8+ T
cell responses. Cancer Immunol
Immunother. 2010;59:1425-33

56
immunology
Publications (continued)
Hawkins RE, Gilham DE, Debets R,
Eshhar Z, Taylor N, Abken H,
Schumacher TN. D evelopment of adoptive
cell therapy for cancer: A clinical perspective.
Hum Gene Ther. 2010;21:665-72
Heemskerk B, Jorritsma A, Gomez-Eerland
R, Toebes M, Haanen JB, Schumacher
TN. Microbead-assisted retroviral
transduction for clinical application.
Hum Gene Ther. 2010;21:1335-42
Hoppes R, Ekkebus R, Schumacher TN,
Ovaa H. Technologies for MHC class I
immunoproteomics. J Proteomics
2010;73:1945-53
Schmid DA, Irving MB, Posevitz V,
Hebeisen M, Posevitz-Fejfar A, Sarria JC,
Gomez-Eerland R, Thome M,
Schumacher TN, Romero P, Speiser DE,
Zoete V, Michielin O, Rufer N. Evidence
for a TCR affi nity threshold delimiting
maximal CD8 T cell function.
J Immunol.2010;184:4936-46
Van den Berg J, Nuijen B, Schumacher
TN, Haanen JB, Storm G, Beijnen JH,
Hennink WE. Synthetic vehicles for DNA
vaccination. J Drug Target 2010;18:1-14
Van den Berg JH, Oosterhuis K, Hennink
WE, Storm G, Van der Aa LJ, Engbersen
JF, Haanen JB, Beijnen JH, Schumacher
TN, Nuijen B. Shielding of charge is
essential for antigen expression and
immunogenicity of nanoparticleformulated
dermal DNA vaccines.
J Contr Rel. 2010;141:234-40
Van Heijst JW, Gerlach C, Schumacher
TN. Tracing the life histories of T cells.
Nat Rev Immunol. 2010;10:621-31
Velthuis JH, Unger WW, Abreu JR,
Duinkerken G, Franken K, Peakman M,
Bakker AH, Hadrup SR, Keymeulen B,
Drijfhout JW, Schumacher TN, Roep BO.
Simultaneous detection of circulating
autoreactive CD8+ T-cells specifi c for
different islet cell-associated epitopes using
combinatorial MHC-multimers. Diabetes
2010;59:1721-30
Wensveen FM, Van Gisbergen KPJM,
Derks IAM, Gerlach C, Schumacher TN,
Van Lier RA, Eldering E. During
interclonal competition following T cell
activation, the Noxa/Mcl-1 axis constitutes
a survival threshold set by TCR affi nity and
IL-2 signaling. Immunity 2010;32:754-65
of an asymmetry during the fi rst cell division, a model put forward by Reiner and
colleagues. To address this possibility, we have now performed experiments in which
genetic tags are introduced only after the fi rst cell division. These data suggest that
while the fi rst cell division of T cells upon their activation may be asymmetric, the
fate of the resulting daughter cells is purely symmetric.
The vast majority of studies – including those described above – primarily assess the
development of antigen-specifi c T cell responses within the blood compartment. To
also allow the analysis of T cell reactivity at the sites of action, the Haanen group and
our group have developed an intravital imaging system in which virus-specifi c T cell
responses can be followed in skin. Mathematical modeling of the data (collaboration
with the group of Dr R de Boer, Utrecht University) has revealed by which migration
pattern antigen-specifi c T cells within the periphery locate virus-infected cells.
Furthermore, recent work on the imaging of memory T cells that remain in skin
following clearance of infection indicates that the antigen-specifi c T cells that stay
put take on a dendritic morphology and show a remarkable crawling behavior in
between skin keratinocytes. The continuous migration of memory T cells allows
these cells to contact large numbers of skin cells through time, and we propose that
memory T cell crawling serves to allow the rapid detection of renewed infection at a
previously infected site.
Figure 2: Mathematical atical
modeling of the effect ect of
directional migration on of effector
T cells on detection of virus-infected sites. Left: cell tracks of in silico cells that migrate using
experimentally determined distributions of angles-to-infection. Right: tracks of in silico cells that
migrate using randomly drawn angles-to-infection. Open circles indicate the simulated sites of infection.
Closed circles indicate cell positions at the end of the simulation.
Adoptive T cell therapy (collaboration with Haanen lab) The cornerstone of our
translational work is the development and evaluation of adoptive T cell therapies
for human cancer. The MHC-based monitoring strategies described above will be
utilized to evaluate T cell reactivity in the randomized trial for TIL therapy that will
be carried out by the Haanen group. Furthermore, the material that is obtained
in these analyses forms a very useful starting point for the further development
of our second approach for adoptive T cell therapy; the genetic engineering of T
cell reactivity. Specifi cally, in the past years our group has developed the retroviral
introduction of antigen-specifi c T cell receptors into peripheral T cells as a means
to induce tumor-specifi c T cell immunity. We have now produced a clinical grade
batch retrovirus encoding a melanoma-reactive TCR for the gene modifi cation of T
cells of patients with metastatic melanoma. The pharmaceutical process that will be
utilized to generate gene-modifi ed T cells for this fi rst NKI TCR gene therapy trial
has been established. To evaluate which antigens can best be targeted in TCR gene
therapy, we are at the same time preparing for a substantially larger clinical program
in which we intend to test a series of different T cell receptors that target various
tumor-associated antigens. The TCRs that we aim to use in this program will be
derived from the antigen-reactive T cell populations identifi ed in our TIL monitoring
program, and we are currently establishing novel technologies to retrieve such TCR
genes. Furthermore, we are evaluating alternative, transposon-based gene transfer
strategies to create the TCR-modifi ed T cells that can be used in these studies.

IMMUNOTHERAPY AND IMMUNOMONITORING
The main objective of this line of research is the development of novel T cell
immunity-based strategies that can be translated into clinical application. The focus
is on treatment of patients with solid tumors, especially melanoma and renal cell
carcinoma. A second line of research concerns immunomonitoring. This work is
performed under supervision of Dr F Vyth-Dreese. Its primary aim is to evaluate
specifi c- and cytokine-based immunotherapies, using advanced technologies for
characterization of immune responses in peripheral blood and at the tumor site.
These studies are conducted together with the Schumacher lab, NKI-AVL, national
and international collaborators.
DNA vaccination for the treatment of cancer
Phase I study in melanoma patients
Induction of immunity following DNA vaccination is generally considered a slow
process. We have shown that DNA delivery to the skin results in a highly transient
pulse of antigen expression. Based on this information, we developed a novel, rapid
and potent intradermal DNA vaccination method. By short-interval intradermal
DNA delivery, robust T cell responses, of a magnitude suffi cient to reject established
subcutaneous tumors, are generated within 14 days. These results were confi rmed
in a non-human primate model (in collaboration with BPRC, Rijswijk). We could
show that DNA tattooing results in 10-100-fold increase in vaccine-specifi c T cells
compared to intramuscular vaccination.
Together with Dr. B Nuijen (Pharmacy) and Profs. W Hennink and G Storm
(University of Utrecht) we have developed an ex vivo human skin model to optimize
DNA tattoo vaccination for human skin and to create a platform for testing novel
DNA formulations for transfection and targeting of human skin cells. Results from
these studies have been crucial for clinical application of this strategy. A clinical
grade DNA vaccine has been produced in the GMP DNA plasmid production unit
(Amsterdam-Biotherapeutics Unit) at the NKI-AVL/Slotervaart Hospital pharmacy
department. In 2009 and 2010, six stage IV melanoma patients have been enrolled
in a fi rst phase I clinical trial with DNA tattoo vaccination. The study was amended
in 2010 and recently opened in the Leiden University Medical Center as well.
DNA vaccination for the treatment of high risk human papilloma virus (HPV) associated
cancers
Human papilloma virus infection (serotypes 16 and 18) is strongly associated with
the development of squamous cell cancer of the cervix, but also of penis, vulva, anus
en oropharynx. Because the persistence of oncogenic high-risk HPV proteins E6
and E7 is required for carcinogenesis, these viral antigens are exquisite targets for
immunotherapeutic interventions. Indeed, therapeutic vaccinations targeting these
viral antigens have shown some promise in woman suffering from cervical cancer.
In the next years (in collaboration with Prof. G. Kenter, Dr. M. van Beurden en Prof.
S. Horenblas (all Division of Surgical Oncology), we will perform a phase I/II study
in patients with HPV 16-positive squamous cell cancer of the penis and cervix, using
our novel and potent intradermal DNA vaccination strategy. In preclinical studies,
we have developed highly immunogenic and safe HPV 16 E6- and E7-containing
DNA vaccines for which we have started GMP production in 2010 for a fi rst clinical
trial.
Adoptive immunotherapy program
TIL therapy
Adoptive therapy with Tumor-infi ltrating Lymphocytes (TIL) is based on results
from the Surgery Branch, NIH (Bethesda, MD, USA) and results from the Sheba
Medical Center (Tel Aviv, Israel) showing a 50% objective response rate in heavily
pretreated stage IV melanoma patients. This treatment combines the ex vivo culture
of melanoma-reactive T cells from resected metastases with non-myeloablative
chemotherapy and high dose bolus IL-2. Our goals are to show that this treatment
can be given safely at the NKI-AVL, to demonstrate in an randomized controlled
phase II trial that this treatment improves progression-free survival compared to
standard chemotherapy and to perform a comprehensive analysis of the T cells
Group leader John Haanen
57
immunology
John Haanen MD PhD Group Leader
Florry Vyth-Dreese PhD Senior staff scientist
Joost van den Berg PhD Post-doc
Bianca Heemskerk PhD Post-doc
Annelies Jorritsma PhD Post-doc
Susanne Quaak PhD Post-doc
Esther Tjin PhD Post-doc
Slivia Ariotti MSc PhD student
Iris van der Heijden PhD student
Koen Oosterhuis MSc PhD student
Raquel Gomez MSc Technical staff
Trees de Jong Technical staff
Willeke van de Kasteele Technical staff
Martin van der Maas Technical staff
Publications
Bendle GM, Linnemann C, Hooijkaas AI,
Bies L, de Witte MA, Jorritsma A,
Kaiser AD, Pouw N, Debets R, Kieback E,
Uckert W, Song JY, Haanen JB,
Schumacher TN. Lethal graft-versus-host
disease in mouse models of T cell receptor
gene therapy. Nat Med. 2010;16:565-70
Borkner L, Kaiser A, van de Kasteele W,
Andreesen R, Mackensen A, Haanen JB,
Schumacher TN, Blank C. RNA
interference targeting programmed death
receptor-1 improves immune functions of
tumor-specifi c T cells. Cancer Immunol
Immunother. 2010;59:1173-83
Heemskerk B, Jorritsma A, Gomez-
Eerland R, Toebes M, Haanen JB,
Schumacher TN. Microbead-assisted
retroviral transduction for clinical
application. Hum Gene Ther.
2010;21:1335-42
Kim Y-H, Vyth-Dreese FA, Schrama E,
Pavel S, Bajema I, Goulmy E, Spierings E.
Dendritic cells facilitate minor H antigen
HA-1-specifi c skin graft-versus-host
reactivity ex vivo. Biology of Blood and
Marrow Transplantation, 2010 (in press)
Oosterhuis K, van den Berg JH,
Schumacher TN, Haanen JB. DNA
Vaccines and Intradermal Vaccination by
DNA Tattooing. Curr Top Microbiol
Immunol. 2010

58
immunology
Publications (continued)
Quaak SG, Haanen JB, Beijnen JH,
Nuijen B. Naked plasmid DNA
formulation: effect of different
disaccharides on stability after
lyophilisation. AAPS PharmSciTech.
2010;11:344-50
Van den Berg JH, Nuijen B, Schumacher
TN, Haanen JB, Storm G, Beijnen JH,
Hennink WE. J Drug. Synthetic vehicles
for DNA vaccination. J Drug Target.
2010;18:1-14
Van den Berg JH, Oosterhuis K, Beijnen
JH, Nuijen B, Haanen JB. DNA
vaccination in oncology: current status,
opportunities and perspectives. Curr Clin
Pharmacol. 2010;5:218-25
Van den Berg JH, Oosterhuis K, Hennink
WE, Storm G, van der Aa LJ, Engbersen
JF, Haanen JB, Beijnen JH, Schumacher
TN, Nuijen B. Shielding the cationic
charge of nanoparticle-formulated dermal
DNA vaccines is essential for antigen
expression and immunogenicity. J Control
Release. 2010;141:234-40
Vyth-Dreese FA, Sein J, Van De Kasteele
W, Dellemijn TA, Van Den Bogaard C,
Nooijen WJ, De Gast GC, Haanen JB, Bex
A. Lack of anti-tumour reactivity despite
enhanced numbers of circulating natural
killer T cells in two patients with
metastatic renal cell carcinoma. Clin Exp
Immunol. 2010;162:447-459
specifi cities of the melanoma-reactive TIL prior and after adoptive transfer.
We expect to start treatment of the fi rst patients early 2011.
T-cell receptor gene therapy
In close collaboration with the Schumacher lab, we have selected a highly avid T
cell receptor (TCR) specifi c for melanocyte differentiation antigen MART-1 26-35.
This TCR, called 1D3, has been cloned into a retroviral vector (MP-71) and has been
produced by a German GMP manufacturer. This TCR has been equipped with extra
safe guards to prevent mispairing with endogenous TCR chains after transduction
of peripheral T cells to prevent potential side effects. The process of clinical grade
culturing and transduction of peripheral T cells with the 1D3-MP-71 retrovirus
is now being validated step-by-step in our GMP facility in order to start a phase I
clinical study in melanoma patients in 2011.
Immunomonitoring of DNA tattoo vaccination trial Recently, we have analysed
samples from the fi rst two cohorts of 6 melanoma patients treated in the DNA
tattoo vaccination trial. Biopsies, taken from the vaccination site, were partly used
to culture and recover T lymphocytes and partly for immunohistological analysis.
Culture of skin pieces in the presence of high dose IL-2 resulted in outgrowth of
MART-1-specifi c CD8 T lymphocytes in 10-500 fold higher numbers compared
to healthy donor skin samples. In 3 out of 5 cases, yields of 30-50% of MART-1
specifi c CD8 T cells were observed compared to less than 0.1% in control skin.
Immunohistological analysis showed the presence of CD8 T lymphocytes and
activated DCs near the basal membrane. This indicated that MART-1 DNA tattoo
vaccination induced migration of MART-1 specifi c CD8 T cells to the vaccination
site. Virtually no therapy-induced MART-1 specifi c CD8 T cells were detected in
the peripheral blood. ELISpot analysis showed therapy-induced IFNg responses to
MART-1 in 1out of 6 and to TTFc peptides in 2 out of 6 patients. These results show
that DNA tattoo vaccination can induce local CD8 T cell responses. In this trial 2
more cohorts, including 9 patients, will be treated.
Immune infiltrates in renal cell carcinoma (RCC) treated with anti-angiogenic
agents Patients with metastatic RCC are currently treated with targeted therapy
consisting of tyrosine kinase and mTOR inhibitors, and anti-VEGF mAb. These
therapies are based on inhibition of angiogenesis, as well as direct tumor-targeting
and may potentiate anti-tumor immune responses. Tumor specimens obtained
from RCC patients treated with Sunitinib, Avastin or IFNa show increased immune
cell infi ltration and apoptosis of tumor and vasculature compared with untreated
patients. These analyses will be extended to additional patients.
Detection of mHag-specific T lymphocytes in human tissues
We have successfully applied in situ tetramer staining for the detection of minor
Histocompatibility antigen (mHag)-specifi c T cells in a human ex vivo in situ skin
explant model of Graft versus Host reactivity (in collaboration with groups at the
Leiden University Medical Center and Utrecht University). Recently, we have
developed and validated a method, using so-called MHC-dextramers, to directly
enumerate specifi c T lymphocytes in cryopreserved tissues. Currently, we are
implementing this technique in Graft versus Host Disease to detect mHag-directed
T lymphocytes in patient skin lesions.
Human ex vivo in situ skin model for vitiligo and melanoma In collaboration
with the Amsterdam Academical Medical Center and Leiden University Medical
Center, a human in situ skin model has been developed to study immune factors
involved in the development of vitiligo and potential therapy of melanoma. Using
melanocyte specifi c T cell clones or bulk T cells obtained from vitiligo lesions cocultured
with normal skin tissues, the induction of vitiligo could be mimicked ex
vivo. Currently, presence of tumor specifi c T cells in peripheral blood of melanoma
patients is compared to tumor infi ltration profi les.

DNA DAMAGE TOLERANCE, PROGRAMMED MUTAGENESIS
AND LYMPHOMAGENESIS
B cells have the unique capacity to mutate their immunoglobulin genes by
programmed mutagenesis. Our research is focused on the following aspects: 1) To
unravel the molecular mechanism of programmed mutagenesis, 2) To determine
the targeting specifi city of the mutation process and its contribution to lymphoma
development, and 3) To understand the decision making between repair and
mutagenesis.
In vivo binding preferences of the mutator protein AID in the genome
To improve adaptive immunity, the activation induced cytidine deaminase (AID)
initiates somatic hypermutation (SHM) and class switch recombination (CSR)
in immunoglobulin (Ig) genes. As shown by selective mutation analysis, non-Ig
genes are targeted by AID, although the genome-wide impact of aberrant targeting
remains to be determined. The aim of this study was to establish a genome-wide
AID binding profi le and identify molecular parameters controlling the targeting
of AID. To accomplish these goals, we apply the DamID technique for AID in a
hypermutation-competent Burkitt lymphoma cell line. This unbiased approach
revealed that AID is targeted throughout the genome and preferentially to actively
transcribed genes. Moreover, AID binding is favored in regions enriched in their
G/C content and enriched for G-stretches in the coding strand. Furthermore, AID
does not exhibit an intrinsic preference for binding to RGYW motifs, suggesting
that these hot spots of SHM are deamination- rather than binding hot spots of AID.
Future studies should allow us to provide a detailed, genome-wide binding profi le
of AID. This profi le will be critical in identifying aberrant AID target sites (ATS),
which will help to identify cancer-associated ATS causal to the development of GC-
and post-GC derived B cell lymphomas. ATS are presently used to identify molecular
predictors that favor AID binding.
The Fanconi Anemia Core Complex is dispensable during Somatic
Hypermutation To generate high affi nity antibodies during an immune response,
B cells undergo somatic hypermutation (SHM) of their Ig genes. Error-prone
translesion synthesis (TLS) DNA polymerases have been reported to be responsible
for all mutations at template A/T and at least a fraction of G/C transversions. In
contrast to A/T mutations, which depend on PCNA ubiquitination, it remains
unclear how G/C transversions are regulated during SHM. Several lines of evidence
indicate a mechanistic link between the Fanconi Anemia (FA) pathway and TLS.
To investigate the contribution of the FA pathway in SHM we analyzed B cells
defi cient for FancG, an essential member of the FA core complex. These B cells
were hypersensitive to treatment with cross-linking agents, but the frequencies and
nucleotide exchange spectra of SHM remained comparable between wild-type and
FancG-defi cient B cells. These data indicate that the FA pathway is not involved in
regulating the outcome of SHM in mammals. In addition, the FA pathway appears
dispensable for class switch recombination.
Allelic exclusion at the immunoglobulin heavy chain locus initiates at the
level of transcription Developing lymphocytes generate their antigen receptor
genes by DNA recombination. Once a gene has been productively assembled, the
receptor is expressed and terminates rearrangement of the second allele to ensure
mono-specifi city. Using specifi c, non-functional IgH knock-in and transgenic
mice, we showed in collaboration with Dr J Lutz and Dr H-M Jäck (University of
Erlangen, Germany) that stable expression of a non-coding µ heavy chain (HC)
mRNA in B cells reduces DNA recombination at the IgH locus and impairs early
B cell development. Functional µHC mRNA serves therefore not only as a classical
messenger, but also as a sensor for productive IgH rearrangements and as a
regulator of allelic exclusion during B cell development.
Group leader Heinz Jacobs
59
immunology
Heinz Jacobs PhD Group leader
Richard Heideman MSc PhD student
Peter Krijger MSc PhD student
Marc Hogenbirk MSc PhD student
Niek Wit MSc PhD student
Paul van den Berk Technical staff
Publications
Krijger PHL, Storb U, Jacobs H. Errorprone
and error-free resolution of AID
lesions in SHM. (invited book chapter)
DNA deamination and the Immune
System. Editors: Fugmann S, Diaz M,
Papavasiliou N. Publisher: Imperial
College Press. 2010
Krijger PHL, Wit N, van den Berk
PCM, Jacobs H. The Fanconi anemia
core complex is dispensable during
somatic hypermutation and class switch
recombination. PLoS ONE 2010 (in press)
Wilting RH, Yanover E, Heideman
MR, Jacobs H, Horner J, van der
Torre J, DePinho RA, Dannenberg
JH. Overlapping functions of Hdac1
and Hdac2 in cell cycle regulation and
haematopoiesis. EMBO J. 2010;29:2586-
97
Krijger PHL, Lee K-Y, Wit N, van den Berk
PCM, Wu X, Roest HP, Maas A, Ding H,
Hoeijmakers JHJ, Myung K and Jacobs
H. HLTF and SHPRH are not essential
for PCNA Polyubiquitination, survival
and somatic hypermutation: existence of
an alternative E3 ligase. DNA Repair 2010
(in press)
Wit N, Krijger PHL, van den Berk PCM
and Heinz Jacobs. Lysine residue 185 of
Rad1 is a topological but not a functional
counterpart of lysine residue 164 of PCNA.
PLoS ONE (in press)

60
immunology
Group leader Christian Blank
Christian Blank MD PhD Group leader
Andrew Kaiser PhD Post-doc
Anna Hooijkaas MSc PhD student
Aurelie Guislain Technical staff
Jules Gadiot Technical staff
Publications
Gadiot J, Hooijkaas AI, Kaiser ADM,
Tinteren H, van Boven H, Blank C.
Overall survival and PD-L1 expression
in metastasized malignant melanoma.
Cancer 2010 (in press)
Borkner L, Kaiser A, van de Kasteele W,
Andreesen R, Mackensen A, Haanen
JB, Schumacher TN, Blank C. RNA
interference targeting programmed death
receptor-1 improves immune functions of
tumor-specifi c T cells.. Cancer Immunol
Immunother. 2010;59:1173-83
COMBATING TUMOR IMMUNE ESCAPE AND
IMMUNOTHERAPY
We aim to identify mechanisms of tumors immune escape and to develop
therapeutic protocols to overcome these. Tumor immune escape mechanisms
include inhibitory molecules on tumor cells or on antigen presenting cells and
immune regulatory cells in the tumor environment. The functional characterization
of inhibitory molecules, exploration of their inhibition and the examination
of possible synergy with small molecule based targeted therapies may help in
designing novel approaches to improve anticancer immunotherapy.
Development of an inducible spontaneous murine melanoma model It is
crucial to test new therapeutic approaches in appropriate in vivo models that
simulate the human cancer reality. Transplantable tumor models often do not mimic
the complex interaction between the tumor cell and the tumor microenvironment
and therefore may have little predictive value for the treatment of cancer patients.
Spontaneous murine melanoma models are more physiological, but due to the
late onset of tumor formation less practical for long-term immunotherapeutic
experiments. By crossing mice that inducibly express the in human melanoma often
observed mutations BRAFV600E and loss of PTEN, we could induce melanoma in
these mice at a high penetrance and at short time to onset. We found TIL within the
tumor environment and could culture TIL and tumor cell lines, making this model
highly valuable for testing small molecule approaches as well as immunotherapeutic
approaches.
Role of co-inhibitory molecules during tumor immune escape Appropriate
T cell activation depends on TCR ligation and a positive secondary signal. Recent
work revealed that this secondary signal is not an on-off phenomenon but a signal
of modulated intensity, which is orchestrated by several co-stimulatory and coinhibitory
molecules. We and others have shown that one of the ligands (PD-L1) of
one such a co-inhibitory molecule (PD-1) is highly expressed on tumor cells and
leads to impaired immune responses. We found an increased PD-L1 expression on
metastases compared to primary melanoma in human, but no infl uence on overall
survival, raising the question in which situations PD-L1 inhibits tumor specifi c T
cells, which is a current project.
Homeostatically proliferating T cells for the treatment of cancer One approach
in immunotherapy is the adoptive transfer of tumor-reactive T cells. For effective
tumor growth control, tumor-reactive T cells should suffi ciently expand and survive,
without exhaustion. Transfer of naïve peripheral T cells into lymphopenic murine
recipients results in a slow cytokine-driven proliferation of these T cells. During this
homeostatic proliferation (HP), T cells acquire effector functions (IFN-production,
lytic activity), while keeping characteristics of naïve T cells. This results in better
CD62L-mediated lymph node homing, less anergy induction and better tumor
growth control compared to naïve or effector T cells. As induction of lymphopenia
by chemo- or chemoradiotherapy is accompanied by serious adverse events, we
aim at in vitro induction of HP. We can expand T cells in vitro with superior tumor
growth control capabilities in vivo (in mice) and characterize them at the moment in
comparison to naïve, primed and memory T cells.
Generation of RCC TIL after tyrosine kinase inhibitor pretreatment
Neoadjuvant treatment of patients improves the outcome of surgery and
radiotherapy. We are currently exploring such an approach to improve the expansion
of tumor infi ltrating lymphocytes (TIL) from renal cell carcinoma (RCC). TIL
therapy is a promising immunotherapeutic approach in melanoma inducing
long lasting clinical responses currently tested at several institutes. Culture of
TIL generated from RCC has been described before, but failed to induce clinical
responses. Reason for this is that former protocols lacked preconditioning of the
patients to induce lymphopenia. We aim at expanding TIL from pretreated RCC
patients to higher numbers, with more gentle expansion protocols to prevent
exhaustion and negative selection.

DIVISION OF MOLECULAR BIOLOGY
Genetic instability and deregulated cell cycle control are hallmarks of human cancer.
Our research involves both aspects focusing on (1) the role of DNA mismatch repair
and the Fanconi anemia genome maintenance pathway in mutation avoidance
and (2) the role of cell cycle checkpoints in tumor suppression. The principle tools
include gene modifi cation in murine embryonic stem cells (ESC) and analyses of the
phenotypic consequences in ESC, mutant mice and cell lines derived thereof.
DNA MISMATCH REPAIR
Lynch syndrome/HNPCC (hereditary non-polyposis colorectal cancer) is caused
by inherited defects in DNA mismatch repair (MMR). The primary function of
MMR is correction of DNA replication errors, which is initiated by MSH2/MSH6 or
MSH2/MSH3 protein complexes that recognize mismatches in DNA. Subsequent
steps involve recruitment of another protein complex, MLH1/PMS2, activation of
exonucleolytic activity to remove the error-containing DNA strand and resynthesis
of a new strand. Mismatches can also arise by replication of damaged bases such as
O 6 -methylguanine. This lesion elicits futile cycles of mismatch repair, ultimately
leading to cell death. Thus, DNA MMR acts anti-mutagenic and mediates the toxicity
of methylating agents.
Oligonucleotide-directed gene modification We have shown that single-stranded
oligo-deoxyribonucleotides (ssODNs) can be used for subtle gene modifi cation in
ESC. The ssODN are identical to the chromosomal target sequence except for one or
a few centrally located nucleotides that comprise a desired modifi cation. However,
the incomplete base paring between the ssODN and its chromosomal complement
activates the MMR system, leading to abortion of the gene modifi cation reaction
(Dekker et al., NAR 2003;31:e27). We found that ESC can be rendered permissive for
‘oligo targeting’ by transient suppression of MSH2 protein level by RNA interference
(Aarts et al., NAR 2006;34:e147).
To study the mechanism of oligo targeting we generated ESCs carrying singlecopy
chromosomally-located neomycin (neo) or EGFP reporter genes that were
disabled by a mutation in the start codon. The appearance of G418-resistant or
green-fl uorescent cells serves as readout for ssODN-mediated restoration of the
start codon. The highest targeting frequencies were obtained with ssODNs of 35-40
nucleotides. Remarkably, the effi cacy of oligo targeting was not affected by any DNA
repair mechanism other than MMR, nor by transcription of the target locus (Aarts
and Te Riele, JCMM 2010;14-6b:1657). Using the EGFP reporter we found that antisense
ssODN yielded green-fl uorescent cells already 24h after exposure, however,
the frequency of targeted cells declined upon prolonged culturing to stabilize at day
4. In contrast, with sense ssODN, green fl uorescent cells appeared at 48h and the
frequency of targeted cells remained stable upon culturing (Aarts and Te Riele, NAR
2010;38:6956). These observations support a model in which the ssODN physically
integrates into the host genome during DNA replication (fi gure 1).
Remarkably, ssODN containing phosphorothioate (PTO) linkages (to protect them
from degradation) induced DNA double-strand breaks impeding proliferation
of targeted cells. Unmodifi ed ssODN were harmless to cells and yielded higher
targeting effi ciencies than PTO-ssODN.
Unclassified variants of MMR genes We have developed a protocol for ssODNdirected
gene modifi cation to target in principle any site in the ESC genome (Aarts
et al., Methods Mol Biol 2009;530:79-99). We use this technology to recreate
suspected variants of MMR genes in ESC in order to assess their capacity to sustain
MMR functions. MMR gene mutations affecting a single codon are widespread
in the human population and in (suspected) Lynch syndrome patients but the
consequences of single amino acid substitutions are often diffi cult to predict. We
have thus far analyzed fi ve of such ‘Variants of uncertain signifi cance’ (VUS) of
MSH2 and found one fully and another partially abrogating MMR activity. As we use
61
molecular biology
Division head, group leader Hein Te Riele
Hein Te Riele PhD Group leader
Rob Dekker PhD Post-doc
Camiel Wielders PhD Post-doc
Kamila Wojciechowicz-Grzadka PhD Post-doc
Marieke Aarts MSc PhD student
Sietske Bakker MSc PhD student
Hellen Houlleberghs MSc PhD student
Tinke Vormer MSc PhD student
Eva Wielders MSc PhD student
Marleen Dekker Technical staff
Elly Delzenne-Goette Technical staff
Sandra De Vries MSc Technical staff
Anja Van der Wal Technical staff
Publications
Aarts M, Te Riele H. Parameters
of oligonucleotide-mediated gene
modifi cation in mouse ES cells. J Cell
Mol Med 2010;14-6b:1657-67
Aarts M, Te Riele H. Subtle gene modifi cation
in mouse ES cells: evidence for incorporation
of unmodifi ed oligonucleotides
without induction of DNA damage.
Nucleic Acids Res 2010;38:6956-67
Aarts M. Gene targeting by single-stranded
DNA oligonucleotides in mouse embryonic
stem cells. PhD thesis, VU University
Amsterdam, April 2010
Aarts M, Te Riele H. Progress and
prospects: oligonucleotide-directed gene
modifi cation in mouse embryonic stem
cells: a route to therapeutic application.
Gene Ther 2010;doi:10.1038/gt.2010.161
Van Harn T, Foijer F, van Vugt M,
Banerjee R, Yang F, Oostra A, Joenje
H, Te Riele H. Loss of Rb proteins
causes genomic instability in the absence
of mitogenic signaling. Genes Dev
2010;24:1377-88
Vormer TL. Tumor suppression by
retinoblastoma proteins: interacting
proteins and cooperating pathways.
PhD thesis, VU University Amsterdam,
November 2010

62
molecular biology
Figure 1. ssODN-mediated correction of
mutant EGFP.
(A) Model I: the sense ssODN (fat line with
white cross) anneals to its chromosomal
complement and serves as a template for
repair of the transcribed strand (white x),
allowing immediate EGFP expression.
Model II: the sense ssODN integrates into the
non-transcribed strand during replication or
recombination (D-loop). A second round of
replication is needed to alter the transcribed
strand and allowing EGFP expression.
Our fi ndings are compatible with Model II.
NT, non-transcribed strand; T, transcribed
strand. (B) Incorporation of sense ssODNs
(fat line) into the non-transcribed strand
during S phase requires an extra round of
replication to transmit the corrected EGFP
sequence to the transcribed strand (day 2).
(C) Incorporation of anti-sense ssODNs (fat
line) into the transcribed strand immediately
provides a template for EGFP production (day
1. However, subsequent semi-conservative
replication will dilute the number of EGFPpositive
cells, theoretically four-fold.
mouse ESC, the latter could be introduced into the germ line of mice. We found this
variant to induce tumour formation as strongly as a full Msh2 knockout, identifying
it as a deleterious mutation. Three variants showed normal MMR capacity, which is
remarkable in view of the evolutionary conservation of the affected amino acids.
THE FANCONI ANEMIA PATHWAY
Another example of cancer predisposition by inherited defects in DNA repair
is Fanconi anemia (FA), a recessive disorder characterized by malformations,
progressive anemia and high incidence of AML and epithelial tumors. FA is caused
by bi-allelic defects in either one of 13 genes, FANCA, B, C, D1, D2, E, F, G, I, J, L,
M, N. At the cellular level, malfunctioning of these genes causes hypersensitivity to
cross-linking agents as manifested by G 2 arrest, chromosomal aberrations and cell
death.
To assess the signifi cance of the FA pathway in suppression of cancer, we have
generated Fancf- and Fancm-defi cient mice. FANCF and FANCM are part of the FA
core complex that mono-ubiquitinates FANCD2 and FANCI. Both, Fancf and Fancm
defi ciency caused hypogonadism in mice and hypersensitivity to cross-linking
agents in mouse embryonic fi broblasts (MEFs), thus phenocopying other FA mouse
models. However, Fancm defi ciency also caused features atypical for FA, including
under-representation of females and decreased overall and tumor-free survival
(Bakker et al., Hum Mol Genet 2009;18:3484). The latter may be correlated to the
role of FANCM in the suppression of spontaneous sister chromatid exchanges as
we observed in MEFs. Thus, FANCM may function both inside and outside the FA
core complex to maintain genome stability. The Fancf and Fancm knockout alleles
have been crossed into cancer prone Apc +/- mice to study whether defects in the FA
pathway accelerate tumorigenesis.
CELL CYCLE CHECKPOINTS
Loss of G 1/S control is a frequent if not mandatory event in tumor development. G 1/S
control relies on the pocket proteins pRB, p107 and p130 that collectively regulate the
activity of E2F transcription factors. We use MEFs devoid of pocket proteins (TKO
MEFs) to study residual cell cycle control mechanisms and to identify events that
promote oncogenic transformation.
Growth-factor independence TKO MEFs still rely on mitogens for survival and
proliferation. In the absence of mitogens, many TKO MEFs undergo apoptosis
whereas the surviving fraction arrests in the G 2 phase of the cell cycle. G 2 arrest was
effectuated through inhibitory interactions of the cyclin-dependent-kinase inhibitors
p21 CIP1 and p27 KIP1 with Cyclins A and B1 (Foijer et al., Cancer Cell 2005;8:455).
G 2 arrested cells showed high levels of DNA double strand breaks, which were only
partially repaired when TKO cells were stimulated to enter mitosis by mitogen readdition.
Moreover, mitotic chromosomes showed a ‘railroad’ appearance indicative
of defects in centromeric sister-chromatid cohesion (Van Harn et al., Genes Dev
2010;24:1377). Furthermore, we found aneuploidy in cell cultures derived from
TKO cells that had been transiently mitogen deprived. These results suggest that
combined lack of pocket proteins and mitogenic signaling can lead to genomic
instability, which may contribute to tumor progression.
Anchorage independence We have also shown that pocket protein ablation
was not suffi cient to sustain anchorage-independent growth, even not upon
expression of RAS V12 , although TKO MEFs were immortal. Apparently, a cell cycle
mechanism still operates to restrict proliferation of these cells in soft agar. By
performing a gain-of-function screen for genes that permit anchorage-independent
growth of RAS V12 -expressing, pocket protein defi cient MEFs, we identifi ed the
immortalizing oncogene TBX2 that was shown to suppress the p53 pathway through
downregulation of p19 ARF (Vormer et al., MCB 2009;28:7263). Furthermore, we
have developed a new technique to enzymatically produce shRNA libraries from
cDNAs. Screening of these libraries yielded several novel suppressors of anchorageindependent
growth.

DNA BASE J
-glucosyl-hydroxymethyluracil (base J), which we discovered in African
trypanosomes in 1993 (Gommers-Ampt et al., Cell 1993;75:1129-1136), is a base
present in kinetoplastid fl agellates and in Euglena. It replaces 1% of thymine in
nuclear DNA and is predominantly located in repetitive sequences, such as telomeric
repeats. We have characterized a J-binding protein (JBP1) that binds to J-containing
duplex DNA (Cross et al. EMBO J 1999;18:6573-6581). We have shown that JBP1 is a
thymidine hydroxylase that catalyses the fi rst step of J biosynthesis, the conversion
of T in DNA into hydroxymethyluracil. JBP1 belongs to the family of Fe 2+ and
2-oxoglutarate-requiring dioxygenases, as does a second putative hydroxylase, JBP2.
In the kinetoplastid Leishmania, a JBP1 KO is lethal. In contrast, JBP2 is dispensable
in Leishmania, but JBP2 KO strains are hypersensitive to bromodeoxyuridine
(BrdU). During growth in BrdU, Leishmania loses its J, which is located for > 98% in
telomeric repeats in this organism. How J loss leads to cell death is unclear. We do
not fi nd alterations in DNA integrity or cell cycle blocks. A recent breakthrough came
form the discovery by R. Sabatini (University of Georgia, US) that trypanosomes
have J at transcriptional start and stop sites. Using immuno-precipitation of J-DNA
and deep sequencing, we have also found the 1% of non-telomeric J in Leishmania at
specifi c chromosome-internal positions, partly at transcriptional stops (collaboration
with NKI-AVL deep sequencing unit and Peter Myler, Seattle). We have shown
that loss of this internal J leads to massive readthrough of RNA Polymerase
II transcriptional stops, as shown in fi gure 2, suggesting that transcriptional
termination is a major function of J. We have also found an interesting interaction
between J and histone H3/H3V (H3 variant) and the tools are on board to determine
the role of the chromatin environment in J function. With Anastassis Perrakis
(NKI-AVL) we are trying to determine the structure of JBP1-J-DNA complexes
by crystallography. In 2010 the structure of the DNA-binding domain of JBP1
was solved. Interestingly, this domain has a unique structure not seen before in
DNA-binding proteins and the specifi c binding of JBP1 to J-DNA was shown to be
dependent on a single aspartate residue interacting with the glucose-moiety of base J.
Figure 2:
Readthrough of
RNA Polymerase
II transcription
termination stops
upon loss of J
in Leishmania
tarentolae. With a
ChIP experiment
we determined the
distribution of J
along chromosome
4. Internal J is located at the transcription termination site of the two long convergent polycistronic
transcription units. J is lost from this location in the JBP1KO cell line grown in the presence of BrdU.
We sequenced and mapped the small RNA degradation products present in WT and mutant cell lines
and plotted the ratio of sense/antisense transcript. When J is lost, antisense transcripts spread from the
transcription stop region to both sides of the chromosome due to readthrough of the stop site.
DRUG TRANSPORTERS
We are interested in mechanisms of drug resistance in cancer cells and focus on
resistance caused by increased ATP-dependent transport of drug out of the cell,
mediated by ATP-binding cassette (ABC) transporters. We have isolated genes for
these transporters and are characterizing their substrate specifi city and sensitivity
to inhibitors in transfected cells. To study the physiological function of these
transporters, we have inactivated genes for several drug transporters by targeted
gene disruption in mice. We are mainly studying the Multidrug Resistance Protein
(ABCC) family members MRP2, 3, 4, 5 and 6.
We have initiated a systematic search for compounds conjugated to glucuronide
Group leader Piet Borst
63
molecular biology
Piet Borst MD PhD Group leader
Henri Van Luenen PhD Academic staff
Sven Rottenberg DVM PhD Dipl ECVP
Senior post-doc
Koen Van de Wetering DVM PhD
Senior post-doc
Charlotte Guyader PhD Post-doc
Robert Jansen PhD Post-doc
Pankaj Tripathi PhD Post-doc
Nikola Banishki MSc PhD student
Petra Krumpochova MSc PhD student
Guotai Xu MSc PhD student
Serge Zander DVM MSc PhD student
Marcel De Haas Technical staff
Liesbeth Van Deemter Technical staff
Sabrina Jan Technical staff
Ariena Kersbergen Technical staff
Sunny Sapthu Technical staff
Wendy Sol Technical staff
Publications
Beedholm-Ebsen R, Van de Wetering K,
Hardlei T, Nexo E, Borst P, Moestrup
SK. Identifi cation of multidrug resistance
protein 1 (MRP1/ABCC1) as a molecular
gate for cellular export of cobalamin. Blood
2010;115:1632-9
Borst P, Wessels L. Do predictive
signatures really predict response to
cancer chemotherapy? Cell Cycle 2010
2010;9:4836-40
Issaeva N, Thomas HD, Djurenovic
T, Jaspers JE, Stoimenov I, Kyle S,
Pedley N, Gottipati P, Zur R, Sleeth K,
Chatzakos V, Mulligan EA, Lundin C,
Gubanova E, Kersbergen A, Harris AL,
Sharma RA, Rottenberg S, Curtin NJ,
Helleday T. 6-thioguanine selectively kills
BRCA2-defective tumors and overcomes
PARP inhibitor resistance. Cancer Res
2010;70:6268-76
Pajic M, Kersbergen A, van Diepen F,
Pfauth A, Jonkers J, Borst P, Rottenberg
S. Tumor-initiating cells are not enriched
in cisplatin-surviving Brca1-/-;p53-/mammary
tumor cells in vivo. Cell Cycle
2010;9:3780-91

64
molecular biology
Publications (continued)
Rottenberg S, Pajic M, Jonkers J.
Studying drug resistance using genetically
engineered mouse models for breast cancer.
In: Zhou J (ed). Multi-Drug Resistance
in Cancer. Totowa: Humana Press,
2010:33-45
Vlaming ML, van Esch A, Pala Z,
Wagenaar E, Van de Wetering K, Van
Tellingen O, Schinkel AH. Abcc2 (Mrp2),
Abcc3 (Mrp3), and Abcg2 (Bcrp1) are the
main determinants for rapid elimination
of methotrexate and its toxic metabolite
7-hydroxymethotrexate in vivo. Mol
Cancer Ther 2009;8:3350-9
Zander S, Kersbergen A, Van der
Burg E, De Water N, Van Tellingen O,
Gunnarsdottir S, Jaspers J, Nygren
AOH, Jonkers J, Borst P, Rottenberg
S. Sensitivity and acquired resistance of
BRCA1; -53-defi cient mouse mammary
tumors to the topoisomerase I inhibitor
topotecan. Cancer Res 2010;70:1700-10
Figure 3: Correlation of gene expression
with the response to platinum drugs. (A)
35 mice with Brca1-/- ;p53-/- mammary
tumors were treated with 6mg of cisplatin
per kg i.v. on day 0. The time until relapse
is the time required for the tumors to grow
back to 100%. (B) Signifi cance analysis
of microarrays of highly versus moderately
cisplatin-sensitive Brca1-/- ;p53-/- mouse
mammary tumors using the MEEBO
(D=1.5; FDR=0) or Illumina (D=0.85;
FDR=0) platform. (C) Kaplan Meier
survival curves according to XIST gene
expression levels of patients with HER2negative,
high-risk breast cancer who had
been randomized between conventional
(CONV, grey) and intensive platinumbased
chemotherapy (IPB, black). P values
were calculated using the logrank test.
or sulphate that are transported by MRPs by comparing the derivatives in plasma/
urine of WT and KO mice using Mass Spectrometry (MS). We have identifi ed several
glucuronidated and sulphated phyto-estrogens, derived from food, as novel MRP2
and MRP3 substrates by this approach. In addition, we have found that conjugated
retinoids are MRP2 substrates. We are refi ning the LC/MS analysis to allow the
identifi cation of all compounds altered in plasma/urine of KO mice. This approach
should also be helpful in fi nding substrates of other MRPs and BCRP (ABCG2).
DRUG RESISTANCE IN ‘SPONTANEOUS’ MOUSE TUMORS
In collaboration with Jos Jonkers (NKI-AVL), we are studying resistance mechanisms
in “spontaneous” mammary tumors arising in mice, conditionally defective in p53
and Brca1. When treated with the maximum tolerable dose of doxorubicin, docetaxel
or topotecan, the breast tumors initially respond but eventually always develop
resistance. Resistance is often associated with upregulation of the Mdr1a and Mdr1b
genes (Abcb1), which encode drug-transporting P-glycoproteins (P-gps) and we have
shown with specifi c inhibitors that remarkably low levels of Abcb1 upregulation
(5-fold the levels in sensitive tumors) suffi ce to make the tumor multidrug resistant.
We are also using this mouse model to test new anticancer drugs and drug
combinations. Impressive tumor regression has been obtained with a new inhibitor
of Poly-ADP-ribose polymerase I (PARPI), olaparib, but resistance to this compound
also arises by Abcb1 upregulation. We have crossed disrupted alleles for the Abcb1
genes into our mouse model to further test the importance of these transporters in
drug resistance and to uncover other forms of resistance, - notably to doxorubicin,
doceta docetaxel e aand doapa olaparib b -, , not otmediated edatedby by P-gp. gp.
In contrast to the results
obtained with MDR drugs,
we have been unable to obtain
cisplatin resistance in this
tumor model. The tumors
respond to each new treatment
with cisplatin, but are never
fully eradicated. Although
we have identifi ed a tumorinitiating
cell (“stem cell”) in
this tumor model characterized
by high surface expression
of CD24 and CD49f, this
fraction does not appear to be
enriched in the “remnants”
from which the tumors regrow
after chemotherapy. We are
testing the hypothesis that the
resistance of “remnants” is not
due to specifi c biochemical defense mechanisms of the putative tumor stem cells,
but to the ability of a sub-fraction of the cells to go into “hibernation”, i.e., stop cell
cycle progression until the drug is gone and the DNA damage has been repaired.
We have succeeded in isolating tumor cell lines in low O2 that resemble the original
tumor. These cultured cells have allowed us to study the fraction of cells surviving
cisplatin. Remarkably, this fraction does not contain G2/M cells, but appears to stall
in G1/G0. We are studying how these cells escape cisplatin-induced death.
The initial response to drug treatment of these tumors varies from tumor to
tumor. We can distinguish “good” from “poor” responders. As this tumor model
is genetically so homogeneous, it is in principle ideal for identifying markers that
predict drug response. We have shown in our model that standard gene expression
profi ling is unable to detect drug resistance mechanisms that cause poor drug
response in a minority of the tumors. This explains the failure of this approach in
the clinic. We have found, however, low Xist expression in most mouse tumors with
“good” initial response to cisplatin (fi gure 3). Low Xist expression was subsequently
also found in our clinic to predict long recurrence-free survival in a group of breast
cancer patients treated with platinum-based chemotherapy.

MOUSE MODELS OF BREAST CANCER
The focus of our research group is on the genetic dissection of human breast
cancer through the use of genetically engineered mouse models. For this, we have
developed models for p53-induced breast cancer, BRCA1- and BRCA2- associated
hereditary breast cancer, and E-cadherin-mutated lobular breast cancer. We are
using these models to (1) investigate genotype-phenotype relations in mammary
tumorigenesis; (2) study therapy response and resistance of primary tumors and
metastases; (3) identify genetic changes underlying breast tumorigenesis; (4) study
the role of immunity in breast cancer development and metastasis.
Functional genetic screens and complementation assays in BRCA1-deficient
ES cells We have generated ES cells and mice containing selectable conditional
(SCo) knockout alleles of Brca1 and Brca2. To identify factors rescuing cells from
growth arrest induced by BRCA1 loss we have performed clonal survival screens in
R26cre-ER T2 ;Brca1 SCo/D ES cells using PiggyBac (PB)-based insertional mutagenesis.
We found that inactivating PB insertions in 53BP1 rescued ES cells from BRCA1
defi ciency. Loss of 53BP1 partially restores the homologous-recombination defect of
BRCA1-defi cient cells and reverts their hypersensitivity to DNA-damaging agents.
Notably, loss of 53BP1 expression is also found in subsets of sporadic triple-negative
and BRCA-associated breast cancers.
We have also used our R26cre-ER T2 ;Brca1 SCo/D ES cells to perform functional
complementation assays for testing human BRCA1 variants of unknown clinical
signifi cance (VUS). For this, we have engineered our ES cells to enable rapid
knock-in of human BRCA1 cDNAs by FLP recombinase mediated cassette exchange
(RMCE). Introduction of wild-type hBRCA1 – but not pathogenic hBRCA1 mutants
– rescues the growth defect of switched R26cre-ER T2 ;Brca1 SCo/D cells. We have so far
tested 60 defi ned hBRCA1 VUSs in our functional complementation assay system.
Conditional mouse models for BRCA-associated breast cancer We have
previously generated conditional mouse mutants with K14cre- or WAPcremediated
tissue-specifi c loss of Brca1/2 and p53 to establish models for BRCA1- and
BRCA2-associated breast cancer. The Brca1 –/– ;p53 –/– mammary tumors share
histopathological and molecular features with BRCA1-defi cient breast cancers
in women: they are highly proliferative, poorly differentiated, hormone receptor
and HER2 negative mammary adenocarcinomas with a high degree of genomic
instability. Interestingly, we have found that mammary tumor formation in our
BRCA1 model is still estrogen-dependent. We are currently investigating whether
this estrogen dependence is due to autocrine or paracrine mechanisms.
The central role of BRCA1 and BRCA2 in the DNA damage response (DDR) implies
that BRCA-defi cient tumors are especially sensitive to therapeutics that directly or
indirectly induce DNA double-strand breaks. In collaboration with Sven Rottenberg
and Piet Borst we have used our BRCA1/2 models to test the anti-tumoral effi cacy
of the PARP inhibitor AZD2281 (olaparib) from KuDOS-AstraZeneca, which may
be selectively toxic to BRCA-defi cient cells because it suppresses DNA singlestrand
break repair. Administration of olaparib to mice with Brca1 –/– ;p53 –/– or
Brca2 –/– ;p53 –/– mammary tumors induced tumor regression without signs of
toxicity. However, long-term treatment with olaparib resulted in the development of
drug resistance caused by upregulation of the Mdr1 genes encoding P-glycoprotein
(Pgp) drug effl ux transporters. To study Pgp-independent mechanisms of olaparib
resistance, we have crossed the BRCA1 mammary tumor model onto an Mdr1 –/–
background and transplanted the resulting Brca1 –/– ;p53 –/– ;Mdr1 –/– tumors into
wildtype recipients. Treatment of these mice with olaparib resulted in induction
of resistance without loss of target (PARP) inhibition. Intriguingly, a fraction of
olaparib-resistant Brca1 –/– ;p53 –/– ;Mdr1 –/– tumors showed loss of 53BP1 expression,
highlighting the role of this DDR factor in therapy resistance.
In collaboration with Hein te Riele and Jo Morris (King’s College London), we
have generated Brca1 mouse mutants that mimic three common human founder
mutations, the BRCA1-185delAG, BRCA1-5382insC frameshift mutants and the
Group leader Jos Jonkers
65
molecular biology
Jos Jonkers PhD Group leader
Karin De Visser PhD Associate staff member
Peter Bouwman PhD Post-doc
Gilles Doumont PhD Post-doc
Marco Koudijs PhD Post-doc
Ewa Michalak PhD Post-doc
Petra Ter Brugge PhD Post-doc
Marieke Van de Ven PhD Post-doc
Martine van Miltenburg PhD Post-doc
Metamia Ciampricotti MSc PhD student
Chris Doornebal MSc PhD student
Rinske Drost MSc PhD student
Henne Holstege MSc PhD student
Janneke Jaspers MSc PhD student
Sjoerd Klarenbeek MSc PhD student
Christiaan Klijn MSc PhD student
Hanneke Van der Gulden Technical staff
Ingrid Van der Heijden Technical staff
Ellen Wientjens Technical staff
Ute Boon Research assistant
Tanya Braumuller Research assistant
Tissee Hau Research assistant
Eva Kregel Research assistant
Mark Pieterse Research assistant
Pramudita Prasetyanti MSc Research assistant
Eline Van der Burg Research assistant
Selected publications
Bouwman P, Aly A, Escandell JM, Pieterse
M, Bartkova J, van der Gulden H, Hiddingh
S, Thanasoula M, Kulkarni A, Yang Q, Haffty
BG, Tommiska J, Blomqvist C, Drapkin R,
Adams DJ, Nevanlinna H, Bartek J, Tarsounas
M*, Ganesan S*, Jonkers J*. 53BP1 loss rescues
BRCA1 defi ciency and is associated with triplenegative
and BRCA-mutated breast cancers.
Nat Struct Mol Biol 2010;17:688-695 (* joint
corresponding authors)
Ciampricotti M, Vrijland K, Hau GS,
Pemovska T, Doornebal CW, Speksnijder
EN, Wartha K, Jonkers J, de Visser KE.
Development of metastatic HER2+ breast
cancer is independent of the adaptive immune
system. J Pathol 2010 (in press)
Derksen PW, Braumuller T, van der Burg
E, Hornsveld M, Mesman E, Wesseling J,
Krimpenfort P, Jonkers J. Mammary-specifi c
inactivation of E-cadherin and p53 impairs
functional gland development and leads to

66
molecular biology
Publications (continued)
pleomorphic invasive lobular carcinoma.
Dis Model Mech 2010 (in press)
Evers B, Helleday T*, Jonkers J*. Targeting
homologous recombination repair defects in
cancer. Trends Pharmacol Sci 2010;31:372-380.
(* joint corresponding authors)
Evers B, Speksnijder EN, Schut E,
Ciampricotti M, Smalley MJ, Derksen
PWB, Jonkers J*, de Visser KE. A tissue
reconstitution model to study cancer
cell-intrinsic and -extrinsic factors in
mammary tumorigenesis. J Pathol
2010;220:34-44 (* corresponding author)
Evers B, Schut E, van der Burg E,
Braumuller TM, Egan DA, Holstege H,
Edser P, Adams DJ, Wade-Martins R,
Bouwman P, Jonkers J. A high throughput
pharmaceutical screen identifi es compounds
with specifi c toxicity against BRCA2-defi cient
tumors. Clin Cancer Res 2010;16:99-108
Holstege H, Horlings HM, Velds A,
Langerød A, Børresen-Dale AL, van de Vijver
MJ, Nederlof PM, Jonkers J. BRCA1-mutated
and basal-like breast cancers have similar
aCGH profi les and a high incidence of protein
truncating TP53 mutations. BMC Cancer
2010;10:654
Holstege H, van Beers E, Velds A, Liu X,
Joosse SA, Schut E, Kerkhoven R, Klijn CN,
Wessels LF, Nederlof PM, Jonkers J. Crossspecies
comparison of aCGH data from mouse
and human BRCA1- and BRCA2-mutated
breast cancers. BMC Cancer 2010;10:455
Klijn C, Bot J, Adams DJ, Reinders M,
Wessels LF*, Jonkers J*. Identifi cation of
networks of co-occurring, tumor-related
DNA copy number changes using a genomewide
scoring approach. PLoS Comput Biol
2010;6:(* joint corresponding authors)
Rottenberg R, Pajic M, Jonkers J. Studying
drug resistance using genetically engineered
mouse models for breast cancer. Methods Mol
Biol 2010; 596: 33-45
Varelaa I, Klijn C, Stephens PJ, Mudle LJ,
Stebbingsa L, Galappaththigea D, van der
Gulden H, Schut E, Campbell PJ, Wessels LF,
Stratton MR*, Jonkers J*, Futreal PA*, Adams
DJ*. Somatic structural rearrangements in
genetically engineered mouse mammary
tumors. Genome Biol 2010;11:R100. (* joint
corresponding authors)
BRCA1-C61G missense variant. We have crossed these 3 mutant mouse strains into
our BRCA1 mammary tumor model to study the impact of these defi ned mutations
on tumor development, therapy response and resistance. All 3 mutants are clearly
pathogenic: homozygous Brca1 mutant mice are embryonic lethal and mammary
tumors develop faster in heterozygous Brca1 mutant mice which undergo K14cremediated
tissue-specifi c loss of the conditional p53 F and Brca1 F alleles. Treatment
of the resulting Brca1 185delAG/– ;p53 –/– , Brca1 5382insC/– ;p53 –/– and Brca1 C61G/– ;p53 –/–
mammary tumors revealed remarkable differences in sensitivity to cisplatin and the
PARP inhibitor olaparib. Whereas Brca1 –/– ;p53 –/– tumors never develop resistance
to cisplatin due to the large Brca1 deletion removing exons 5-13, the Brca1 C61G/–
;p53 –/– and Brca1 185delAG/– ;p53 –/– tumors readily become resistant to this drug while
retaining the Brca1 founder mutation, suggesting that BRCA1 RING activity is
required for tumor suppression but dispensable for therapy resistance.
Conditional mouse models for E-cadherin-deficient metastatic breast cancer
Loss of E-cadherin is associated with invasive lobular carcinoma (ILC), which
accounts for 10-15% of all breast cancers. To study the causal role of E-cadherin in
breast oncogenesis, we have generated a mouse model for invasive lobular carcinoma
(ILC) based on epithelium-specifi c inactivation of E-cadherin and p53. Compared to
p53 –/– mammary carcinomas, Ecad –/– ;p53 –/– tumors show a signifi cantly reduced
latency, a morphological switch from ductal to lobular carcinoma, and a phenotypic
change from non-invasive to highly invasive and metastatic tumors. We have used
the E-cadherin mammary tumor model for intervention studies with mTOR and
SRC inhibitors. Whereas mTOR inhibition leads to tumor stasis, SRC inhibition
does not affect primary tumor growth. We are currently testing the effects of mTOR
and SRC inhibition on tumor metastasis.
The inflammatory tumor-microenvironment and its impact on breast
cancer development and therapy Immune cells are one of the most abundant
cell types recruited to the microenvironment of many tumors. Their role during
tumorigenesis is, however, controversial, as both tumor-protective and tumorpromoting
properties have been reported. The overall research goal of Karin de
Visser is to address the role of the adaptive and innate immune system during
spontaneous breast cancer progression and metastasis formation. In addition,
the infl uence of the infl ammatory tumor-microenvironment on response and
resistance of tumors to chemotherapy is addressed. For these studies, the E-cadherin
mammary tumor model is employed. Like human breast cancers, mammary
carcinomas arising in this mouse model are characterized by abundant presence
of immune cells, including degranulating mast cells and macrophages, T- and
B-lymphocytes, antibody depositions and increased levels of pro-infl ammatory
mediators. By genetic elimination and pharmacological inhibition of specifi c subsets
of the adaptive and innate immune system, we are currently investigating their
functional signifi cance in a tumor-stage specifi c manner. We have identifi ed a
critical role for adaptive immune cells in spontaneous metastasis formation, and we
are currently investigating the underlying mechanisms. We are also studying the
ability of the immune system to modulate chemotherapy response and resistance.
These studies may shift therapeutic focus towards a combined cancer cell-intrinsic
and -extrinsic viewpoint.
Genomic analysis of mouse mammary tumors We have performed array-based
comparative genomic hybridization (aCGH) analysis of panels of mammary tumors
derived from our mouse models. We have developed two novel algorithms for multiexperiment
analysis of aCGH data: KC-SMART identifi es regions with signifi cantly
recurrent DNA copy number alterations (CNAs) and comparative KC-SMART
identifi es signifi cant differences in recurrent CNAs between sample groups. We
have used both algorithms in cross-species comparisons to identify conserved
BRCA1- or BRCA2-specifi c CNAs in mouse mammary tumors and human BRCAassociated
breast cancers. Furthermore, we have developed a novel approach to fi nd
co-occurring or mutual exclusive CNAs. Using these methods, we identifi ed several
mutual exclusive amplifi cations in mouse mammary tumors that might represent
redundant oncogenic pathways.

MOLECULAR DISSECTION OF CANCER BY DIFFERENTIAL
DRUG SENSITIVITY
In the clinic, we mainly use anticancer drugs based on outcomes of clinical trials
that have been carried out in the general breast cancer population, whereas little is
known about the molecular mechanisms underlying differential drug sensitivity.
The same holds true for other cancer types, including non-small cell lung cancer
(NSCLC), stomach cancer, ovarian cancer, and colorectal cancer. The focus of our
research line is to unravel these molecular mechanisms in order to develop tests
that may guide treatment decisions in the clinic and ultimately improve survival.
For this purpose we use several genome-wide approaches and molecular techniques,
in order to dissect the mechanisms that divide clinically well-defi ned cohorts of
breast, colorectal, stomach, ovarian and NSCLC patients into resistant and sensitive
to a particular drug. In addition, we have a close collaboration with the groups of
Jos Jonkers and Piet Borst, who use conditional mouse models for breast cancer,
and derived clonal cell lines, to study differential chemosensitivity in a controlled
fashion.
A second research line focuses on the impact of prognostic molecular classifi ers on
adjuvant systemic treatment advice in breast cancer.
The 70-gene prognosis signature and outcome after tamoxifen (Collaboration
with Division Experimental Therapy and Agendia BV)
Node-positive breast cancer patients can have an excellent outcome with tamoxifen
only. It is unclear whether analyzing both the 70-gene-signature and hormone
receptors provides superior prediction of outcome in tamoxifen-treated patients
than standard measures. We evaluated three series: 121 patients (81% node positive;
98% > 50 years; > 80% grade I/II) received adjuvant tamoxifen, 151 patients did
not receive tamoxifen (10% node positive; 44% > 50 years; 70% grade I/II) and
92 patients received tamoxifen for metastatic disease. The 70-gene signature was
analysed using MammaPrint TM . Oestrogen receptor (ER) and progesterone receptor
(PR) immunohistochemistry was evaluated following St. Gallen Consensus (Highly
Endocrine Responsive: ER and PR ≥ 50%, Incompletely Endocrine Responsive: ER
and/or PR low or either one absent). In patients treated with adjuvant tamoxifen,
both the 70-gene signature (adjusted for Endocrine Response Categories HR 2.17) as
well as the Endocrine Response Categories (adjusted for 70-gene signature HR 6.35)
were associated with breast-cancer-specifi c-survival. In metastatic disease, combined
analysis of the 70-gene signature and ER/PR revealed also additional value. In
patients who did not receive tamoxifen, only the 70-gene signature was associated
with outcome. We concluded that the 70-gene signature and ER and PR provide
independent information on outcome after tamoxifen for node positive ER-positive
breast cancer.
Value of 70-gene prognosis signature in T1 breast cancer In collaboration with
the Division of Diagnostic Oncology, Division of Surgical Oncology and Agendia BV,
using a pooled database of ~ 1700 breast cancer patients with data on the 70-gene
prognosis signature, we have demonstrated that the 70-gene prognosis signature
retained its prognostic value in patients with T1 tumors.
Development of a predictive test for tamoxifen resistance in breast cancer
In collaboration with the group of Rob Michalides (Division Cell Biology II) and the
group of Göran Landberg (Lund University, Lund, Sweden and Breakthrough Breast
Cancer Research Unit, Paterson Institute for Cancer Research, Manchester, UK), we
have shown that phosphorylation of ERa at serine 305 (ERaS305-P) causes tamoxifen
resistance. Earlier, p21-activated kinase 1 (PAK1) nuclear expression has also been
associated with tamoxifen resistance. We have collected primary breast cancer
material of ~ 1000 postmenopausal patients who participated in a randomized trial
of adjuvant tamoxifen versus no endocrine therapy started in the eighties, in order
to confi rm our earlier fi ndings in a third, independent series. For ~ 600 patients
we have also isolated tumor DNA to study the role of mutation, amplifi cation and
methylation of selected genes in tamoxifen resistance. Analyses are ongoing.
Based on our earlier fi ndings we are now preparing a prospective randomized study
Group leader Sabine Linn
67
molecular biology
Sabine Linn MD PhD Group leader
Karin Beelen MD PhD student
Rutger Koornstra MD PhD student
Philip Schouten MSc PhD student
Marieke Vollebergh MD PhD student
Mark Opdam Technical staff
Tesa Severson MSc Technical staff
Publications
Knauer M, Mook S, Rutgers EJ, Bender RA,
Hauptmann M, van de Vijver MJ, Koornstra
RH, Bueno-de-Mesquita JM, Linn SC, van
‘t Veer LJ. The predictive value of the 70-gene
signature for adjuvant chemotherapy in
early breast cancer. Breast Cancer Res Treat
2010;120:655-61
Mook S, Knauer M, Bueno-de-Mesquita JM,
Retel VP, Wesseling J, Linn SC, Van ‘t Veer
LJ, Rutgers EJ. Metastatic Potential of T1
Breast Cancer can be Predicted by the 70-gene
MammaPrint Signature. Ann Surg Oncol
2010;17:1406-13
Retèl VP, Joore MA, Knauer M, Linn
SC, Hauptmann M, Harten WH. Costeffectiveness
of the 70-gene signature versus
Sankt Gallen guidelines and Adjuvant
Online for early breast cancer. Eur J Cancer
2010;46:1382-91
Straver ME, Rutgers EJ, Rodenhuis S,
Linn SC, Loo CE, Wesseling J, Russell NS,
Oldenburg HS, Antonini N, Vrancken
Peeters MT. The Relevance of Breast Cancer
Subtypes in the Outcome of Neoadjuvant
Chemotherapy. Ann Surg Oncol 2010;17:2411-
2418
Vollebergh MA, Kappers I, Klomp HM,
Buning-Kager JC, Korse CM, Hauptmann
M, de Visser KE, van den Heuvel MM, and
Linn SC. Ligands of EGFR and the insulin-like
growth factor family as serum biomarkers for
response to EGFR-inhibitors in patients with
advanced NSCLC. J Thorac Onc 2010;5:1939-
1948
Kroep JR, Linn SC, Boven E, Bloemendal HJ,
Baas J, Mandjes IAM, van den Bosch J, Smit
WM, de Graaf H, Schröder CP, Vermeulen
GJ, Hop WCJ, Nortier JWR. Lapatinib:
clinical benefi t in patients with HER2

68
molecular biology
Publications (continued)
positive advanced breast cancer. Neth J Med
2010;68:371-6
Kok M, Linn SC. Gene expression profi les
of the oestrogen receptor in breast cancer.
Neth J Med 2010;68:291-302
Vollebergh MA, Lips EH, Nederlof PM,
Wessels LFA, Schmidt MK, van Beers EH,
Cornelissen S, Holtkamp M, Froklage FE,
de Vries EGE, Schrama JG, Wesseling J,
van de Vijver MJ, van Tinteren H, de Bruin M,
Hauptmann M, Rodenhuis S, Linn SC.
An aCGH classifi er derived from BRCA1mutated
breast cancer and benefi t of
high-dose, platinum-based, chemotherapy in
HER2-negative breast cancer patients.
Ann Oncol 2010
Knauer M, Cardoso F, Wesseling J, Bedard
PL, Linn SC, Rutgers EJ, van ‘t Veer LJ.
Identifi cation of a low-risk subgroup of HER-2positive
breast cancer by the 70-gene prognosis
signature. Br J Cancer, 2010
Kok M, Zwart W, Holm C, Fles R, Hauptmann
M, Van ‘t Veer LJ, Wessels LF, Neefjes J, Stål
O, Linn SC, Landberg G, Michalides R. PKAinduced
phosphorylation of ERalpha at serine
305 and high PAK1 levels is associated with
sensitivity to tamoxifen in ER-positive breast
cancer. Breast Cancer Res Treat 2011;125:1-12
Figure 4: Association of BRCA-likeCGH status with overall survival after HD-
CTC-chemotherapy and conventional
chemotherapy. Kaplan-Meier survival curves
for overall survival were generated separately
for patients with BRCA-likeCGH (A) and
with Non-BRCA-likeCGH (B) tumours, who
had been randomly assigned to HD-CTCchemotherapy
or conventional chemotherapy.
to test whether biomarker-guided adjuvant endocrine therapy improves recurrencefree
survival in premenopausal, hormone-receptor positive breast cancer patients
when compared to standard care.
Molecular mechanisms underlying sensitivity for high dose alkylating
agents The inability of breast cancer cells defi cient in homologous recombination
(HR), such as BRCA1/2-mutated cells, to repair DNA double strand breaks (DSBs)
appears to offer a target for DSB-inducing therapies, such as platinum agents,
intensifi ed alkylating therapy, and poly(ADP)ribose polymerase (PARP) inhibitors.
Our group previously employed array Comparative Genomic Hybridization (aCGH)
to assess the genomic profi les of BRCA1- and BRCA2-mutated breast cancers. We
hypothesized that this so-called BRCAness is present in sporadic breast cancers and
might predict sensitivity to DSB-inducing agents in hormone-receptor positive and
negative patients.
We tested the performance of these BRCA1-like and BRCA2-like classifi ers in the
context of a randomized clinical trial that compared 5 x fl uorouracil-epirubicincyclophosphamide
(FEC) with 4 x FEC followed by 1 x high-dose (HD) carboplatinthiotepa-cyclophosphamide
(CTC) with autologous stem cell rescue for high risk,
primary operable breast cancer [Rodenhuis et al., New Engl J Med, 2003]. Patients
with breast cancers with a BRCA1-like and/or BRCA2-like CGH pattern were labeled
BRCA-like CGH . We had chosen to only evaluate HER2-negative patients as in the
original study HER2-positive patients had appeared not to derive any benefi t from
HD-CTC. We evaluated whether the effect on recurrence-free and overall survival
(RFS, OS) of HD-CTC compared to conventional chemotherapy differed by BRCAlike
CGH status. Approximately one third of patients were scored as BRCA-like CGH
and these had a greater benefi t of HD-CTC compared to conventional chemotherapy
regarding OS (adjusted HR 0.19). In patients with Non-BRCA-like CGH tumors this
benefi t was not signifi cant (adjusted HR 0.90). The difference observed between
treatment arms was signifi cantly different (p-interaction: 0.004) (fi gure 4). Using
aCGH we were able to select hormone-receptor positive and triple negative breast
cancer patients who had selective benefi t of intensifi ed DSB-inducing chemotherapy.
(Collaboration with Petra Nederlof, Division of Diagnostic Oncology, and Sjoerd
Rodenhuis, Division of Medical Oncology).
In collaboration with the Jonkers and Borst groups we are now evaluating potential
biomarkers that may cause resistance to DSB-inducing agents in patients with
BRCA-like CGH tumors.
Translation of the BRCA-like CGH classifier to other cancer types HR defi ciency
is not limited to breast cancer. Although less obvious than breast and ovarian cancer
risk, other cancers also occur at increased rates in hereditary breast and ovarian
cancer families with germline BRCA1 and -2 mutations. These other cancers include
gastric (BRCA2-associated, RR:2.59), pancreatic (BRCA2-associated, RR:3.51), and
colon cancer (BRCA1-associated, RR:2.03) (Thompson and Easton; The Breast
Cancer Linkage Consortium). In collaboration with the NKI Familial Cancer Clinic
possible BRCA-associated tumors will be collected to investigate a causal effect
as well as generate tumor type specifi c array CGH profi les. These profi les will
subsequently be tested in defi ned cohorts of patients who had been treated with
either DSB-inducing therapy or other therapy and outcome correlated with BRCAlike
CGH status.
Identification of druggable targets in lobular breast cancer and triple
negative breast cancer As participant of the FP7 RATHER consortium, together
with the Bernards group (Division of Molecular Carcinogenesis), we have initiated
a project to assess the presence of druggable targets in 150 lobular and 150 triple
negative breast cancers. We are using high-throughput next generation sequencing,
together with exon capture technology, to sequence genomic regions of interest
in breast cancer in all 300 tumor samples. The fi nal goal is to deliver diagnostic
tests along with the right targeted therapy – chemotherapy combination to improve
treatment options and outcome for these diffi cult to treat breast cancer subtypes.

GENES AND PROTEINS INVOLVED IN ANTICANCER DRUG
RESISTANCE AND PHARMACOKINETICS
Our research focuses on genes and proteins that cause drug resistance or drug
susceptibility in tumors, or infl uence the pharmacological and toxicological behavior
of anticancer and many other drugs and toxins, including carcinogens. Insight into
these systems may: i) improve chemotherapy and more generally pharmacotherapy
approaches for cancer and other diseases; ii) increase insights into factors
determining susceptibility to carcinogens, and; iii) allow elucidation of physiological
functions. To study the physiological, pharmacological and toxicological roles of
the proteins involved, and their interactions, we generate and analyze knockout or
transgenic mice lacking or overexpressing the relevant genes.
Impact of drug transporters We have a long-standing interest in plasma
membrane proteins of the ATP binding cassette (ABC) multidrug transporter
family, including P-glycoprotein (P-gp, ABCB1/MDR1), MRP2 (ABCC2) and BCRP
(ABCG2) (fi gure 6). These proteins actively export a wide range of anticancer, anti-
HIV/AIDS, and many other drugs from cells. This ATP-dependent drug extrusion
can cause multidrug resistance (MDR) in tumor cells. P-gp, MRP2 and BCRP all
localize to the apical membrane of polarized epithelial cells, resulting in apically
directed export of drug substrates, and there is considerable overlap in substrate
specifi city between these transporters. Previous experiments in P-gp and Bcrp1
knockout mice indicated that these transporters can protect an organism against
exogenous toxins and drugs by limiting penetration of substrates into brain, testis,
and fetus, by restricting uptake of orally administered substrates, and by mediating
excretion of substrates via liver and intestine. To extend these analyses we have
generated Mrp2 knockout mice, and crossed these with existing P-gp, Bcrp1 and
Mrp3 knockout mice in order to elucidate the separate and combined contributions
of these transporters to pharmacological, toxicological and physiological functions.
Mrp3 is expressed in the basolateral membrane of hepatocytes and enterocytes, so
transporting in the opposite direction of P-gp, Bcrp1 and Mrp2.
P-gp, Bcrp1, Mrp2 and Mrp3 (compound) knockout mice We generated and
characterized Bcrp1/Mrp2/Mrp3 combination knockout mice, which were viable
and fertile, and had a normal life span. We then used these mice, in conjunction
with existing P-gp/Mrp2, Mrp2/Mrp3, and single knockout mice to investigate the
roles of P-gp, Bcrp1, Mrp2 and Mrp3 in the elimination and toxicity of a number
of different drugs and drug conjugates. We found that Bcrp1 and Mrp2 defi ciency
increases the plasma levels of acyl glucuronide conjugates of the anti-infl ammatory
drug diclofenac, and that Mrp3 defi ciency decreases these levels, presumably
because of their opposite roles in excreting the conjugates from the liver towards
bile and blood, respectively. Bcrp1/Mrp2/Mrp3 defi ciency resulted in mildly
increased liver toxicity of diclofenac, presumably resulting from the increased liver
accumulation of diclofenac conjugates. Rather similar results were found for the
anticancer drug etoposide, which displayed decreased biliary excretion of etoposide
in Mrp2 knockout mice, resulting in increased formation of etoposide glucuronide
in the liver, which was secreted into blood by Mrp3. In Mrp2/Mrp3 combination
knockout mice this resulted in highly increased liver levels of etoposide glucuronide.
P-gp restricted the oral availability of etoposide, and mediated its direct intestinal,
but not its hepatobiliary excretion. The anticancer drug methotrexate showed
dramatically decreased elimination in Bcrp1/Mrp2/Mrp3 knockout mice, and
highly increased liver levels of this drug and its primary nephrotoxic metabolite
7OH-methotrexate. These effects were far more pronounced than observed in the
single or double knockout mice. Collectively, these studies indicate that there can
be substantial overlap and redundancy in the detoxifying (protective) functions of
these drug transporters for a number of anticancer and other drugs. This probably
explains why it is often still comparatively safe to administer these potentially
highly toxic drugs to patients, as chances are low that all of these systems would be
simultaneously compromised by genetic polymorphisms or drug-drug interactions,
thus resulting in toxic overdosing.
Group leader Alfred Schinkel
69
molecular biology
Alfred Schinkel PhD Group leader
Birk Poller PhD Post-doc
Dilek Iusuf MSc PhD student
Evita Van de Steeg MSc PhD student
Selvi Durmus MSc PhD student
Anita Van Esch Technical staff
Els Wagenaar Technical staff
Publications
Van Waterschoot RA, Lagas JS, Wagenaar
E, van der Kruijssen CM, van Herwaarden
AE, Song JY, Rooswinkel RW, van Tellingen
O, Rosing H, Beijnen JH, Schinkel A.H.
Absence of both cytochrome P450 3A and
P-glycoprotein dramatically increases
docetaxel oral bioavailability and risk of
intestinal toxicity. Cancer Res.
2009;69:8996-9002
Van Waterschoot RA, Eman RM, Wagenaar
E, van der Kruijssen CM, Rosing H, Beijnen
JH, Schinkel AH. ABCC2, ABCC3, and
ABCB1, but not CYP3A, protect against
trabectedin-mediated hepatotoxicity. Clin
Cancer Res. 2009;15:7616-23
Vlaming ML, van Esch A, Pala Z, Wagenaar
E, van de Wetering K, van Tellingen O,
Schinkel AH. Abcc2 (Mrp2), Abcc3 (Mrp3),
and Abcg2 (Bcrp1) are the main determinants
for rapid elimination of methotrexate and its
toxic metabolite 7-hydroxymethotrexate in
vivo. Mol Cancer Ther. 2009;8:3350-9
Lagas JS, Fan L, Wagenaar E, Vlaming ML,
van Tellingen O, Beijnen JH, Schinkel AH.
P-glycoprotein (P-gp/Abcb1), Abcc2, and
Abcc3 determine the pharmacokinetics of
etoposide. Clin Cancer Res. 2010;16:130-40
Lagas JS, Sparidans RW, Wagenaar E,
Beijnen JH, Schinkel AH. Hepatic clearance
of reactive glucuronide metabolites of diclofenac
in the mouse is dependent on multiple
ATP-binding cassette effl ux transporters.
Mol Pharmacol. 2010;77:687-94
Lagas JS, van Waterschoot RAB, Sparidans
RW, Wagenaar E, Beijnen JH, Schinkel AH.
Breast cancer resistance protein and P-glycoprotein
limit sorafenib brain accumulation.
Mol Cancer Ther. 2010;9:319-26

70
molecular biology
Publications (continued)
Ciarimboli G, Deuster D, Knief A, Sperling
M, Holtkamp M, Edemir B, Pavenstädt H,
Lanvers-Kaminsky C, am Zehnhoff-
Dinnesen A, Schinkel AH, Koepsell H,
Jürgens H, Schlatter E. Organic cation
transporter 2 mediates cisplatin-induced
oto- and nephrotoxicity and is a target for
protective interventions. Am J Pathol.
2010;176:1169-80
Van Waterschoot RA, Lagas JS, Wagenaar
E, Rosing H, Beijnen JH, Schinkel AH.
Individual and combined roles of CYP3A,
P-glycoprotein (MDR1/ABCB1) and MRP2
(ABCC2) in the pharmacokinetics of
docetaxel. Int J Cancer. 2010;127:2959-64
Van Waterschoot RA, ter Heine R,
Wagenaar E, van der Kruijssen CM,
Rooswinkel RW, Huitema AD, Beijnen JH,
Schinkel AH. Impact of CYP3A and the drug
transporters P-glycoprotein (ABCB1) and
MRP2 (ABCC2) on the pharmacokinetics of
lopinavir. Br J Pharmacol. 2010;160:1224-33
Van de Steeg E, Wagenaar E, van der
Kruijssen CM, Burggraaf JE, de Waart DR,
Oude Elferink RP, Kenworthy KE, Schinkel
AH. Organic anion transporting polypeptide
1a/1b-knockout mice provide insights into
hepatic handling of bilirubin, bile acids and
drugs. J Clin Invest. 2010;120:2942-52
Teunissen SF, Vlaming ML, Rosing H,
Schellens JH, Schinkel AH, Beijnen JH.
Development and validation of a liquid
chromatography-tandem mass spectrometry
assay for the analysis of 2-amino-1-methyl-6phenylimidazo[4,5-b]pyridine
(PhIP) and its
metabolite 2-hydroxyamino-1-methyl-6phenylimidazo[4,5-b]pyridine
(N-OH-PhIP)
in plasma, urine, bile, intestinal contents,
faeces and eight selected tissues from mice.
J Chromatogr B Analyt Technol Biomed Life
Sci. 2010;878:2353-62
Sparidans RW, Iusuf D, Schinkel AH,
Schellens JH, Beijnen JH. Liquid
chromatography-tandem mass spectrometric
assay for pravastatin and two isomeric
metabolites in mouse plasma and tissue
homogenates. J Chromatogr B Analyt
Technol Biomed Life Sci. 2010;878:2751-9
Van de Steeg, E. Physiological and
pharmacological functions of OATP1A/1B
transporters. PhD thesis, University of
Utrecht, April 2010.
Figure 5: Putative structure of
a prototypic ABC drug transporter.
Insights from Cyp3a, Cyp3a/P-gp and Cyp3a/P-gp/Mrp2 combination knockout
mice Cytochrome P450 3A (CYP3A) enzymes metabolize >50% of prescribed drugs,
and represent one of the most important detoxifying systems. As CYP3A activity
shows high inter- and intra-patient variability, it can have a profound infl uence
on variable drug behavior (pharmacodynamics) and drug toxicity. Moreover, its
substrates overlap extensively with those of the drug transporters P-gp, BCRP
and MRP2. To investigate the physiological and pharmacological roles of CYP3A
in combination with P-gp, we previously generated Cyp3a knockout mice, and
showed a pronounced effect of Cyp3a defi ciency on the oral bioavailability, i.v.
clearance and toxicity of the anticancer drug and CYP3A substrate docetaxel.
In combination Cyp3a/P-gp knockout mice the oral availability of docetaxel was
disproportionately increased, suggesting a partly overlapping and compensatory
activity of both detoxifying systems in docetaxel handling. We now extended these
studies to lopinavir, an anti-HIV/AIDS drug known to be a substrate of both CYP3A
and P-gp, and possibly MRP2. We found that lopinavir was transported by MDR1
but not by MRP2 in vitro. Accordingly, the lopinavir AUC oral was signifi cantly
increased in P-gp knockout mice (~9-fold vs. wild-type) but not in Mrp2 knockout
mice. A more than 2000-fold increase in lopinavir AUC oral was observed in Cyp3a
knockout mice compared to wild-type mice. Interestingly, no signifi cant difference
in AUC oral between Cyp3a knockout and Cyp3a/P-gp/Mrp2 knockout mice was
observed. CYP3A4 activity in the intestine or liver could each already reduce
the lopinavir AUC oral by more than 100-fold compared to the Cyp3a knockout
situation. The results demonstrate that CYP3A is the major determinant of lopinavir
pharmacokinetics, far more than P-gp. Both intestinal and hepatic CYP3A activity
contribute profoundly to the low oral bioavailability of lopinavir. The impact of P-gp
on oral lopinavir kinetics was only detectable in the presence of CYP3A, suggesting
saturation of P-gp in the absence of CYP3A activity.
Insights from Oatp1a/1b (Slco1a/1b) knockout mice Organic anion transporting
polypeptides (OATP, gene name: SLCO) belong to the superfamily of the solute
carrier class of organic anion transporters. OATPs facilitate sodium-independent
uptake transport of a wide variety of organic endo- and exogenous compounds, such
as bile salts, steroid and thyroid hormones and their conjugates, and numerous
drugs and toxins. Members of the OATP1A/1B family have a broad substrate
specifi city and are highly expressed in the sinusoidal membrane of hepatocytes, or
in the apical membrane of enterocytes where they might affect liver or intestinal
uptake, respectively, of drugs, xenobiotics, and endogenous substances. They might
thus play an important role in drug disposition. To investigate the physiological
and pharmacological roles of Oatp1a/1b transporters in vivo, we generated mice
lacking all (5-7) Slco1a/1b genes. Slco1a/1b knockout mice were viable and fertile but,
surprisingly, suffered from marked conjugated hyperbilirubinemia, due to impaired
hepatic (re-)uptake of bilirubin glucuronide, and increased plasma unconjugated bile
salt levels. The resulting hypothesis that sinusoidal secretion, presumably by MRP3
and related transporters, and subsequent re-uptake of glucuronidated compounds
into hepatocytes by OATPs occurs under physiological conditions might alter
our perspective on normal liver functioning. It may prevent saturation of biliary
excretion and other hepatocyte detoxifi cation processes by spreading these processes
from the most heavily exposed periportal hepatocytes over the entire liver lobule.
Slco1a/1b knockout mice further showed drastically decreased hepatic uptake and
consequently increased systemic exposure
of the drugs methotrexate and fexofenadine
upon i.v. and oral administration. Slco1a/1b
knockout mice did not display reduced
intestinal absorption of oral methotrexate or
fexofenadine. Rifampicin was an effective
and specifi c Oatp1a/1b inhibitor in controlling
methotrexate pharmacokinetics. Our results
indicate that Oatp1a/1b transporters play
an essential role in hepatic (re-)uptake of
conjugated bilirubin, unconjugated bile salts
and drugs.

BIOINFORMATICS AND STATISTICS
The Bioinformatics and Statistics group provides leadership on the collection and
analysis of data for the research programs of the institute, by conducting research
in bioinformatics and statistics and by performing state of the art analyses of a wide
array of data types. Research topics include stratifying tumors into groups with
distinct and homogeneous outcome and therapy response; the characterization
of genes and pathways involved in tumorigenesis and understanding molecular
regulatory mechanisms. A number of exemplary projects are presented below in
more detail.
Systems Biology In collaboration with the Beijersbergen, Bernards and Jonkers
groups we have established the Cancer Systems Biology Center. The aim of this
center is to develop a strategy to tackle the complexity of molecular networks that
govern breast tumorigenesis. This strategy is rooted in a modeling and experimental
validation cycle spanning multiple levels of complexity including cell lines, mouse
models and patients. As a start we are focusing on breast cancers for which no
effective targeted therapies exist: ‘triple negatives’ and invasive lobular carcinomas.
Since there are strong indications for the involvement of PI3K and MAPK signaling
pathways in these subtypes, we are generating in silico models of therapy response by
employing 1) normal and tumor cell lines as in vitro model systems; 2) quantifi cation
of functional activation of pathway components and associated cellular phenotypes;
3) computational modeling to create quantitative models of pathway behavior and
resistance mechanisms; and 4) mouse tumor models as in vivo model systems. The
identifi ed models will be directly validated in proof-of-concept pre-operative trials.
We hope that this strategy will yield improved diagnostic tools and tumor-specifi c
treatments resulting in more tailored cancer therapy.
Oncogene discovery by direct association of insertion features with gene
expression Insertional mutagenesis is a potent forward genetic screening technique
to identify novel putative cancer genes. An important – yet unresolved – issue is
to determine which genes are affected by the viral insertions retrieved from these
screens. To address this, we developed RBM (rule-based mapping), an approach that
automatically determines the most appropriate target genes for a set of integration
sites based on a collection of literature derived rules. RBM exploits properties of
individual insertions and genes, such as orientation and distance to the gene, and
allows insertion mapping without prior knowledge of gene expression.
For both MuLV and SB, RBM produced superior association of insertions with gene
expression compared to selecting the nearest gene as target. We also developed
KC-RBM (kernel convolved RMB), a mapping approach that combines the power of
the rules captured in RBM with data-driven common insertion site calling based on
Gaussian kernel convolution. We demonstrate the superiority of RBM and KC-RM
over existing approaches in recovering true positives from a list of independently,
manually curated targets.
Extracting oncogenic pathways from insertional mutagenesis screens
We have developed combinatorial association logic networks (CALs): an approach to
extract simple Boolean logic circuits which employ combinations of insertion loci to
predict the expression pattern of downstream targets. In classical one-dimensional
analyses, direct interactions between the insertion patterns and transcription levels
across tumors are detected. However, when the insertion loci themselves interact,
direct associations between the individual loci and transcript levels may become
undetectable. Therefore, our method detects associations between transcript levels
and the outputs of small Boolean logic networks that combine multiple genetic loci.
The detection of logic networks requires solving a demanding optimization problem.
By reformulating the objective function and applying a customized branch and
bound algorithm, we obtain runtimes of up to four orders of magnitude faster than
exhaustive search. We demonstrated our method on an insertional mutagenesis
dataset, combining insertion data with transcriptional information from the same
sample, fi nding known and novel associations between genes involved in Notch
signaling.
71
molecular biology
Group leader Lodewyk Wessels
Lodewyk Wessels PhD Group leader
Michael Hauptmann PhD Academic staff
Marta Lopez-Yurda PhD Academic staff
Wilma Heemsbergen PhD Academic staff
Nicos Angelopoulos PhD Post-doc
Sander Canisius PhD Post-doc
Andreas Schlicker PhD Post-doc
Hayssam Soueidan PhD Post-doc
Jordi Vidal Rodriguez PhD Post-doc
Ewald Van Dyk MSc PhD student
Johann De Jong MSc PhD student
Christiaan Klijn MSc PhD student
Wouter Meuleman MSc PhD student
Jeroen De Ridder MSc PhD student
Jorma De Ronde MSc PhD student
Christine Staiger MSc PhD student
Bram Gerritsen Bioinformatician
Jelle Ten Hoeve Bioinformatician
Publications
De Ridder J, Gerrits A, Bot J, de Haan
G, Reinders M, Wessels L. Inferring
combinatorial association logic networks
in multimodal genome-wide screens.
Bioinformatics. 2010;26:i149-57
Peric-Hupkes D, Meuleman W, Pagie
L, Bruggeman SW, Solovei I, Brugman
W, Gräf S, Flicek P, Kerkhoven RM, van
Lohuizen M, Reinders M, Wessels L,
van Steensel B. Molecular maps of the
reorganization of genome-nuclear lamina
interactions during differentiation. Mol Cell.
2010;38:603-13
Klijn C, Bot J, Adams DJ, Reinders M,
Wessels L, Jonkers J. Identifi cation of
networks of co-occurring, tumor-related
DNA copy number changes using a genomewide
scoring approach. PLoS Comput Biol.
2010;6:e1000631
De Ronde JJ, Hannemann J, Halfwerk H,
Mulder L, Straver ME, Vrancken Peeters
MJ, Wesseling J, van de Vijver M, Wessels
LF, Rodenhuis S. Concordance of clinical
and molecular breast cancer subtyping in
the context of preoperative chemotherapy
response. Breast Cancer Res Treat.
2010;119:119-26

72
molecular biology
Publications (continued)
De Ronde JJ, Klijn C, Velds A, Holstege
H, Reinders MJ, Jonkers J, Wessels LF.
KC-SMARTR: An R package for detection
of statistically signifi cant aberrations in
multi-experiment aCGH data. BMC Res
Notes. 2010;3:298
Horlings HM, Lai C, Nuyten DS,
Halfwerk H, Kristel P, van Beers E,
Joosse SA, Klijn C, Nederlof PM,
Reinders MJ, Wessels LF, van de Vijver
MJ. Integration of DNA copy number
alterations and prognostic gene expression
signatures in breast cancer patients. Clin
Cancer Res. 2010;16:651-63
Kool J, Uren AG, Martins CP, Sie D,
de Ridder J, Turner G, van Uitert M,
Matentzoglu K, Lagcher W, Krimpenfort
P, Gadiot J, Pritchard C, Lenz J, Lund AH,
Jonkers J, Rogers J, Adams DJ, Wessels
L, Berns A, van Lohuizen M. Insertional
mutagenesis in mice defi cient for p15Ink4b,
p16Ink4a, p21Cip1, and p27Kip1 reveals
cancer gene interactions and correlations
with tumor phenotypes. Cancer Res.
2010;70:520-31
Heemsbergen WD, Al-Mamgani A, Witte
MG, Van Herk M, Pos FJ, and Lebesque
JV. Urinary obstruction in prostate cancer
patients from the Dutch trial (68 Gy vs.
78 Gy): relationships with local dose, acute
effects and baseline characteristics. Int J
Radiat Oncol Biol Phys 2010;78:19-25
He Y, Van ‘t Veer L, Lopez-Yurda MI, Van
de Velde C, Marijnen C. Do rectal cancer
patients with PIK3CA mutations benefi t
from preoperative radiotherapy with regard
to local recurrences? Clin Cancer Res,
2010;16: 6179
Rasch CR, Hauptmann M, Schornagel J,
Wijers O, Buter J, Gregor T, Wiggenraad
R, De Boer JP, Ackerstaff AH, Kroger R,
Hoebers FJ, and Balm AJ. Intra-arterial
versus intravenous chemoradiation for
advanced head and neck cancer: results
of a randomized phase 3 trial. Cancer
2010;116:2159-2165
Retel VP, Joore MA, Knauer M, Linn SC,
Hauptmann M, and Van Harten WH.
Cost-effectiveness of the 70-gene signature
versus Sankt Gallen guidelines and
Adjuvant Online for early breast cancer.
Eur J Cancer 2010;46:1382-1391
Identification of networks of co-occurring oncogenic gains and losses
Collaborating oncogenic events can also be induced by copy number alterations.
To detect such events in aCGH data, we developed a scoring framework to separate
truly co-occurring aberrations from passenger mutations and dominant single
signals present in the data. Analysis of high-resolution DNA copy number data
from a panel of 95 hematological tumor cell lines correctly identifi ed co-occurring
recombinations at the T-cell receptor and immunoglobulin loci in T- and B-cell
malignancies, respectively. In addition, our analysis revealed networks of cooccurring
genomic losses and gains that are highly enriched for functional
relationships. The co-occurring losses we fi nd are independent of the canonical
cancer genes within the network. Our fi ndings suggest that large-scale, low-intensity
copy number changes may be an important feature of cancer development or
maintenance by affecting the gene dosages of a large interconnected network of
functionally related genes. We have adapted this approach to be applicable to aCGH
profi les derived from human tumors. This led to the discovery of co-occurrences
uniquely associated with the triple negative subtype in breast cancer.
Evaluation of biomarkers predicting treatment response Biomarkers
predicting treatment response are useful for tailoring treatment. Before evaluation
in prospective randomized trials, the fi rst clinical evaluation often takes place in
relatively small retrospective patient series or older trials. We investigated whether
a classifi er based on comparative genomic hybridization and initially developed to
identify mutations in the BRCA1 gene of breast cancer patients might also detect
loss-of-function of this gene due to causes other than mutation and could therefore
predict sensitivity to double strand break inducing agents. The hypothesis was
evaluated in 230 stage III breast cancer patients from an available clinical trial who
had been randomized between adjuvant high-dose platinum-based and conventional
anthracycline-based chemotherapy. We observed greater benefi t from high-dose
versus conventional chemotherapy among patients identifi ed by the classifi er based
on a signifi cant interaction between classifi er and treatment. In a study of advanced
non-small cell lung cancer, we demonstrated that epidermal growth factor (EGFR)
ligands may predict response to EGFR-inhibitor treatment based on a clinical sample
of 60 patients treated with EGFR-inhibitors and 60 matched control patients. Low
concentrations of transforming growth factor-alpha and high concentrations of
amphiregulin were associated with a better disease-specifi c survival among patients
treated with EGFR-inhibitors versus control patients.
Long-term effects of therapeutic and diagnostic radiation exposure After the
introduction of modern radiotherapy and chemotherapy, many cancers have become
curable malignancies, e.g., Hodgkin lymphoma or testicular cancer. However, the
life expectancy of survivors is compromised by the occurrence of late complications
of treatment such as second malignancies and cardiovascular disease. In a cohort
of about 500 long-term childhood cancer survivors treated with different potentially
cardiotoxic therapies we showed that a high percentage of patients (27%) have
an abnormal left ventricular shortening fraction, and that anthracycline dose,
cardiac irradiation, and younger age at diagnosis were associated with subclinical
cardiac dysfunction. In another project, we are evaluating the risk of cancers
of the stomach and pancreas as second malignancies related to radiation (and
chemo-) therapy received by survivors of testicular cancer. This will be performed
in a multinational cohort with detailed assessment of radiation doses and the
corresponding uncertainties. Although radiation doses from diagnostic procedures
are usually much lower than therapeutic doses, some diagnostic imaging procedures
may deliver doses high enough to present a non-negligible risk for sensitive
individuals. Within a multi-national pilot study, we determined that it is feasible in
the Netherlands to set up a nationwide cohort of approximately 100,000 children
who have been exposed to ionizing radiation from diagnostic computed tomography
(CT) scans, to estimate the radiation doses to different organs, and to evaluate their
subsequent risk of childhood cancer, particularly leukemia. Funding has been
obtained to conduct such studies in eight European countries on about one million
children, including the Netherlands.

DIVISION OF MOLECULAR
CARCINOGENESIS
FUNCTIONAL GENOMICS
My group uses functional genomics technologies to identify mechanisms of
resistance to cancer drugs and to fi nd novel cancer-relevant genes. We use various
types of genetic screens to achieve these goals.
Identification of mechanisms of drug resistance Unresponsiveness to therapy is
a recurring problem in the treatment of cancer. It is therefore important to identify
the molecular pathways that contribute to unresponsiveness to cancer therapeutics.
We use loss-of-function genetic screens with large sets of shRNA vectors to identify
genes that contribute to drug resistance, in particular to the new classes of targeted
therapeutics. In the past year, we have focused on the identifi cation of genes whose
suppression contributes to resistance to receptor tyrosine kinase (RTK) inhibitory
drugs and their downstream signaling pathways. To do this, we used the H3122 nonsmall
cell lung cancer cell line, which harbors the EML4-ALK translocation. This
translocation renders these cells highly sensitive to the small molecule ALK kinase
inhibitors TAE684 and PF02341066 (also known as Crizotinib). In a genomewide
shRNA screen, we identifi ed both components of the SWI/SNF chromatin
remodeling complex as well as components of the MEDIATOR complex as genes
whose inhibition confers resistance to ALK inhibition. Interestingly, knockdown of
these genes also conferred resistance to crizotinib in MET-amplifi ed lung cancer
cell lines and to the EGFR inhibitory drug gefi nitib in lung cancers having an
activated version of the EGFR. These data suggest that the MEDIATOR and SWI/
SNF complexes play critical roles downstream of RTK signaling. Consistent with
this, knockdown of either of these genes caused a signifi cant increase in phospho-
ERK and phospho-AKT, even in the presence of RTK-inhibitory drugs. We also
found that knockdown of the MEDIATOR or SWI/SNF complex components caused
a marked increase in phospho-EGFR, even in the presence of RTK inhibitory drugs
like gefi tinib. We are currently investigating how knockdown of key components
of the MEDIATOR and SWI/SNF complexes can cause activation of the ERK and
AKT signaling pathways and affect EGFR activity. Importantly, high throughput
sequencing of these MEDIATOR and SWI/SNF components suggests that mutations
in these genes may occur in lung cancer. Whether these mutations are associated
with resistance to RTK inhibitory drugs remains to be investigated.
Functional studies on de-ubiquitinating enzymes The Androgen receptor
(AR) belongs to the nuclear receptor super-family and is essential for male sexual
development and maturation, as well as prostate cancer development. Regulation of
AR signalling activity depends on several post-translational modifi cations, one of
these being ubiquitination. We screened a short hairpin library targeting members
of the de-ubiquitination enzyme (DUB) family and identifi ed the X-linked DUB
USP26 as a novel regulator of AR signalling. USP26 is a nuclear protein that binds
to AR via three important nuclear receptor interaction motifs, and modulates
AR ubiquitination, consequently infl uencing AR activity and stability. Our data
suggest that USP26 assembles with AR and other co-factors in sub-nuclear foci, and
serves to counter-act hormone-induced AR ubiquitination, thereby contributing to
regulation of AR transcriptional activity.
Histone ubiquitination has emerged as an important epigenetic mark in regulating
gene expression and thereby plays an essential role in the development of higher
organisms. Moreover, aberrant histone ubiquitination is frequently seen in cancer.
However, most of these studies have been carried out in yeast and have focused on
mRNA coding genes. The Drosophila ubiquitin protease USP36 is a H2B specifi c
DUB. Using a human USP36 specifi c antibody we found that USP36 is strictly
nucleolar in localization. We then looked at the pre-rRNA levels by quantitative RT
PCR using primers specifi c for 5’ETS (rRNA External Transcribed Spacer) upon
USP36 knock down. A dramatic reduction in the pre-rRNA levels in the absence of
73
molecular carcinogenesis
Division head, group leader René Bernards
René Bernards PhD Group leader
Katrien Berns PhD Academic staff
Annette Dirac PhD Senior Post-doc
Michiel van der Heijden MD PhD Senior
Post-doc
Michael Hölzel MD PhD Senior Post-doc
Sidong Huang PhD Senior Post-doc
Prasanth Kumar PhD Post-doc
Ian Majewski PhD Post-doc
Rianne Oosterkamp MD Clinical fellow
Ernst-Jan Geutjes MSc PhD student
Floris Groenendijk MD PhD student
Guus Heynen MSc PhD student
Jasper Mullenders Msc PhD student
Anirudh Prahallad MSc PhD student
Chong Sun MSc PhD student
Astrid Bosma Technical staff
Annemiek Gennissen Technical staff
Marielle Hijmans MSc Technical staff
Wipawadee Grernrum Technical staff
Publications
Ashworth A, Bernards R. Using functional
genetics to understand breast cancer biology.
Cold Spring Harb Perspect Biol. (Polyak,
Rosen and Bissell, eds). 2010 (in press)
Bernards R, Filipowicz W, Livingston DM,
Mihich E. Twenty-second Annual Pezcoller
Symposium: RNA Biology and Cancer.
Cancer Res. 2010 (in press)
Bernards R. It’s diagnostics, stupid.
Cell 2010;141:13-7
Dirac AMG, Bernards R. The de-ubiquitinating
enzyme USP26 is a regulator
of androgen receptor signaling. Mol. Can.
Res. 2010;8:844-54
Epping MT, Meijer LAT, Krijgsman O,
Bos JL, Pandolfi PP, Bernards R. TSPYL5
suppresses p53 levels and function by physical
interaction with USP7. Nature Cell Biol.
2010 (in press)

74 molecular carcinogenesis
Publications (continued)
Hölzel M, Huang S, Koster J, Øra I,
Lakeman A, Caron H, Nijkamp W, Xie
J, Callens T, Asgharzadeh S, Seeger RC,
Messiaen L, Versteeg R, Bernards R. NF1
is a tumor suppressor in neuroblastoma
that determines retinoic acid response and
disease outcome. Cell 2010;142:218-29
Mullenders J, Fabius AWM, van Dongen
MMW, Kuiken HJ, Beijersbergen RL,
Bernards R. Interleukin-1R–Associated
Kinase2 Is a Novel Modulator of the
Transforming Growth Factor Signaling
Cascade. Mol. Can. Res. 2010;8:592-603
Van der Heijden MS, Bernards R.
Inhibition of the PI3K pathway:
hope we can believe in? Clinical Cancer
Res. 2010;16:3094-9
endogenous USP36 was observed, indicating that USP36 has a role in the regulation
of mammalian ribosomal RNA (rRNA) gene transcription. We are currently asking
how USP36 control rRNA gene transcription.
Figure 1: TSPYL5 regulates p53 turnover.
The degradation of the p53 tumor
suppressor protein is regulated by both
ubiquitination by the ubiquitin ligase
MDM2 and deubiquitination by the
DUB USP7. When TSPYL5 is overexpressed
in breast cancer as a result
of gene amplifi cation, TSPYL5 binds
USP7, thereby preventing it from
interacting with p53. Consequently, the
balance between ubiquitination and
deubiquitination is shifted towards
increased polyubiquitination of p53,
causing an increase in p53 destruction
by the proteasome. As a result, TSPYL5
over-expressing breast cancer cells are
functionally defi cient in p53.
TSPYL5 is a gene of unknown function located at a region on chromosome 8q22,
which is frequently amplifi ed in breast cancer. Using mass spectrometry, we
found that TSPYL5 physically interacts with ubiquitin-specifi c protease 7 (USP7)/
herpesvirus-associated ubiquitin-specifi c protease (HAUSP). USP7 is the DUB
for the p53 tumor suppressor and we fi nd that TSPYL5 opposes the activity of
USP7 towards p53, resulting in increased p53 ubiquitination (fi gure 1). We found
that TSPYL5 reduces p53 protein levels and inhibits activation of p53 target genes.
Moreover, expression of TSPYL5 overrides p53-dependent proliferation arrest and
oncogene-induced senescence in multiple assays. These data identify TSPYL5 as a
novel breast cancer oncogene that critically modulates the p53-USP7 network.
High throughput kinome sequencing Cancer cells often contain multiple genomic
alterations that together are responsible for the deregulated growth. Cancer cells
often depend on the continued presence of these genomic alterations and sudden
inhibition of the signals that emanate from these genomic alterations frequently
results in death of the cancer cells, a phenomenon coined “oncogene addiction”.
Conversely, the presence of downstream activating mutations (e.g. in KRAS) confers
potent resistance to EGFR inhibitory drugs in colon cancer. The presence of specifi c
changes in the genomes of cancer cells can therefore have strong predictive value for
responsiveness to therapies that target these mutations. Since most of the mutations
identifi ed to date that predict responses to targeted therapies are in kinase genes
or their downstream signaling pathways, we set out to develop a dedicated highthroughput
sequencing platform to identify kinase mutations in large numbers
of tumors. In collaboration with Agilent Technologies (Santa Clara, CA USA),
we developed and validated a “kinome capture” platform that allows us to rapidly
sequence 586 kinase and kinase-related genes through the enrichment of their
coding sequences by hybridization selection. We are currently using this platform to
fi nd novel mutations in subgroups of breast cancer that are currently diffi cult to treat
with conventional therapies. We aim to identify novel kinase targets for therapy that
can be exploited to improve the therapy of these types of breast cancer.

THE RNAi STRATEGY IN TARGET DISCOVERY
The research in my laboratory continues to evolve around the identifi cation of novel
drug targets in cancer, using large-scale cell-based screening technologies. We apply
genome wide siRNA collections as well as large shRNA collections with the goal of
identifying essential components in disease-related pathways that can be explored
as drug targets in cancer therapy. Furthermore we use these RNAi technologies
to search for synthetic lethal interactions with specifi c tumor-associated genetic
alterations. We have begun to use RNAi technologies in combination with
phosphoproteome profi ling and cell-based assays to generate predictive models for
therapy response to pathway targeted therapeutics in breast cancer.
Synthetic lethal interactions For the effective treatment of cancer, there is a
great need for drugs that specifi cally target tumor cells without affecting normal
cells. With the use of RNA interference, we explore synthetic lethal phenotypes
in mammalian cells. Synthetic lethal phenotypes are defi ned as a combination of
two mutations, which by themselves are non-lethal, but together result in a lethal
phenotype. These interactions can lead to the identifi cation of novel cancer drug
targets that are only cytotoxic in the context of a tumor specifi c alteration and
represent “genotype specifi c” drug targets. We have generated a panel of isogenic
cell lines derived from primary human BJ fi broblasts that contain single or multiple
defi ned genetic alterations that together are required for tumorigenic transformation
of these primary cells. These genetic alterations include among others, loss-of-
TP53, activation of RAS or activation of PI3K. These cell lines have been used in
high throughput single well assays in combination with large siRNA collections
targeting over 8000 genes to identify siRNAs that result in enhanced lethality only
in the background of these tumor specifi c genetic alterations. We have completed
these screens and have identifi ed several siRNAs whose lethality is dependent the
activation of RAS, PI3K or loss of TP53 in our isogenic BJ model. For RAS synthetic
lethal interactions, we have shown that the phenotypes caused by the siRNAs are
on-target and cause a synthetic lethal phenotype also in the presence of HRAS,
NRAS and KRAS oncogenic activation (see fi gure 2). These results indicate that
these synthetic lethal phenotypes are associated with RAS pathway activation. We
are extending the analysis of the TP53, PIK3CA and RAS dependent interactions in
a panel of wild type and mutant cancer cell lines of different tissue origin to exclude
context dependent effects. We are in the process of establishing the underlying
biological mechanism for the synthetic lethal phenotypes.
Figure 2: Identifying synthetic
lethal interactions with RAS
Viability scores for 8000 siRNA
smartpools in BJETp53KD
and BJETp53KD/RASV12 cell
lines. Each dot represents an
individual siRNA smartpool.
Depicted in dark grey, in
the middle under the cloud,
are siRNA smartpools that
display an enhanced reduction
in viability of the RASV12
expressing cells without effect on
the control BJETp53KD cell line.
Generation of computational models for predicting response to therapy in
breast cancer PI3K and MAPK signaling pathways are strongly implicated in triple
negative breast cancer and invasive lobular carcinomas. At present, many drugs are
75
molecular carcinogenesis
Group leader Roderick Beijersbergen
Roderick Beijersbergen PhD Group leader
Pasi Halonen PhD Post-doc
Helena Aguilar PhD Post-doc
Armida Fabius MSc PhD student
Johan Kuiken MSc PhD student
Jeroen Nijwening MSc PhD student
Klaas de Lint MSc PhD student
Jordi Vidal-Rodriguez PhD Bioinformatician
Cor Lieftink MSc Bioinformatician
Bram Gerritsen MSc Bioinformatician
Wouter Nijkamp Technical staff
Ben Morris Technical staff
Publications
Nijwening JH, Beijersbergen RL. Using
large-scale RNAi screens to identify
novel drug targets for cancer. IDrugs.
2010;13:772-7
Aguilar H, Solé X, Bonifaci N, Serra-
Musach J, Islam A, López-Bigas N,
Méndez-Pertuz M, Beijersbergen RL,
Lázaro C, Urruticoechea A, Pujana MA.
Biological reprogramming in acquired
resistance to endocrine therapy of breast
cancer. Oncogene. 2010;29:6071-83
Mullenders J, Fabius AW, van Dongen
MM, Kuiken HJ, Beijersbergen RL,
Bernards R. Interleukin-1R-associated
kinase 2 is a novel modulator of the
transforming growth factor beta signaling
cascade. Mol Cancer Res. 2010;8:592-603
Heeg S, Hirt N, Queisser A, Schmieg H,
Thaler M, Kunert H, Quante M, Goessel
G, Von Werder A, Harder J, Beijersbergen
R, Blum HE, Nakagawa H, Opitz OG.
www.ncbi.nlm.nih.gov/pubmed/21156006
EGFR overexpression induces activation
of telomerase via PI3K/AKT-mediated
phosphorylation and transcriptional
regulation through Hif1-alpha in a cellular
model of oral-esophageal carcinogenesis.
Cancer Sci. 2010.
Kuiken HJ, Beijersbergen RL.
www.ncbi.nlm.nih.gov/pubmed/21142664
Exploration of synthetic lethal interactions
as cancer drug targets. Future Oncol.
2010;6:1789-802

76
molecular carcinogenesis
in clinical development targeting specifi c components of these pathways although
their successes are limited. Complicated interactions with other signaling pathways
and unexpected feedback loops can limit the effectiveness of treatment and, in
some instances, even accelerate the tumorigenic process. The understanding and
modeling of these complex pathways using a systems approach can yield predictive
models for therapy responses to pathway-targeted therapeutics in breast cancer. We
are developing in silico models of therapy response in breast cancer by exploiting
normal and tumor cell lines as in vitro model systems for the quantifi cation of
functional activation of pathway components and associated cellular phenotypes. We
use RNAi for perturbation experiments in combination with a Luminex platform for
quantifi cation of the activation status of individual pathway components (fi gure 3).
Figure 3: Phosphoproteome
analysis of the PI3K and
MAPK pathways
Quantifi cation of levels
of phosphorylation of
pathway components
involved in PI3K/MAPK
signaling using the
Luminex platform. Human
mammary epithelial cells
were starved for 24 hours
after which they were restimulated
with 10ng/ml human epidermal growth factor.
Phosphorylation of ERK, p70S6, p38 and AKT were measured at different time-points.
The data obtained from these experiments are used to create and validate a dynamic
and quantitative computational model of pathway behavior and phenotypical
outcome in relation to drug response. With this approach we anticipate to maximize
the success rate of available targeted therapies against MAPK and PI3K pathway
components.

MECHANISMS OF CELL CYCLE PROGRESSION IN NORMAL
AND CANCER CELLS
In my group we aim to understand how cell division works in both normal and
cancer cells. We use biochemistry, molecular genetics and automated live-cell
fl uorescence imaging in human cells for this purpose. We are focussing on three
larger biological processes in the cell cycle: i) how cells decide to enter mitosis or
stay in G2 phase; ii) how cells use regulated protein destruction to exit mitosis and
divide successfully and iii) how cells that exit mitosis re-initiate DNA replication.
Increasingly, we are also studying the mechanisms by which cells respond to insults
or mistakes in G2 or mitosis, which could be critical for cancer therapy.
Mitosis: Activation of cyclin B1-CDK1 and other mitotic kinases Cells should
delay mitotic entry when the conditions are unfavourable for safe division, such
as after DNA damage, or when cells are too small. The decision to enter mitosis is
controlled by signalling pathways that direct the activation of the principal mitotic
kinase, cyclin B1-CDK1. Cyclin B1-CDK1 activity is intensely regulated by changing
protein levels, intracellular localisations and intrinsic activities of the WEE1/MYT1
inhibitory kinases and CDC25 activating phosphatases. We aim to gain further
insights into the mechanisms that trigger abrupt cyclin B1-CDK1 activation in late
Figure 4: Distinct requirement of Cdk1 for mitotic entry and mitotic progression in human cells
(A) Cells selected for Cdk1 shRNA expression were synchronized in G2/M by thymidine release and
mitotic cells were isolated. Separated G2 and mitotic pools were analyzed for Cdk1 expression by
Western blotting. Cdc20 protein levels serve as loading control. The percentage of remaining Cdk1
protein is indicated in the fi gure.(B) Cells collected by mitotic shake-off were lysed (lanes 1 and 2) or
released from nocodazole and incubated in fresh medium for 3 h, recollected, and lysed (lanes 3 and4).
Differences in mitotic phosphorylation shift of APC3 (human Cdc27 ortholog) and Cdc25C, depending
on the Cdk1 levels, are shown (lanes 1 and 2).(C) The impaired phosphorylation of APC3 in Cdk1attenuated
mitotic cells (lane 1) was rescued by coexpression of a Cdk1-YFP construct containing a
silent mutation in the RNAi targeting region (lane 2). Lane 3 are mitotic cells transfected with a control
shRNA, revealing normal endogenous Cdk1 levels.(D) Distribution of metaphase duration, measured as
time between chromosome alignment at the metaphase plate and onset of sister chromatid separation,
in Cdk1 RNAi cells (right) or Cdk1 RNAi cells rescued by coexpression of non–RNAi-sensitive Cdk1-YFP
(left). (E) Time-lapse microscopy analysis of mitotic progression after entry with normal or impaired
Cdk1 levels. Bottom panels are consecutive images of tubulin-YFP in a pS-control cell in mitosis; top
panels show delayed chromosome alignment (frames 2 and 3) and stalled metaphase (frames 4–6)
after Cdk1 shRNA. From ‘Cyclin B1-Cdk1 activation continues after centrosome separation to control
mitotic progression.’ (2007) Lindqvist A, van Zon W, Karlsson Rosenthal C, Wolthuis RMF. PLoS
Biology:e123.
77
molecular carcinogenesis
Group leader Rob Wolthuis
Rob Wolthuis PhD Group leader
Michiel Boekhout MSc PhD student
Linda Clijsters MSc PhD student
Erik Voets MSc PhD student
Janneke Ogink Technical staff
Bas ter Riet Technical staff
Publications
Van Zon W, Ogink J, ter Riet B, Medema
RH, te Riele H, Wolthuis RMF. The APC/C
recruits cyclin B1-Cdk1-Cks in prometaphase
before D box recognition to control mitotic
exit. J. Cell Biol. 2010;190:587-602
Voets E, Wolthuis RMF. MASTL is the
human orthologue of Greatwall kinase
that facilitates mitotic entry, anaphase and
cytokinesis. Cell Cycle 2010;9:3591-3601
Gurden MD, Holland AJ, van Zon W, Tighe
A, Vergnolle MA, Andres DA, Spielmann
HP, Malumbres M, Wolthuis RMF,
Cleveland DW, Taylor SS. Cdc20 is required
for the post-anaphase, KEN-dependent
degradation of centromere protein F. J. Cell
Sci. 2010;123:321-30
Van Zon W, Wolthuis RMF. Cyclin A and
Nek2A: APC/C-Cdc20 substrates invisible to
the mitotic spindle checkpoint. Biochem. Soc.
Trans. 2010;38:72-7

78
molecular carcinogenesis
G2 and the parameters of an ‘activation threshold’ for mitosis (fi gure 4). Various
other kinases and phosphatases participate in controlling the positive feedback loop
that leads to cyclin B1-CDK1 activation and the G2-M transition. A recent project led
to the identifi cation a novel human orthologue of the Greatwall kinases, MASTL.
MASTL may reduce the mitotic entry threshold and drive mitotic entry of G2 cells by
inhibiting PP2A activity. By determining the mechanisms that direct mitotic entry,
we aim to understand how normal and cancer cells may differ in their decisions to
enter mitosis or stay in G2 phase.
Mitotic Exit: coordination of events by protein destruction Once cells are in
mitosis, they need to ensure that all the duplicated chromosomes are attached to
the mitotic spindle. This is safe-guarded by the mitotic Spindle Checkpoint, which
inhibits another key enzyme of our interest, a large ubiquitin ligase called the
Anaphase-Promoting Complex (APC/C) and its activator CDC20. Mitotic entry fi rst
requires accumulation of regulatory proteins, such as cyclin B1, but these proteins
must be destroyed again at distinct, consecutive time points for correct cell division
(fi gure 5). We found that degradation of cyclin A and Nek2A, followed by loss of
cyclin B1, GEMININ and SECURIN, coordinates mitosis and prepares cells for the
next S phase. As such, precise regulation of protein destruction by the APC/C is
essential to guarantee cell viability and genomic integrity.
An important question that remains is how the APC/C acts with CDC20 to
recognize a critical substrate at the right time in mitosis. Our recent work revealed
new roles for APC/C-cooperating E2 ubiquitin conjugating enzymes, mechanisms
by which APC/C substrates are targeted for destruction, and roles of the spindle
checkpoint in connecting mitosis to the following S phase. An important future
research goal will be to understand how genetic modifi cations in cancer cells may
impinge on critical steps in mitotic progression and mitotic exit. Furthermore, we
are trying to resolve the factors that determine the fate of cells that are arrested in
mitosis as a result of prolonged spindle checkpoint activity. It is anticipated that
answers to these questions could create exciting opportunities to improve anticancer
therapies.
Figure 5: Measuring APC/C activity by time-lapse fl uorescence microscopy of Cyclin B1 destruction
A plasmid encoding for fl uorescent cyclin B1 was injected into G2-phase cells, a few hours before
they entered mitosis. The top panel shows a cell undergoing mitotic division, followed over time by
differential interference contrast (DIC). The bottom panel shows the localisation and quantitative levels
of cyclin B1 in the fl uorescence channel of the same cells. The cell undergoing division rapidly degrades
cyclin B1, as measured by a decrease in fl uorescence signal. This assay allows the quantitation of
ubiquitin-dependent protein destruction in live cells undergoing division and was fi rst established in the
laboratory of Jon Pines, Gurdon Institute, Cambridge, UK.

DIVISION OF MOLECULAR GENETICS
ROLE OF POLYCOMB-GROUP GENES IN TRANSCRIPTIONAL
REPRESSION, STEM CELL FATE AND TUMORIGENESIS
Our lab has a long-standing interest in epigenetic gene regulation dictated by
chromatin modifi cations. We study the mechanism of transcriptional repression
by Polycomb-group (Pc-G) protein complexes, and the effects of deregulation
of Pc-G genes on development, Cell cycle control, cancer formation and stem
cell maintenance. In addition, we are performing large-scale genetic screens in
primary cells and in cancer-predisposed mice to identify cancer-relevant networks
of oncogenes and tumor-suppressor genes. Model organisms comprise Mouse and
Drosophila.
Functional characterization of Pc-G protein complexes Repressive Pc-G proteins
and counteracting Trithorax-group (Trx-G) of nucleosome remodeling factors are
involved in maintenance of proper gene expression patterns during development at
the level of chromatin structure. Pc-G protein complexes control large sets of genes
including Hox gene clusters and the INK4a/ARF tumor suppressor locus. At least
two biochemical distinct evolutionary highly conserved Pc-G protein complexes
can be distinguished. The fi rst (PRC2) contains EzH1/EzH2 (SET domain proteins
acting as Histone H3 methylases), Su(z)12, Eed and histone deacetylases. The
second large complex (PRC1) encompasses Bmi1/Mel18, M33/MPc2, Mph1/Mph2
and Ring1b/Ring1a together with other more loosely associated proteins is required
throughout development. To study Pc-G, function we focus on representative
members of PRC1 and PRC2 in gain- and loss-of-function studies in mice and
Drosophila. In genetic and biochemical experiments we identifi ed the Bmi1/
Ring1b heterodimer as an E3 ubiquitin ligase for monoubiquitination of histone
H2A (collaboration with G. Buchwald and T. Sixma, Division II). Conditional
Ring1b loss-of-function experiments indicate an essential role for maintenance
of Pc-G repression in development and stem cell maintenance. An outstanding
question is how the activity of PcG enzymes is regulated; we recently obtained
evidence that phosphorylation of Bmi1 is required for E3 ligase activity of PRC1
and by mutating the essential phosphorylation sites demonstrated that these are
important for Bmi1’s oncogenic capacity. In addition we are studying the function of
the deubequitinating enzyme Usp3 that binds to mono-ubiquinated H2A and can
remove this mark. A major unresolved question is where and how Pc-G complexes
bind to chromatin. We have performed genome-wide surveys of where PRC1 and
PRC2 complexes bind to the Drosophila and mammalian genomes using DAMid
profi ling on tilling arrays (collaboration with B. van Steensel, Department of
Gene Regulation). This highlighted binding of both PRC1 and PRC2 to distinct
domains of 10-140 Kb containing ± 400 target genes in Drosophila and identifi ed
± 800 mammalian targets. These comprise conserved developmental regulators
that control differentiation. In recent in vivo 4C (chromatin conformation capture
on Chip) experiments we demonstrated that these domains interact in vivo in 3D
nuclear space in Drosophila neural tissues.
Connections between Pc-G gene repression, control of stem cell fate and
cancer formation We originally identifi ed Bmi1 as an oncogene that cooperates
with cMyc in the induction of B and T-cell lymphomas in mice, underscoring the
connection between deregulation of Pc-G repression and cancer. In contrast, Bmi1
knockout mice show severe progressive proliferation defects and increased apoptosis
of lymphoid and myeloid cells, resulting in severe lymphopenia. In addition, also
Bmi1-defi cient primary embryo fi broblasts (MEFs), neural precursors and many
other primary cell types show proliferation defects. We demonstrated that these
defects are in part due to increased levels of the tumor suppressors p16INK4a
and p19ARF, that are critical regulators of the RB and the p53 tumor suppressor
pathways. As such, the INK4a/ARF locus acts as an important tumor-prevention
mechanism in normal cells and stem/precursor cells. Using the mammary fat pad
Division head, group leader
Maarten Van Lohuizen
79
molecular genetics
Maarten Van Lohuizen PhD Group leader
Elisabetta Citterio PhD Senior Post-doc
Jean Bernard Beaudry PhD Post-doc
Jaap Kool PhD Post-doc
Karim Nacerdine PhD Post-doc
Inka Pawlitzky PhD Post-doc
Gaetano Gargiulo PhD Post-doc
Anke Sparmann PhD Post-doc
Bas Tolhuis PhD Post-doc
Nienke de Vries PhD Post-doc
Bart Westerman PhD Post-doc
Cesare Lancini PhD Post-doc
Waseem Akthar PhD Post-doc
Michela Serresi PhD Post-doc
Joep Vissers MSc PhD student
Martijn Koppens PhD student
Marleen Blom Technical staff
Paulien Cornelissen Technical staff
Danielle Hulsman Technical staff
Ellen Tanger Technical staff
Els Verhoeven Technical staff
Huub van Vugt Technical staff
Figure 1: Mammary fat pad
transplantation assay. Ductal outgrowth is
severely impaired upon transplantation of
Bmi1-/- (KO) when compared to Bmi1+/-
(Het). This suggests a severe mammary
epithelial precursor (stem) cell defect in
Bmi1 defi cient mice.

80
molecular genetics
Publications
Kool J, Uren AG, Martins CP, Sie D,
de Ridder J, Turner G, van Uitert M,
Matentzoglu K, Lagcher W, Krimpenfort
P, Gadiot J, Pritchard C, Lenz J, Lund
AH, Jonkers J, Jane Rogers J, Adams DJ,
Wessels L, Berns A, van Lohuizen M.
Insertional mutagenesis in mice defi cient
for CDK inhibitors p15Ink4b, p16Ink4a,
p21Cip1, p27Kip1 reveals cancer gene
interactions and correlations with tumor
phenotypes. Cancer Res. 2010;70:520-31
Mattison J, Kool J, Uren AG, de Ridder J,
Wessels L, Jonkers J, Bignell GR, Butler
A, Rust AG, Brosch M, Wilson CH, van
der Weyden L, Largaespada DA, Stratton
MR, Futreal PA, van Lohuizen M, Berns
A, Collier LS, Hubbard T, Adams DJ.
Novel candidate cancer genes identifi ed
by a large-scale cross-species comparative
oncogenomics approach. Cancer Res.
2010;70:883-95
De Vries NA, Bruggeman SW, Hulsman
D, de Vries HI, Zevenhoven J, Buckle T,
Hamans BC, Leenders WP, Beijnen JH,
van Lohuizen M, Berns A, van Tellingen
O. Rapid and robust transgenic highgrade
glioma mouse models for therapyintervention
studies. Clin Cancer Res.
2010;16:3431-41
Peric-Hupkes D, Meuleman W, Pagie L,
Bruggeman SW, Solovei I, Brugman W,
Gräf S, Flicek P, Kerkhoven RM, van
Lohuizen M, Reinders M, Wessels L,
van Steensel B. Molecular maps of the
reorganization of genome-nuclear lamina
interactions during differentiation. Mol
Cell. 2010;28:603-613
transplantation model, we revealed the essential role of Bmi1 in mammary epithelial
stem cells and precursors and ductal tree development (fi gure 1). Genetic studies
showed that the proliferative defects but not the observed premature differentiation
upon loss of Bmi1 in mammary epithelial precursors is in part mediated via INK4a/
ARF. Importantly, these studies revealed a dual role for Bmi1/Pc-G: controlling
both proliferation and differentiation. A key characteristic of cancer cells is their
unlimited self-renewal. In this respect, cancer cells resemble stem cells, and
accumulating evidence suggests that many forms of cancer may indeed contain cells
carrying stem cell markers. In studying the proliferation defects in Bmi1 defi cient
mice we discovered that Bmi1 is required for proliferation and self-renewal of neural
stem cells. Importantly, loss of the INK4a/ARF locus rescues the proliferation
& renewal defects, indicating it also is a critical Pc-G target in neural stem cells.
Using a transplantable Glioma model we demonstrated a critical role for Bmi1 in
brain tumor maintenance (fi gure 2). Interestingly, Bmi1 acts in this tumor setting
in an Ink4a/ARF-independent manner on cell adhesion and migration. These
results, together with the recently established role of Bmi1 in hemapoietic stem cells
and leukaemic stem cells, suggest a common conserved role for Bmi1-containing
Polycomb complexes in maintenance and expansion of stem cells or committed
progenitors and in the pathogenesis of tumors originating from the neoplastic
transformation of these cells. The possible broader relevance of these fi ndings for
human cancer is further underscored by the amplifi cation of BMI1 in Mantle cell
lymphomas and a subset of brain cancers and the overexpression of BMI1 in various
tumor types including non-small cell lung cancer, breast cancer, prostate cancer
and liver cancer. Conditional transgenic- and knockout models are currently used
to investigate the role of Pc-G genes in various tissue stem/progenitors and in solid
cancers that develop in these tissues.
Figure 2: Severely reduced
Glioma formation of
Bmi1-/- transformed
astrocytes. Survival curves
indicate that astrocytes
oncogenically transformed
by loss of INK4a/ARF
and activation of EGFreceptor
signaling rapidly
form agressive gliomas whereas tumor formation is delayed upon transplantation of Bmi1-defi cient
transformed astrocytes orthotopically transplanted in the forebrain of recipient mice.
In vivo genetic screens to identify new groups of collaborating oncogenes
or tumor suppressors In close collaboration with A Berns (this Division), J
Jonkers (Division of Molecular Biology) and D Adams and A Bradley/The Sanger
Centre, Hinxton, UK, we have developed high-throughput insertional mutagenesis
techniques and are now extending and optimizing these types of screens to other
cancer relevant models such as breast cancer. The power of this approach as a cancer
gene discovery platform is highlighted by the fi rst completed screens in hemapoietic
tumors induced in wild type, p53 or p19Arf defi cient mice. We recently extended
these screens to p15Ink4b, p21 and p27 defcient mice and to Pten-defi cient mice
prone to MMTV-induced mammary tumorigenesis.These screens yielded over
10.000 insertion sites implicating over 300 loci in tumorigenesis and uncovered
new pathway-specifi c oncogenes and candidate tumor-suppressors. Cross species
comparative analysis with a large array-CGH dataset of human cancer cell lines
revealed both new and novel candidate oncogenes and tumor-suppressor genes.
The role and mechanism of action of several of these new putative oncogenes or
tumor suppressors, is under investigation hemapoietic- and mammary fat pad celltransplantation
systems.

MOUSE MODELS FOR CANCER
The mouse is used as a model organism for establishing the role of oncogenes and
tumor suppressor genes in tumor development. By utilizing Cre/Lox mediated
switching and taking advantage of somatic gene transfer methods, expression
of multiple oncogenes and tumor suppressor genes can be regulated in a tissuespecifi
c and spatial-temporal fashion. This permits a more accurate modeling of
tumorigenesis as it occurs in man. It provides the opportunity to carefully defi ne
relevant genotype-phenotype correlations. The models also permit us to identify
new oncogenes and tumor suppressor genes involved in tumor progression using
a variety of techniques, such as array CGH, expression profi ling and proviral
insertional mutagenesis. Finally, these models constitute an excellent experimental
system to test prevention and intervention strategies.
Functional analysis of oncogenes and tumor suppressor genes We utilize mice
carrying combinations of different oncogene and conditional tumor suppressor
gene alleles to model a range of tumors. Our current focus is on several lung cancer
subtypes and mesotheliomas. To achieve (sporadic) activation of oncogenes and
inactivation of tumor suppressor genes we use Adeno-Cre or Lentivirus-mediated
somatic gene transfer to switch the conditional oncogenes and tumor suppressor
gene alleles in the desired tissues. Subsequently, tumor initiation and progression is
monitored in longitudinal studies using noninvasive imaging techniques.
Lung tumors We focus on small cell lung cancer (SCLC) and non-small cell
lung cancer (NSCLC). When Rb and p53 are inactivated specifi cally in lung, SCLC
develops in nearly 100% of the mice. The marker profi le of these tumors closely
resembles that of human SCLC. Even similar genomic aberrations are found such
as the amplifi cation of the L-Myc gene. These tumors are often heterogeneous
consisting of different cell types that either grow as spheres in suspension or
attached to substrate. Cells growing in suspension carry neuroendocrine markers
whereas the adherent non-neuroendocrine cells show more progenitor-like features.
Interestingly, these phenotypically very diverse cell lines share distinct genetic
aberrations indicating that they were derived from a common progenitor. We
wondered why these different cell types persisted in the tumor. Subcutaneous
grafting each of the cell types independently gave rise to localized tumors that
retained the features of the inoculated cells. However, grafting mixtures resulted in
local growth as well as metastasis of the neuroendocrine cells to liver indicating that
the non-neuroendocrine cells in the graft empowered the neuroendocrine cells to
metastasize. Interestingly, mutant Hras can convert the neuroendocrine cells into
non-neuroendocrine cells In spite of an intensive effort we have not yet identifi ed
the underlying mechanism that enables the neuroendocrine tumor cells to form
metastases.
There is increasing interest in the cell of origin of a tumor as this is likely a
key factor in determining the behavior of the tumor with respect to malignant
progression and response to therapy. To gain insight into the cell of origin of SCLC
and NSCLC we have designed a series of cell-type specifi c Adeno-Cre viruses that
enable us to switch oncogenes and tumor suppressor genes in distinct lung cell
types in vivo. We equipped adenoviruses with specifi c promoter constructs that
would upon infection of lung cells drive Cre expression specifi cally in Clara cells,
Alveolar type II cells, neuroendocrine cells or basal epithelial cells. In this way we
can assess which cell type can give rise to which tumor type and which oncogenes
and tumor suppressor gene mutations are needed catalyze this process. It appeared
that Cre driven from a neuroendocrine-specifi c promoter in conditional Rb/p53
mutant mice gives rise to SCLC with high effi ciency, whereas Cre driven from the
alveolar-specifi c promoter SPC shows a much lower incidence and delayed onset of
SCLC. Expression from a Clara cell-specifi c promoter caused mostly hyperplasias
in the bronchial epithelial lining and resulted only rarely to SCLC. When similar
experiments are performed in conditional mutant KrasG12D mice NSCLC is most
effi ciently induced with AdenoCre carrying an alveolar type II specifi c promoter.
Interestingly, Clara cell specifi c expression resulted in tumors with different features
indicating that the cell of origin is an important factor for the tumor that arises.
Group leader Anton Berns
81
molecular genetics
Anton Berns PhD Group leader
Paul Krimpenfort PhD Academic staff
Margriet Snoek PhD Academic staff
Hilda De Vries PhD Post-doc
Jitendra Badhai PhD Post-doc
Ivo Huijbers PhD Post-doc
Andor Kranenburg PhD Post-doc
Min-chul Kwon PhD Postdoc
Kate Sutherland PhD Post-doc
Anthony Uren PhD Post-doc
Andrej Alendar MSc PhD student
Erwin Van Montfort MSc PhD student
Colin Pritchard MSc Research assistant
Rahmen Bin Ali Technical staff
Jan Paul Lambooij Technical staff
Natalie Proost Technical staff
Johanna Blitz Technical staff
Fina Van de Ahé Technical staff
John Zevenhoven Technical staff
Publications
Calbo J, van Montfort E, Proost N, van
Drunen E, Beverloo H, Meuwissen R,
Berns A. A functional role for tumor cell
heterogeneity in a mouse model of Small
Cell Lung Cancer. Cancer Cell 2010 (in
press)
Nawijn M, Alendar A, Berns A. For better
or worse. The role of Pim in oncogenesis.
Nat Rev Cancer 2010 (in press)
Berns A. The blind spot of p53. Nature
2010;468:519-20
De Vries NA, Bruggeman SW, Hulsman
D, de Vries H, Zevenhoven J, Buckle
T, Amans B, Leenders WP, Beijnen JH,
van Lohuizen M, Berns A, van Tellingen
O. Rapid and robust transgenic highgrade
glioma mouse models for therapy
intervention studies. Clin. Cancer Res.
2010;16:3431-41

82
molecular genetics
Publications (continued)
Sutherland K, Berns A. Cell of origin of
lung cancer. Mol Oncol 2010 (in press)
Dannenberg JH, Berns A. Drugging drug
resistance. Cell. 2010;141:118-20
Kool J, Uren AG, Martins C, Sie D,
de Ridder J, Turner G, van Uitert M,
Maentzoglu K, Lagcher W, Krimpenfort
P, Gadiot J, Pritchard C, Lenz J, Lund A,
Jonkers J, Rogers J, Adams D, Wessels
L, Berns A, van Lohuizen M. Insertional
mutagenesis in mice defi cient for p15Ink4b,
p16Ink4a, p21Cip1, and p27Kip1 reveals
cancer gene interactions and correlations
with tumor phenotypes. Cancer Res.
2010;70:520-31
Mattison J, Kool J, Uren AG, de Ridder J,
Wessels L, Jonkers J, Bignell GR, Butler
A, Rust AG, Rosch M, Wilson C, van der
Weyden L, Largaespada DA, Stratton
M, Futreal PA, van Lohuizen M, Berns
A, Collier LS, Hubbard T, Adams DJ.
Novel candidate cancer genes identifi ed
by a large-scale cross-species comparative
oncogenomics approach. Cancer Research
2010;70: 883-95
Mesotheliomas Previously we have shown that inactivation of Nf2 and Ink4a/Arf
or Nf2/p53/Ink4a by intrathoracic Adeno-Cre injection of compound conditional
knockout mice gives rise to mostly sarcomatoid mesotheliomas and that Ink4a plays
an important role in the aggressiveness of the tumor. Also germline Ink4b/Ink4a/
Arf triple knockout mice show a high incidence of mesotheliomas. Furthermore,
sporadic local inactivation of these genes in combination with activation of mutant
KiRas results in mesotheliomas with an epitheloid phenotype, closely resembling
the predominant mesothelioma subtype found in man. We have made this model
more versatile. First, we use normal mesothelial cells isolated from the various
mutant mice as a starting point. These cells can be propagated in vitro. This not
only permits effi cient in vitro switching of conditional alleles but also easily allows
introduction of other genes or shRNA constructs. Subsequent injection of these
cells in recipient mice gives rise to mesotheliomas. This permits us to test responses
to specifi c inhibitors both in vitro and in vivo. Interestingly, we noted that also in
this system different cells of origin appear to give rise to different mesothelioma
subtypes, each with a different response profi le to the various drugs. We will focus
on specifi c resistance mechanisms underlying these differences. This is particularly
interesting since these differences are not caused by specifi c mutations but by the
characteristics of the cell of origin.
Parallel to these experiments we have begun to establish cultures from
mesothelioma specimen of human mesotheliomas. So far it has been diffi cult to
achieve growth after grafting in immunodefi cient mice. We are continuing these
experiments now with Nod;commonGammaKO;Rag2KO mice.
Ink4 proteins We have produced Ink4-less mice. Mice lacking all the four Ink4
genes are viable but specifi c compound Ink4 mutants show a higher incidence
of distinct tumors. We are currently making a complete inventory of increased
susceptibilities of the different strains and will focus on specifi c tumor types in
which loss of multiple Ink4 alleles likely play an important role.
ES cell lines from compound mutant mice We have also invested in the
development of methods to generate swiftly complex compound mutant mice.
The concept is to re-derive embryonic stem (ES) cells from available compound
mutant strains and use these ES cells to introduce additional genetic changes by
gene targeting or the exchange of expression cassettes. This approach has become
within reach by the description of defi ned media (2i and 3i) that prevent the
differentiation of ES cells. Using this approach we have successfully derived a series
of ES cell lines from the strains we routinely use. Injection of these ES cells in preimplantation
embryos results in the majority of cases in chimeras with extensive
ES cell contribution. We are testing whether these chimeras can be directly used
for tumorigenesis studies. If this is the case we can quickly assess the relevance of
distinct genetic alterations on tumor incidence and tumor characteristics without
having to embark on lengthy and extensive breeding protocols. It also would
substantially reduce the number of mice needed for most of our tumor studies.

DISSECTING CANCER CELL SIGNALLING NETWORKS AND
IDENTIFYING THERAPEUTIC CANCER TARGETS
Are there any intrinsic cellular mechanisms that protect us against cancer? How
can we dissect tumor-suppressing genetic networks? How can we effectively identify
novel cancer genes? Can we use our laboratory results to make a difference in
the clinic? For example, how can we identify novel and specifi c drug targets? In a
nutshell, these are the fundamental as well as clinically relevant questions that we
are taking up in the Peeper laboratory, which has two main focuses. First, we are
dedicated to dissecting signaling mechanisms that protect mammalian cells against
oncogenic transformation, aiming to increase our understanding of the molecular
principles of cancer. Second, we are determined to identify novel and specifi c
therapeutic targets as well as anticancer drugs.
Both aims are based on the premises that non-malignant cells require multiple
(epi-)genetic alterations to transform into metastatic tumor cells, and that tumor
cells, in turn, become dependent on activated oncogenes but also several normal
genes for their survival and proliferation. To achieve these goals, we make use of
advanced techniques, including screens with 100.000-vector RNAi libraries and
next-generation sequencing, but we also use classical biochemical and genetic
approaches.
While we are studying a diverse array of tumor types, there is a particular focus
on melanoma (a highly aggressive skin cancer) and metastatic breast cancer.
At the cellular level, we have a special interest in several cancer-relevant events:
(i) override of Oncogene-Induced cellular Senescence (‘OIS’, a potent tumor
suppressor mechanism limiting the proliferative capacity of incipient cancer cells);
(ii) suppression of ‘anoikis’ (apoptosis resulting from lack of adhesion, suppression
of which may contribute to metastasis); and (iii) (non-)oncogene addiction (in
the context of ‘Synthetic Lethality’ screens). Our results over the past decade
demons trate that these approaches, together, lead to the identifi cation of signaling
networks of gene products, deregulation of which allows for tumorigenesis and
metastasis. Our more recent results indicate that among these, several factors exist
that can be exploited for therapeutic intervention of cancer.
Ongoing
I. Breast cancer metastasis: mechanism and drug target identification
Metastasis commonly underlies the malignancy of cancers, representing the
principal cause of cancer-treatment failure and patient death. Tumor spread and
seeding are prevented by several physiologic barriers, including ‘anoikis’: apoptosis
resulting from lack of adhesion. Indeed, acquiring resistance to anoikis has been
proposed to represent a general prerequisite for survival of metastases during
circulation. Based on this cancer hallmark, and with the help of a genome-wide
functional screen, we previously identifi ed TrkB, a neurotrophic tyrosine kinase
receptor, as a potent suppressor of anoikis. Whereas non-malignant intestinal
epithelial cells underwent caspase-associated anoikis in vitro, this was completely
prevented by active TrkB, allowing formation of spheroid aggregates growing in
suspension. Mice readily cleared parental cells, but TrkB-expressing cells formed
metastases in several organs. We are currently exploring what the role is of TrkB in
metastasizing tumors and whether it can serve as a therapeutic target. We have also
begun to identify downstream signaling targets of this receptor. We found several
transcription factors, that is, Twist, Zeb1 and Snail, whose RNAi-mediated silencing
strongly suppressed the metastatic potential of tumor cells.
Further taking advantage of this experimental system, we have recently discovered a
novel and critical mediator of breast cancer metastasis, the Fra-1 transcription factor.
Fra-1 depletion from human breast cancer cells reduced their metastatic potential by
>3 orders of magnitude. Further, by analyzing the expression of a set of Fra-1 target
genes we can make accurate predictions on the clinical outcome of breast cancer. We
are currently using this so-called prognostic Fra-1 signature to screen for factors that
may be amenable to targeted inhibition. Eventually, this should lead to combined
diagnosis/treatment options for the patient.
Group leader Daniel Peeper
83
molecular genetics
Daniel Peeper PhD Group leader
Gireesh Anirudhan PhD Post-doc
Tristan Gallenne PhD Post-doc
Kylie Greig PhD Post-doc
Cornelia Hömig PhD Post-doc
Christelle Lenain MD PhD Post-doc
Katrin Meissl PhD Post-doc
Patricia Abrao Possik PhD Post-doc
Naoki Uno MD PhD Post-doc
Celia Vogel MSc Post-doc
Sedef Iskit MSc PhD student
Joanna Kaplon MSc PhD student
Marjon Smit MSc PhD student
Sirith Douma MSc Technical staff
Figure 3: A genome-wide screen for
oncogenes that bypass OIS identifi es
LRF. Shown are proliferation assays
demonstrating the oncogenic activity of
LRF when essayed alone or in conjunction
with other oncogenes like RAS and MYC.
Below is a quantifi cation of this effect.

84
molecular genetics
Publications
Kuilman T, Michaloglou C, Mooi WJ,
Peeper DS. The essence of senescence.
Genes Dev. 2010 15;24:2463-79
Smit MA, Peeper DS. Epithelialmesenchymal
transition and senescence:
two cancer-related processes are crossing
paths. Aging (Albany NY).
2010;2:735-41
Blagosklonny MV, Campisi J, Peeper DS,
Rando TA, Rudolph KL, Sassone-Corsi P,
Serrano M, Sharpless NE, Skulachev VP,
Tilly JL, Tower J, Verdin E, Vijg J. Impact
papers on aging in 2009. Aging (Albany
NY). 2010;2:111-21
Peeper DS. PICS-ure this: prosenescence
therapy? Cancer Cell. 2010 16;17:219-20
Vredeveld LC, Rowland BD, Douma
S, Bernards R, Peeper DS. Functional
identifi cation of LRF as an oncogene that
bypasses Rasv12-induced senescence via
upregulation of cyclin E. Carcinogenesis.
2010;31:201-7
Figure 4: Identifi cation of Zeb1 as a
critical metastasis gene. Shown is the
effect of RNAi-mediated depletion of Zeb1
in TrkB-expressing cancer cells: silencing
of Zeb1 (with two independent vectors)
strongly suppresses the ability of tumor cells
to produce metastases in the lung, relative
to control vectors.
II. Oncogene-Induced cellular Senescence (OIS): mechanism and drug target
identification
We are identifying novel OIS pathways whose deregulation contributes to cancer
in general and melanoma in particular. Also in this setting, we are combining
function-based, genome-wide screens with classical molecular biological
approaches. For example, we study the genetic basis for malignant progression of
benign moles (nevi) to malignant melanoma. We, in a longstanding collaboration
with prof. Wolter Mooi (VUmc), were the fi rst to discover that moles display many
of the classical hallmarks of OIS in vivo. In subsequent investigations, using a
combination of genetic profi ling and laser capture microdissection of compound
clinical nevus-melanoma specimens, we have identifi ed a mechanism by which nevi
can escape from senescence, thereby progressing to melanoma. These results may
have clinical implications, an interesting possibility that we are currently pursuing.
We have also designed several functional genomic screens with retroviral and
lentiviral cDNA expression and RNA interference libraries to identify OIS escape
mechanisms. For example, gene-expression microarray expression analysis of
senescent cells and their ‘escapers’ revealed various signaling networks that are
induced specifi cally in BRAF E600 -expressing, senescent human cells. Among
the top outliers were interleukins 6 (IL-6) and IL-8. RNAi-mediated knockdown
of either abrogated the senescence response. C/EBPb was identifi ed as an OIS
integrator, acting with IL-6 to amplify the activation of the infl ammatory network.
In collaboration with prof. Lucien Aarden (Sanquin) we found that IL-6 has
antagonistic functions in OIS and proliferation, as a function of the genetic context.
And together with W. Mooi, we observed that in human colon adenomas, IL-8
specifi cally co-localized with arrested, p16 INK4A -positive epithelium. Our results
suggest a model in which the antagonistic functions of IL-6 contribute to connect
senescence with an infl ammatory phenotype and cancer. We propose that the
dozens of cytokines that are being secreted by senescent cells serve to allow for
communication between them and their microenvironment. Hence, we termed this
the Senescence-Messaging Secretome, or SMS.
We have also recently discovered a candidate melanoma susceptibility gene,
TSC22D1. Only the short TSC22D1 transcript was upregulated by the melanoma
driver BRAF E600 , whereas the abundance of the large protein variant was suppressed
by proteolytic degradation. The TSC22D1 protein variants, in complex with their
dimerization partner THG1, exerted opposing functions, as selective depletion of the
short form, or conversely, overexpression of the large variant, resulted in abrogation
of OIS. This was accompanied by the suppression of several infl ammatory factors
and p15 INK4B , with TSC22D1 acting as a critical effector of C/EBPb. These results
demonstrate that the differential regulation of antagonistic TSC22D1 variants is
required for the establishment of OIS and suggest a contribution of TSC22 family
members to the progression of BRAF E600 -driven neoplasia.
III. Screening for novel therapeutic targets in melanoma
We have set up several screens to identify novel therapeutic targets for melanoma.
Cancer cells commonly depend on activated oncogenes, as well as certain nononcogenes,
for their proliferation and/or survival, a phenomenon known as (non-)
oncogene addiction. In that context, exploiting the phenomenon of synthetic
lethality (SL) as a mean of therapeutic intervention is gaining increasing interest.
For example, set out to screen for synthetic lethal partners of BRAF E600 . We
performed an siRNA screen using the Dharmacon kinome siRNA library that
contains 779 smart pool siRNAs targeting all human RNAs encoding kinases and
kinase-associated proteins, in a single well format. 384-well plates were imaged by
high-throughput fl uorescence microscopy to determine the cell count for each well.
This screen yielded a list of 61 genes whose knockdown resulted in selective toxicity
to BRAF E600 -expressing cells, which are currently being validated.
Along these lines, we are setting up several additional screens, both in vitro and in
vivo, to fi nd genes that are essential for tumor progression. For this purpose, we
make use of large-scale RNAi libraries and massive parallel sequencing. Together,
these approaches should lead to the identifi cation of novel therapeutic targets for
cancer, and melanoma in particular.

GENES INVOLVED IN BREAST CANCER PROGRESSION
AND METASTASIS
Cancer is the result of sequential accumulation of multiple genetic changes affecting
oncogenes and tumor suppressor genes in somatic cells. Despite recent advances in
understanding of the molecular basis of oncogenic transformation only a limited
number of key players involved have been identifi ed and extensively studied, while
this is essential for the development of more effective novel therapeutic strategies.
The aim of our laboratory is to identify genes involved in breast cancer by using
mouse mammary tumor virus (MMTV) induced insertional mutagenesis in mouse
models and characterize these genes and the oncogenic pathways they act in.
Identifi cation of mammary cancer genes in mouse models for breast cancer by
MMTV insertional mutagenesis screens MMTV proviral insertion in the genome
of murine mammary epithelial cells can activate fl anking proto-oncogenes leading
to mammary tumor induction. In recent years, we identifi ed approximately 50
common insertion sites (CISs) in mammary tumors in a series of approximately
400 tumors from wild-type Balb/c mice, Balb/c mice conditionally defi cient for
Tp53 in the mammary gland and MMTV-NeuNT transgenic mice, using the
splinkerette PCR and conventional capillary sequencing. Many of the CISs affect
genes not previously implicated in cancer or breast cancer in particular. Together
with the Jonkers lab, most of the collected MMTV induced mammary tumors were
reanalyzed using the new parallel sequencing platforms. Preliminary results indeed
show additional proviral insertion sites and some novel CISs.
The proviral insertion mutagenesis screen performed to date, revealed that Wnt
and Fgf genes were activated in almost all wild type BALB/c tumors, less frequently
in Tp53 defi cient tumors and rarely in ErbB2 overexpressing tumors. These results
suggest that the initial oncogenic event determines the subsequent oncogenic
changes. Two genes belonging to the R-spondin gene family, Rspo2 and Rspo3, and
members of the Odz gene family were also frequently activated in tumors from wild
type and conditionally defi cient Tp53 mice but rarely or not activated in tumors from
ErbB2 transgenic mice. In contrast, other genes like Eras were found to be more
frequently tagged in tumors from MMTV infected ErbB2 transgenic mice than in
wild type mice.
Analysis of genes collaborating with ErbB2. We have identifi ed Eras, Irs4 and
Igf2 as frequent MMTV target genes in mammary tumors from ErbB2 TG
mice. Overexpression of Eras was found to be most signifi cantly associated with
tumors from MMTV infected ErbB2 transgenic mice compared with tumors
from MMTV infected wild-type FVB mice. Overexpression of Eras and Irs4 in
immortalized normal mouse mammary epithelial cells (NMuMG cells) strongly
increased the growth rate in vitro, anchorage independent growth and rendered
these cells tumorigenic in nude mice, validating the mammary tumor inducing
capacity of these genes. Moreover, both genes strongly accelerated ErbB2 induced
tumorigenesis when co-expressed in NMuMG cells and strongly activated the PI3kinase
pathway. It has been shown that ERBB2 preferentially forms a heterodimer
with ERBB3. In this complex, ERBB2 mainly activates the MAPK pathway, while
ERBB3 activates the PI3K pathway. Since ERBB2 driven mammary tumorigenesis is
highly dependent on PI3-kinase activity, we hypothesize that genes like Eras and Irs4
are targeted by MMTV in ErbB2 tumors that lack ERBB3 mediated PI3K activation
(see model in fi gure 5). Indeed, ErbB3 is highly expressed in all tumors that arise in
non-MMTV-infected ErbB2 transgenic mice but only in 57% of the MMTV infected
ErbB2 transgenic mice. Thus activation of the PI3K-pathway via ERAS can act as an
alternative for the activation of this pathway through ERBB3, PTEN inactivation or
PIK3CA mutations. Although Eras is highly homologous to H-Ras and K-Ras, the
latter Ras oncogenes do not synergize with ErbB2. Eras mRNA was also expressed
to a variable degree in a small percentage of human breast cancers suggesting a
possible involvement of this normally silent gene in human breast carcinogenesis.
Group leader John Hilkens
85
molecular genetics
John Hilkens PhD Group leader
Gerjon Ikink MSc PhD student
Mandy Boer Technical staff
Figure 5: Model of PI3K signaling
pathways in MMTV induced mammary
tumors from ErbB2 transgenic mice.

86
molecular genetics
Group leader Jacqueline Jacobs
Jacqueline Jacobs PhD Group leader
Paul-André Genest PhD Post-doc
Marieke Peuscher MSc PhD student
Jaco Van der Torre MSc PhD student
Janet Van Noord Technical staff
Figure 6: Loss-of-function genetic
screening strategy aimed at identifying
genes that contribute to telomere-induced
genome instability. Mouse embryo
fi broblasts with genetic disruption of the
telomere component Trf2, but expressing
a temperature sensitive Trf2 allele,
lose telomere protection on all their
chromosome ends when cultured at a nonpermissive
temperature that inactivates
Trf2. This results in severe fusion of
chromosome ends and cells are unable to
divide or die. However, inhibition of
Nbs1 by a shRNA isolated in this screen,
allows cells to survive and divide in the
presence of uncapped telomeres.
CHROMOSOME END PROTECTION BY TELOMERES
Telomeres are protein-DNA complexes that protect natural chromosome ends
from being treated as damaged DNA. Telomeres progressively shorten with every
cell division until they become too short to function properly. The subsequent
recognition of chromosome ends as broken DNA has important consequences for
cellular and organismal life span but also for tumor development, and telomere
maintenance is therefore target of several recently developed anti-cancer strategies.
Our main aim is to increase our understanding of how mammalian cells precisely
perceive and respond to loss of telomere function, how telomere maintenance is
controlled and how factors involved in the response to telomere dysfunction affect
cellular transformation and tumor development.
Telomere-induced cellular senescence Loss of telomere function triggers a
DNA damage-like response that causes cells to die or stop proliferating indefi nitely
(senesce). This response limits the replicative life span of cells and thereby
contributes to organismal aging. In addition, it represents an important tumor
suppressor mechanism as it prevents unlimited outgrowth of potentially cancerous
cells. To investigate the consequences of loss of telomere protection we have
performed micro-array analysis of the gene expression changes induced by loss of
telomere function upon TRF2 inhibition. Next to genes involved in cancer, cell death
and cell cycle, genes involved in infl ammatory/immune responses represented gene
groups with the most signifi cant changes in expression upon telomere dysfunction.
We have subsequently focused on common upstream transcriptional regulators
of these genes and have investigated whether these regulators respond directly to
telomere dysfunction and contribute to telomere-induced senescence.
As an alternative unbiased approach to identify novel factors involved in DNA
damage and/or telomere damage response activation, we performed an siRNA
screen to fi nd factors that are involved in ATM activation following DNA damage.
Out of 254 different chromatin remodelers, modifi ers or DNA helicases, we found
26 genes that upon knockdown led to reduced activation of ATM in a primary
screen. We are in the process of validating and characterizing several of these genes.
Telomere maintenance and telomere-induced chromosome instability
If cells with uncapped telomeres fail to senesce or die and continue proliferating
in the absence of a mechanism that replenishes telomeric repeats, DNA repair
activities acting on chromosome ends cause chromosome fusions, anaphase bridges
and nonreciprocal translocations. Such cells are at high risk of developing into
cancer cells. Although telomere dysfunction is thought to be a major mechanism
underlying chromosomal instability in human cancers, little is known about the
precise structure of an uncapped telomere, how it is recognized, what precise
processing events occur and how these events are controlled. To identify novel
factors that contribute to telomere-induced genome instability we have developed an
RNAi loss-of-function genetic screen in mouse cells in which we can instantly and
reversibly uncap telomeres (see fi gure 6). The degree of telomere fusion induced
in this system is so severe that cells stop proliferating or die. In this screen we
have obtained multiple cell clones that survived severe telomere uncapping. The
isolation and characterization of shRNA vectors is ongoing, but has already led to
the discovery of several genes who’s inhibition by RNAi is able to confer resistance
to lethal telomere-induced genome- instability. One of these is Nbs1, a component
of the MRN complex which has recently also been shown by others to indeed
contribute to the processing of uncapped telomeres by non-homologous end-joining
(see fi gure), and which validates our screen. The other genes identifi ed so far have
not previously been implicated in the response to uncapped telomeres. They include
genes that encode for proteins involved in protein ubiquitination and methylation.
Their mechanism of action in the telomere damage response is now being
investigated, as well as their potential roles in the response to DNA double-strand
breaks and in cellular transformation and tumor formation.

THE DEVELOPMENTAL ROLES OF ONCOGENES AND TUMOR
SUPPRESSORS IN ZEBRAFISH
Our research focuses on understanding how vertebrates sense and respond to
energetic stress, using zebrafi sh as our central experimental model. We aim to
provide insights into how tumors adapt their metabolism to survive, proliferate and
grow under nutrient and oxygen-poor conditions. We are following two main lines of
research:
Analysis of energy metabolism and hypoxia during development and cancer
During development of an organism, mechanisms that sense nutrient availability
are intimately linked with growth control pathways in order to coordinate energy
conditions with organ growth and tissue homeostasis. Tumor cells employ several
of the same mechanisms that coordinate nutrient availability with growth during
development to ensure tumor cell proliferation. To improve cancer therapy, we need
a better understanding of the molecular pathways that link energy metabolism with
growth control during development and how are those affected in cancer.
Zebrafi sh (Danio rerio) is an ideal system to dissect molecular mechanisms and
pathways, largely owing to the rapid development and optical clarity of the embryos.
Furthermore, the large progeny size (females typically lay 200 eggs at a time), and
availability of readily accessible transparent embryos combine to render the zebrafi sh
an excellent model for high-throughput genetic and chemical screens in the intact
organism. The major metabolic pathways are very well conserved between humans
and zebrafi sh.
Mutations in the serine-threonine kinase LKB1 in humans lead to a gastrointestinal
polyposis disorder with highly increased predisposition to cancer. Recently, somatic
mutations in LKB1 have been found in about 30% of lung carcinomas as well as in
endometrial cancer. LKB1 activates AMP-activated kinase (AMPK) the “energy switch”
of the cell and that leads to growth suppression through several pathways including
inhibition of the mTOR pathway. Since mouse models of lkb1 defi ciency are embryonic
lethal, the role of energy metabolism during development has not been explored.
To study control of metabolism during development, we generated and characterized
lkb1-defi cient zebrafi sh. Importantly, the zebrafi sh lkb1 mutants are embryonic
viable -unlike their mouse counterparts- and exhibit deregulated metabolism.
We demonstrated that zebrafi sh LKB1 is required for AMPK activation and
found that lkb1 mutants are unable to sense and respond to energetic stress. Lkb1
mutant zebrafi sh display a fast metabolic rate and exhaust their energy reserves
prematurely. They exhibit hallmarks of premature starvation such as abnormal lipid
accumulation in the liver (fi gure 7). We showed that attenuation of metabolic rate in
lkb1 mutants, by application of the TOR inhibitor rapamycin, suppresses key aspects
of the lkb1 phenotype.
We are currently exploring the connection between LKB1, energy metabolism and
hypoxia. This investigation will lead to a better understanding of the adaptation of
metabolic processes in order to meet energy demand that tumor cells employ in
order to cope with decreasing nutrient and oxygen supply within the tumor. We aim
to gain insight into these processes at the organim level during normal development
and during cancer formation.
Analysis of the developmental roles of the Polycomb group proteins
(collaboration with Maarten van Lohuizen)
Polycomb group (PcG) protein complexes, which function in the epigenetic
regulation of gene expression, control numerous developmental processes.
Epigenetic silencing mediated by PcG is implicated in stem-cell fate maintenance
and cancer. The PcG member Ring1b is an E3 ubiquitin ligase that ubiquitinates
histone H2A. This mark correlates with the repression of gene expression of PcG
target genes. Targeted inactivation of Ring1b in mice leads to very early lethality
precluding analysis of the role of Ring1b in embryonic development. We have
recently succeeded in generating a stable mutant in Ring1b by using Zinc Finger
Endonuclease technology. We have established that Ring1b protein levels and
ubiquitination of H2A are dramatically reduced in the mutants. Ring1b mutant
embryos lack fi ns and all jaw cartilage elements. We are currently characterizing the
mechanism underlying these specifi c defects.
87
molecular genetics
Group leader Anna-Pavlina Haramis
Anna-Pavlina Haramis PhD Group Leader
Yme van der Velden MSc PhD student
Liqin (Bruce) Wang Research assistant
Publication
Brennan C, Dosch R, Haramis AP,
Luckenbach T, Martinez-Morales JR,
Moro E, Polok B, Ramesh TM, Russell
C, Argenton F, Strähle U. Report of the
European zebrafi sh principal investigator
meeting in Padua, Italy, March 18-22
2010. Zebrafi sh. 2010;7:305-10
Figure 7: Zebrafi sh larvae at day 7 postfertilization
stained with Oil Red O, a red
dye that binds lipids. No lipids are detected
in wt larvae; abnormal lipid accumulation
in the liver of lkb1 mutants (arrow).

88
epidemiology
Division head, group leader Flora van Leeuwen
Group leader Matti Rookus
Flora Van Leeuwen PhD Group leader
Matti Rookus PhD Group leader
Marjanka Schmidt PhD Group leader
Berthe Aleman MD PhD Academic staff
Frans Hogervorst PhD Academic staff
Nicola Russell PhD Academic staff
Emiel Rutgers MD PhD Academic staff
Michael Schaapveld PhD Academic staff
Laura Van ’t Veer PhD Academic staff
Senno Verhoef PhD Academic staff
Anouk Pijpe PhD Post-doc
Suzanne Rebers PhD Post-doc
Sandra Van den Belt-Dusebout PhD Post-doc
Martijn Verheus PhD Post-doc
Eric Vermeulen PhD Post-doc
Naomi Boekel MSc PhD student
Richard Brohet MSc PhD student
Anja van Eggermond MSc PhD student
Lieske Schrijver MSc PhD student
Mandy Spaan MSc PhD student
Rianne van Nimwegen MSc PhD student
Sandra van den Broek MSc PhD student
Janneke Verloop MSc PhD student
Cherita Sombroek MSc Junior scientifi c
researcher
Thea Mooij MSc Statistical analyst
Marie-José Blom MSc Data manager
Juliet Boessenkool-Pape MSc Data manager
Ellen Engelhardt MSc Data manager
Eduard Ivanov MSc Data manager
Cora Knol MSc Data manager
Judith de Lange MSc Data manager
Harmke Groot MSc Research assistant
Esther Janssen Research assistant
Kiki Jeanson MSc Research assistant
Kim Kersten MSc Research assistant
Marianne Kuenen Research assistant
Gabey Ouwens Research assistant
Saskia Pelders MSc Research assistant
Frederiek Diemer Research assistant
DIVISION OF PSYCHOSOCIAL
RESEARCH AND EPIDEMIOLOGY
EPIDEMIOLOGY
The cancer epidemiology group is currently concentrating on two principal research
lines: (1) the etiology of hormone-related cancers, with a focus on gene-environment
interactions; (2) the long-term health consequences of cancer treatment, particularly
in terms of the risk of developing second malignancies or cardiovascular disease.
Risk factors for hormone-related cancer In our nationwide cohort study in
families with a BRCA1/2 mutation (GEO-HEBON study), we are studying whether
1) hormonal/life-style factors modify cancer risk in BRCA1/2 families, 2) common
genetic alterations are associated with the risk of breast cancer among BRCA1/2
carriers.
We continued the coordination of the data collection of the International BRCA1/2
Carrier Cohort Study (IBCCS), a follow-up study on 3823 BRCA1/2 mutation carriers,
and the harmonization of the epidemiologic risk factor data for the Consortium of
Investigators of Modifi ers of BRCA1/2 (CIMBA). The CIMBA risk factor database
now includes 10,031 BRCA1/2 carriers. We coordinate the data cleaning process that
is being conducted by eight international groups for specifi c risk factors and by us
for exogenous hormones. The CIMBA DNA database contains more than 20,000
BRCA1/2 carriers. Three approached were taken to fi nd new genetic modifi ers of
BRCA1- and BRCA2- related cancer risk: 1. targeted single nucleotic polymorphisms
(SNPs), found to be related with cancer risk in the general population or among
BRCA1/2 carriers in earlier studies, 2. SNPs with highest signifi cance in GWA
studies in the general population, and 3. new GWA studies within CIMBA. The
fi rst approach, though generally based on plausible biological pathways, was not
very successful in general. One of the exceptions was the fi nding that the minor
allele of CASP8 D302H, a key enzyme involved in the initiation of apoptosis, was
associated with a reduced risk of breast cancer and ovarian cancer for BRCA1, but
not for BRCA2 mutation carriers. As an example of the second approach, the top 350
SNP list of two GWAS studies in the general population were examined as genetic
risk modifi ers for BRCA1 and BRCA2. A total of 8 SNPs in BRCA1 mutation carriers
and 12 SNPs in BRCA2 carriers were associated with breast cancer risk. SNPs in
SNRPB and CAMK1D were strongest in BRCA1, whereas SNPs in LOC134997 and
FBXL7 were most signifi cant in BRCA2. Furthermore, the SNP at 9p22.2 (rs3814113)
that was identifi ed in a GWAS on ovarian cancer in the general population showed
a reduced risk of ovarian cancer in BRCA1 as well as in BRCA2 carriers. Thirdly,
two new GWAS studies, with several replication phases, were conducted in BRCA1
and BRCA2, respectively. In BRCA1 carriers fi ve SNPs on 19p13 were associated
with breast cancer risk, two of which showed independent associations (rs8170
and rs2363956). Genotyping these SNPs in population-based cases and controls
identifi ed a similar association with estrogen receptor–negative breast cancer and
an association with estrogen receptor–positive disease in the opposite direction. The
fi ve SNPs were also associated with triple-negative breast cancer. The GWA study in
BRCA2 indicated that SNPs that modify BRCA2 penetrance are similar to variants
that modify risk of sporadic breast cancer. Functional studies are ongoing to identify
the biological pathway related with the newly discovered SNPs in BRCA1, while more
indepth genetic analyses are being conducted for BRCA2.
To increase power for the assessment of the risk of hormone-related cancers after
fertility treatment, such as ovarian stimulation for in vitro fertilization (IVF), we
expanded our nation-wide OMEGA-cohort of 25,152 women treated for subfertility
(collaboration with CW Burger, Erasmus MC Rotterdam). The original cohort
comprises 19,145 women who received ovarian stimulation for IVF between 1983
and 1995 and 6,007 subfertile women not treated with IVF. For the expansion of the
IVF cohort (n = 8,800), we collected detailed data regarding IVF treatment (number
of cycles, drug doses, number of oocytes retrieved) from the 12 collaborating IVFclinics
through computerized databases and medical charts. We also identifi ed
4,100 subfertile women to enlarge the comparison group of women not treated

with IVF, comprising women who underwent tubal surgery or intra-uterine
insemination, or who withdrew from the IVF waiting list. The original cohort has
recently been linked with the Netherlands Cancer Registry to update cancer risk
and analyses of breast cancer risk (n = 609 cases) are ongoing. For the expanded
cohort 50% of the addresses have been updated. Three pilot studies including 643
IVF-treated women have been performed; the average response rate of 51% was not
infl uenced by the use of an internet questionnaire, the collection of DNA (toenails),
or the study region. More responders fi lled in the internet questionnaire (55%)
compared to the paper questionnaire (45%). To increase the response rates, the lay-out
of the questionnaire, the study leafl et and the website have been adapted, and media
attention has been sought. In total, approximately 20,000 women will be invited,
starting November 2010. Data will be collected regarding life-style, reproductive
factors, and health outcomes through (internet and paper) questionnaires.
A new prospective cohort study (the Nightingale Study) among nurses in the
Netherlands was initiated in collaboration with the Institute for Risk Assessment
Sciences (IRAS, Utrecht University) and the BIG-register (of the Ministry of Health,
Welfare and Sport). Exposure to light-at-night has been suggested as a contributing
cause of breast cancer (IARC classifi cation ‘probable human carcinogen, 2A’).
Since shift- and night-time work is prevalent and increasing in modern societies,
this exposure may contribute to the continuing increase of breast cancer incidence
and may be of public health concern. This study will provide insight into, amongst
others, the potential association between occupational exposures (e.g. shift work,
electromagnetic fi elds) and the risk of cancer and other diseases, and on potential
biological mechanisms. Women will be asked to complete a (web-based or paper)
questionnaire, sign an informed consent, and donate toenails for DNA analyses
(e.g. clock genes). Specifi c attention will be directed to potential shift-work related
selection bias.
Late effects of cancer treatment Now that curative treatment is available for a
substantial group of cancer patients, it is increasingly important to evaluate how the
occurrence of late complications of treatment affects their long-term survival. We
aim to evaluate the risk of second cancers and cardiovascular disease (CVD) after
radio- and chemotherapy (CT) for Hodgkin’s lymphoma (n=3,400), testicular cancer
(n=3,745) and breast cancer (n~80,000) over a period of up to 30 years after primary
treatment.
In 2010, we evaluated the long-term risk of colorectal cancer following Hodgkin’s
lymphoma treatment. Results show that Hodgkin’s lymphoma patients have a
3.7-fold (95% confi dence interval (CI) 2.6-5.0) increased standardized incidence
ratio (SIR) of developing colorectal cancer compared with the general population,
with an absolute excess risk of 7.8 colorectal cancer cases per 10,000 patients/year.
After a median follow-up of 20 years, 40 colorectal cancer patients were identifi ed
(23 colon, 17 rectum). The highest SIR (7.5, 95% CI 3.4-14.3) was seen for patients
treated before age 25. Cumulative incidence was 1.9% (95% CI 1.3-2.7) at 30 years
of follow-up. Especially for colon cancer, the SIR increased with longer follow-up
duration (9.3, 95% CI 3.4-20.2 in 30-year survivors). No increased SIR was found
for colorectal cancer in patients treated with RT alone. However, a signifi cantly
increased SIR was found for patients treated with RT and CT; the SIR for patients
treated with CT and RT below the diaphragm was (6.9 95% CI 3.9-11.2). We also
found that a 40-year old HL-survivor treated before age 25 has the same CRC risk as
a 55-60-year old person from the general population.
After we published our results on treatment-related risk factors for stomach cancer
after Hodgkin’s lymphoma we contributed our data to an international multicenter
case-control study of stomach cancer after Hodgkin’s lymphoma, coordinated by the
U.S. National Cancer Institute. The study includes 89 cases with stomach cancer
and 190 matched controls. Preliminary results show that stomach cancer risk
increases with larger radiation doses to the mean stomach, with 5.3-fold increased
risk for radiation doses of 30-39 Gy compared with less than 0.5 Gy. The study
confi rms our fi nding that procarbazine increases the risk of stomach cancer.
By the end of 2009 we established the nationwide Dutch BETTER (Better care after
Hodgkin lymphoma: Evaluation of long-Term Treatment Effects and screening
Key publications
89
epidemiology
Antoniou AC, Sinilnikova OM, Rookus MA,
Chenevix-Trench G and 133 other authors.
Common variants in LSP1, 2q35 and 8q24
and breast cancer risk for BRCA1 and
BRCA2 mutation carriers. Hum Mol Genet
2009;18:4442-4456
Antoniou AC, Wang X, Rookus MA, Couch
FJ and 168 other authors. A locus on 19p13
modifi es risk of breast cancer in BRCA1
mutation carriers and is associated with
hormone receptor-negative breast cancer in
the general population. Nat Genet 2010;
42:885-892
Blows FM, Driver KE, Schmidt MK, Broeks
A, van Leeuwen FE, Huntsman D and 38
other authors. Subtyping of breast cancer
by immunohistochemistry to investigate a
relationship between subtype and short and
long term survival: a collaborative analysis
of data for 10,159 cases from 12 studies. PLoS
Med. 2010;7:e1000279
Braat DDM, Schutte JM, Bernardus RE,
Mooij TM, Van Leeuwen FE. Maternal
death related to IVF in the Netherlands
1984-2008. Hum Reprod 2010;25:1782-86
Broeks A, Braaf LM, Wessels LF, van
de Vijver M, De Bruin ML, Stovall M,
Russell NS, Van Leeuwen FE, Van ‘t Veer.
Radiation-associated breast tumors display a
distinct gene expression profi le. Int J Radiat
Oncol Biol Phys 2010;76:540-7
Figure 1: Risk of colorectal cancer after
treatment of Hodgkin’s lymphoma in 2667
patients (Relative risk is compared to the
general population)

90
epidemiology
Publications (continued)
Cardous-Ubbink MC, Geenen MM, Schade
KJ, Heinen RC, Caron HN, Kremer LCM,
Van Leeuwen FE. Hypertension in Long-
Term Survivors of Childhood Cancer: A
Nested Case-Controle Study. Eur J Cancer
2010;46:782-90
Devilee P, Rookus M. A Tiny Step Closer
to Personalized Risk Prediction for Breast
Cancer. New England Journal of Medicine
2010;362:1043-1045
Enciso-Mora V, Broderick P, van Leeuwen
FE, Houlston RS. and 41 other autors. A
genome-wide association study of Hodgkin’s
lymphoma identifi es new susceptibility loci at
2p16.1 (REL), 8q24.21 and 10p14 (GATA3).
Nat Genet 2010;42:1126-1130
Engel C, Versmold B, van Leeuwen FE,
Schmutzler RK and 46 other authors.
Association of the variants CASP8 D302H
and CASP10 V410I with breast and ovarian
cancer risk in BRCA1 and BRCA2 mutation
carriers. Cancer Epidemiol Biomarkers Prev
2010;19:2859-2868
Fles R, Hoogendoorn WE, Platteel I,
Scheerman CE, Leeuw-Mantel G, Mourits
MJ, Hollema H, van Leeuwen FE, van
Boven HH, Nederlof PM. Genomic
profi le of endometrial tumors depends on
morphological subtype, not on tamoxifen
exposure. Genes Chromosomes Cancer
2010;49:699-710
Gaudet MM, Kirchhoff T, Rookus MA, van
Leeuwen FE, Chenevix-Trench G and 170
other authors. Common genetic variants
and modifi cation of penetrance of BRCA2associated
breast cancer. PLoS Genet
2010;6:e1001183
Kaas R, Verhoef S, Wesseling J, Rookus
MA, Oldenburg HS, Peeters MJ, Rutgers
EJ. Prophylactic mastectomy in BRCA1
and BRCA2 mutation carriers: very low
risk for subsequent breast cancer. Ann Surg
2010;251:488-492
Marees T, Van Leeuwen FE, Schaapveld M,
Imhof SM, De Boer MR, Kors WA, Ringens
PJ, Moll AC. Risk of third malignancies
and death after a second malignancy in
retinoblastoma survivors. Eur. J. Cancer
2010:46:2052-58
Recommendations) consortium, consisting of hemato-oncologists and radiation
oncologists of the NKI, all 8 University Medical Centers and 3 large peripheral
hospitals. The consortium aims to increase the life expectancy and enhance the
quality of life of HL survivors in the Netherlands by reducing morbidity and
mortality from late adverse effects of HL treatment. In 2010, two meetings have
been organised, in which draft national screening guidelines were developed.
Furthermore we are also developing a website for HL survivors with information
about late effects of treatment and possibilities to reduce these risks. An evaluation
of the website focussing on knowledge, risk perception and worries about late
effects, is in progress. In UMCN, UMCU and Medisch Spectrum Twente we
also started identifying HL survivors, who are eligible to be recalled for medical
surveillance, based on the screening guidelines. Figuur 2 Flora van Leeuwen
To further study the effects of radiotherapy, chemotherapy, reproductive and genetic
factors on the risk of breast cancer after Hodgkin lymphoma, we are performing a
nationwide case-control study (collaboration with Division Experimental Therapy,
Annegien Broeks, Laura van ’t Veer). So far we have identifi ed 170 case patients, who
were individually matched to 462 controls. All women who are still alive receive a
questionnaire on lifestyle factors and hormone use and are asked to provide a blood
sample for genetic analyses.
Within our nationwide cohort, comprising 3,745 testicular cancer patients treated
in the Netherlands during 1965-1995, we recently examined the risk of late relapse
after successful treatment of a testicular germ cell tumor. Late relapses, defi ned as
a relapse occurring 2 years or more after treatment of a testicular germ cell tumor
which resulted in a complete response, are rare events. Incidence, characteristics
and tumor-specifi c survival for late relapses have not been well defi ned in previous
studies. Our study cohort had long-term and very complete follow-up. After a
median follow-up of 19 years 79 patients experienced a late relapse. Almost 80% of
these relapses occurred within 10 years after completion of treatment, giving a 10year
cumulative incidence of 2.7%. Beyond the 10th year follow-up the annual rate of
late relapse was very low, i.e. 0.06% per year. Only initial stage predicted late relapse
risk, while age, histology, period of diagnosis and initial treatment did not. The 10

year cumulative incidence reached 11.3% among stage III non-seminoma patients.
The 10-year testis cancer-specifi c survival following a late relapse was 70%. Older
age (≥50 years) at late relapse, a symptomatic presentation or a late relapse detected
by increasing serum markers and no histological confi rmation of the relapse were
all associated with signifi cantly worse testis cancer-specifi c survival. Surgery for late
relapse was associated with better survival.
In a previous cohort study we showed increased risks of cardiac morbidity and
mortality among breast cancer patients treated between 1970 and 1986. To evaluate
the long-term cardiovascular morbidity and mortality in survivors of breast cancer
treated with more contemporary regimens we are conducting two new large cohort
studies.
The fi rst is a population-based cohort of patients with invasive breast cancer
(n=97,747) and ductal carcinoma in situ (n=13,545) of the breast diagnosed between
1989 and 2004 in the Netherlands. Patients have been identifi ed through the
Netherlands Cancer Registry. The cohort has been linked with the heart intervention
registry maintained by the Steering Committee Heart Interventions Netherlands
(BHN) and the Dutch Hospital Discharge registry (LMR) for cardiovascular
disease and with the Dutch general population registry (GBA), the Central Bureau
for Genealogy (CBG) and Statistics Netherlands (CBS) for date and cause of
death, respectively. Additional treatment information is currently being supplied
by hospital registries, nine RT institutes, Pharmo, clinical trials, and regional
documentation projects.
The second cohort is hospital-based and consists of 24,000 patients treated
between 1970 and 2004 in the Netherlands Cancer Institute or the Erasmus MC,
Daniel den Hoed Cancer Center. For this cohort detailed treatment information
and cardiovascular risk factors are being collected from medical fi les and through
general practitioners. Our fi rst analyses were restricted to a sub cohort of 10-year
survivors treated for breast cancer between 1970 and 1989 before the age of 46
years (n=854) We observed an increased risk of cardiovascular disease for patients
irradiated to the internal mammary chain nodes compared to patients not irradiated
to these nodes (HR=3.81; CI=1.80-8.05). Risk was also elevated for patients treated
after 1980 with more recent radiotherapy techniques.
Publications (continued)
91
epidemiology
Marees T, Dommering CJ, Imhof SM, Kors
WA, Ringens PJ, Van Leeuwen FE, Moll
AC. Incidence of retinoblastoma in Dutch
children conceived by IVF: an expanded
study. Human Reprod 2009;24:3220-24
Pijpe A, Manders P, Brohet RM, Collee JM,
Verhoef S, Vasen HF, Hoogerbrugge N, van
Asperen CJ, Dommering C, Ausems MG,
Aalfs CM, Gomez-Garcia EB, Van’t Veer
LJ, van Leeuwen FE, Rookus MA. Physical
activity and the risk of breast cancer in
BRCA1/2 mutation carriers. Breast Cancer
Res Treat 2010;120:235-44
Pijpe A, Manders P, Hooning MJ, Kluijt I,
Vasen HF, Hoogerbrugge N, van Asperen
CJ, Meijers-Heijboer H, Ausems MG, van
Os TA, Gomez-Garcia EB, Brohet RM,
HEBON, van Leeuwen FE, Rookus MA.
Body weight and risk of breast cancer in
BRCA1/2 mutation carriers. Breast Cancer
Res Treat. 2010
Pijpe A, Manders P, Mulder RL, van
Leeuwen FE, Rookus MA. Reliability of
self-reported diagnostic radiation history in
BRCA1/2 mutation carriers. Eur J Epidemiol
2010;25:103-113
Schmidt MK, Tommiska J, Broeks A, Van
Leeuwen FE, Van ‘t Veer LJ, Pharaoah PDP,
Easton DF, Shah M, Humphreys M, Dörk
T, Reincke SA, Fagerholm R, Blomqvist C,
Nevanlinna H. Combined effects of single
nucleotide polymorphisms TP53 R72P and
MDM2 SNP309, and p53 expression in
survival of breast cancer patients. Breast
Cancer Res 2009;11:R89
van den Belt-Dusebout A, Aleman B,
Gietema J, de Wit R, Van ‘t Veer M.B.,
Lugtenburg A.D.G., Krol S., van Leeuwen
F.E. Langetermijncomplicaties na behandeling
voor zaadbalkanker en Hodgkinlymfoorm.
Ned Tijdschr Geneeskd 2010: 154
Van der Pal HJ, Van dalen EC, Hauptmann
M, Kok WE, Caron HN, Van den Bos C,
Oldenburger F, Koning CC, Van Leeuwen
FE, Kremer LC. Cardiac Function in 5-years
survivors of childhood cancer; a long-term
follow-up study. Arch Int Med 2010;170:
1247-55
Voskuil DW, Van Nes JGH, Junggeburt JMC,
Van de Velde CJH, Van Leeuwen FE, De
Haes JCJM. Maintenance of physical activity
and body weight in relation to subsequent
quality of life in postmenopausal breast cancer
patients. Ann. Oncol 2010;10:2094-101

92
psychosocial research
Group leader Neil Aaronson
Neil Aaronson PhD Group leader
Marc van Beurden MD PhD Academic staff
Hester Oldenburg MD PhD Academic staff
Emiel Rutgers MD PhD Academic staff
Gabe Sonke MD PhD Academic staff
Martijn Stuiver MSc Academic staff
Senno Verhoef MD PhD Academic staff
Saskia Duijts PhD Post-doc
Marieke van Leeuwen PhD Post-doc
Eric Vermeulen PhD Post-doc
Willem Eijzenga MSc PhD student
Karin Gehring MSc PhD student
Doranne Hilarius MSc PhD student
Rianne Hoopman MSc PhD student
Ruud Knols MSc PhD student
Chantal Lammens MSc PhD student
Hanna van Waart MSc PhD student
Marijke Wevers MSc PhD student
Chad Gundy MSc Senior statistical analyst
Miranda Gerritsma Research assistant
Marianne Kuenen Research assistant
Jacoline Melis Research assistant
Publications
Douma KFL, Bleiker EMA, Aaronson NK,
Cats A, Gerritsma MA, Gundy CM, Vasen
HF. Long-term compliance with endoscopic
surveillance for familial adenomatous polyposis
(FAP). Colorectal Dis. 2010;12:1198-207
Douma KFL, Aaronson NK, Vasen HFA,
Gerritsma MA, Gundy CM, Janssen EPA,
Vriends AHJT, Cats A, Verhoef S, Eveline
Bleiker. Psychological distress and use of
psychosocial support in familial adenomatous
polyposis. Psychooncology 2010;19:289-98
Douma KFL, Aaronson NK, Vasen HFA,
Verhoef S, Gundy CM, Bleiker EMA.
Attitudes towards genetic testing in childhood
HEALTH-RELATED QUALITY OF LIFE ASSESSMENT AND
BEHAVIORAL INTERVENTIONS IN CLINICAL ONCOLOGY
This research line has two primary foci: (1) development of methods and applications
of health-related quality of life (HRQL) assessment in clinical research and clinical
practice; and (2) development and testing of behavioral and psychosocial interventions
to reduce symptom burden and improve the HRQL of patients with cancer.
Methods and measures for assessing the HRQL of mid- to long-term survivors
of testicular and prostate cancer This collaborative study between the EORTC
Quality of Life and Genito-Urinary Cancer Groups has two primary objectives: (1) to
test the logistics required to conduct survivorship studies within the context of the
EORTC, with specifi c focus on long-term follow-up of patients treated in phase III
clinical trials; (2) to pilot test questionnaires for assessing the HRQL of mid- to
long-term cancer survivors (> 5 years disease free). Approximately 140 patients will be
recruited from each of two EORTC GU Group phase III clinical trials – one in
testicular and one in prostate cancer. The two samples will be drawn from three
broad geographic/cultural regions: (1) Northern Europe; (2) Southern Europe; and (3)
the United Kingdom. HRQL will be assessed at 3 levels: (1) generic (the SF-36 Health
Survey); (2) cancer-specifi c (the EORTC QLQ-C30 plus condition-specifi c modules;
and (3) cancer survivor-specifi c (the Impact of Cancer questionnaire). In 2010, IRB
approval was obtained and data collection was initiated in the Netherlands, Norway,
France, and Italy. IRB approval is pending in Belgium and the UK.
A randomized controlled trial of exercise training in hematological cancer
patients following peripheral blood stem cell transplantation This RCT, carried
out by R. Knols, a PhD student of ours in Zurich, Switzerland, evaluated the effects of
a 12-week outpatient physical exercise program, incorporating aerobic and strength
exercises (n = 64), as compared to a usual care control condition (n = 67) on
hematopoietic stem cell transplantation (HSCT) patients’ physical performance and
psychosocial well-being. Primary outcomes were quantifi ed physical performance and
self-reported physical functioning. Secondary outcomes were body composition
measurement, quantifi ed walking activity and patient-reported outcomes (physical
activity, fatigue and health-related quality of life). Assessments were at baseline,
immediately after program completion and at 3-month follow-up. Signifi cant intervention
effects were observed at both post-treatment and follow-up on physical performance
measures. No other outcomes yielded statistically signifi cant group differences.
A randomized controlled study of the efficacy of graduated compression
stockings for prevention of lymphedema after inguinal lymph node dissection
graduated compression stockings have been advocated for prevention of lymphedema
after inguinal lymph node dissection. Scientifi c evidence of their effi cacy is lacking.
In this RCT, 80 patients (45 with melanoma, 35 with an urogenital tumor) who
underwent ILND were randomly allocated to compression stocking use for six
months or to an usual care control group. No signifi cant differences were observed
between groups in the incidence of edema, the median time to the occurrence of
edema, incidence of genital edema, frequency of complications, HRQL or body
image. This study was unable to demonstrate the effi cacy of prophylactic use of PhDd
compression stockings for prevention of lymphedema after inguinal node dissection.
Cognitive behavioral therapy and physical exercise for climacteric symptoms
in breast cancer patients experiencing treatment-induced menopause
This multicenter, randomized trial is evaluating the effectiveness of cognitive
behavioral therapy (CBT), physical exercise (PE) or the combination of CBT/PE in
alleviating climacteric symptoms in breast cancer patients experiencing treatmentinduced
menopause. 422 women were randomized to one of four groups: CBT, PE,
CBT+PE or usual care. Preliminary results based on short-term (immediate postintervention)
follow-up indicate that both CBT and PE signifi cantly improve
menopausal and urinary symptoms, and sexuality, as compared to usual care. No
signifi cant program effects were observed for secondary outcomes, including body
image, Neither CBT nor PE signifi cantly infl uenced body image, psychological well

eing or other aspects of HRQL. Long-term (6-month follow-up) results are
currently being evaluated.
P hysical exercise during chemotherapy to improve physical fitness and
reduce fatigue (PACES) This multicenter, RCT is evaluating the effectiveness of
two physical exercise interventions in maintaining or enhancing physical fi tness and
in minimizing fatigue in patients undergoing adjuvant chemotherapy for breast or
colon cancer: (1) a low intensity, home-based, self-management program; and (2) a
moderate intensity, structured, supervised program. In total, 360 patients will be
randomized to one of the two intervention groups or to a usual care control group.
All participants undergo performance tests and complete self-report questionnaires
at baseline, at the completion of chemotherapy, and at 6 month follow-up. Patient
recruitment started in the fi rst quarter of 2010.
Behavioral and psychosocial effects of rapid genetic counseling and testing
(RGCT) in newly diagnosed breast cancer patients This multicenter, RCT, carried
out in collaboration with the University Medical Center Utrecht (Dr. Margreet Ausems),
is investigating the uptake of RGCT when offered routinely to newly diagnosed breast
cancer patients who, prior to receiving primary treatment, are identifi ed as having at
least a 10% risk of carrying a mutation in the BRCA1 or BRCA2 gene, and the impact
of RGCT on a range of outcomes. Women (N = 255) recruited from 11 hospitals in the
Amsterdam and Utrecht regions of the Netherlands are randomized to either RGCT
or usual care (2:1 ratio). The study endpoints include: (1) uptake of RGCT; (2) choice
of clinical management strategy, including direct bilateral mastectomy or delayed
preventive contralateral mastectomy; (3) cancer risk perception and cancerrelated
distress; (4) knowledge of genetic aspects of breastcancer; (5) decisional
satisfaction; (6) HRQL; and (6) satisfaction with RGCT. Questionnaires are
administered at study entry, and at 6 and 12 month follow-up. All 255 patients have
now been recruited into the study. Data collection will continue through 2011.
Predictors of neuropsychological improvement following cognitive rehabilitation
in patients with gliomas This study investigated the specifi c patient-related factors
that predict responsiveness to a cognitive rehabilitation program that has previously
been demonstrated to be successful at the group level in patients with gliomas. Four
categories of possible predictors of improvement were selected for evaluation: sociodemographics,
clinical variables, self-reported cognitive symptoms, and objective
neuropsychological test performance. Nearly 60% of the participants were classifi ed
as reliably improved. Reliable improvement was predicted by (younger) age (p = .003)
and (higher) education (p = .011). Cognitive rehabilitation programs should take these
patient characteristics into account and, if possible, adapt programs to increase the
likelihood of improvement among older and less well educated participants.
Chemotherapy-induced nausea and vomiting (CINV) in daily clinical practice:
A community hospital-based study This multicenter, observational study
investigated: (1) the impact of CINV on patients’ (HRQL) in daily clinical practice;
(2) the association between patient characteristics, type of antiemetics and CINV;
and (3) the role of CINV in decisions to modify antiemetic treatment. During three
consecutive chemotherapy cycles, 277 patients used a diary to record emesis episodes
and antiemetic use on the day prior to and 7 days after having received chemotherapy.
Acute and delayed nausea during the fi rst treatment cycle were reported by 39% and
68% of the patients, respectively. The comparable fi gures for acute and delayed
vomiting were 12% and 23%. During the fi rst and subsequent treatment cycles,
approximately one-third of the patients indicated that CINV had a substantial impact
on their daily life. At all treatment cycles, patients receiving treatment with
moderately emetogenic chemotherapy containing anthracyline reported more acute
nausea than patients receiving highly emetogenic chemotherapy. Acute vomiting was
associated signifi cantly with change in (i.e., additional) antiemetic treatment.
93
psychosocial research
Key publications (continued)
and reproductive decision-making for familial
adenomatous polyposis (FAP). Eur J Human
Genetics 2010;18:186-93
Douma KFL, Bleiker EMA, Vasen HFA, Gundy
CM, Gerritsma MA, NK Aaronson. Quality of
life and consequences for social life of familial
adenomatous polyposis. Colorect Dis 2010
Gundy CM, Aaronson NK. Effects of mode of
administration (MOA) on the measurement
properties of the EORTC QLQ-C30: a
randomized study. Health Qual Life Outcomes.
2010;8:35
Lammens CRM, Bleiker EMA, Verhoef S,
Hes FJ, Ausems MGE, Majoor-Krakauer
D, Sijmons RH, van der Luijt RB, van den
Ouweland AMW, van Os T, Hoogerbrugge
N, Gomez-Garcia EB, Dommering CJ, Gundy
C, Nagtegaal T, Aaronson NK. Psychosocial
impact of Von Hippel-Lindau disease: levels and
sources of distress. Clin Genet 2010;77:483-91
Hilarius DL, Kloeg PH, van der Wall E, Komen
M, Gundy CM, Aaronson NK. Cancer-related
fatigue: Clinical practice versus practice
guidelines. J Support Care Cancer 2010
Knols RH, de Bruin ED, Uebelhart D,
Aufdemkampe G, Schanz U, Stenner-Liewen
F, Hitz F, Taverna C, Aaronson NK. Effects
of an oupatient physical exercise program
on hematopeoetic stem-cell transplantation
recipients: a randomized clinical trial. Bone
Marrow Transplant (in press)
Lammens CRM, Aaronson NK, Verhoef S,
Wagner A, Sijmons RH, Ausems MGEM,
Vriends AHJT, Ruijs MWG, van Os TAM,
Spruijt L, Gomez-Garcia EB, Kluijt I, Nagletaal
T, Bleiker EMA. Genetic testing in Li-
Fraumeni Syndrome: Uptake and psychosocial
consequences. J Clin Oncol 2010;28:3008-14
van der Poll-Franse LV, Mols F, Gundy CM,
Creutzberg CL, Nout RA, Verdonck-de Leeuw
IM, Taphoorn MJ, Aaronson NK. Normative
data for the EORTC QLQ-C30 and EORTCsexuality
items in the general Dutch population.
Eur J Cancer (in press)
Taphoorn MJB, Claassens L, Aaronson NK,
Coens C, Mauer M, Osoba D, Stupp R,
Mirimanoff ROM, van den Bent MJ, Bottomley
A. An international validation study of the
EORTC brain cancer module (EORTC QLQ-
BN20) for assessing health-related quality of life
and symptoms in brain cancer patients. Eur J
Cancer 46 2010:1033-1040

94
psychosocial research
Group leader Sanne Schagen
Sanne Schagen PhD Group leader
Willem Boogerd MD PhD Academic staff
Jose Belderbos MD PhD Academic staff
Sabine Linn MD PhD Academic staff
Olaf van Tellingen PhD Academic staff
Rita Roodbergen MSc Academic staff
Michiel de Ruiter PhD Post-doc
Riejanne Seigers MSc PhD student
Christien Schilder MSc PhD student
Vincent Koppelmans MSc PhD student
Sanne Menning MSc PhD student
Myrle Kemperman MSc PhD student
Chad Gundy MSc Senior statistical analyst
Florien Boele Research assistant
Marianne Kuenen Research assistant
Marion Weevers Research assistant
Astrid Klinkenberg Research assistant
Merel van den Ende Research assistant
COGNITIVE FUNCTION IN CANCER PATIENTS
Signifi cant proportions of cancer patients report cognitive changes following
therapy that interfere with their daily life activities and that can persist well into
the survivorship period. The projects and collaborations constituting this research
line center around the investigation of the prevalence, nature and cause of cognitive
problems associated with systemic therapies. Understanding who is at risk and the
emotional, cognitive and biological mechanisms associated with the development
and maintenance of these effects is critical to treatment and prevention.
Late effects of chemotherapy on cognitive functioning in the elderly. NKI-
Erasmus MC It is still largely unknown if chemotherapy induced cognitive changes
observed several years after administration are persistent in the long term. We
conducted a cross-sectional study comparing cognitive functioning of breast cancer
patients who had undergone six cycles of CMF chemotherapy on average 21 years
ago (n=196) to a population-based sample of women without a history of cancer
(n=1509). All participants were between 50 and 80 years of age. Exclusion criteria
were endocrine treatment, development of a secondary malignancy after the
primary breast cancer, breast cancer relapse and metastasis. Chemotherapy-exposed
women performed signifi cantly less well than the reference group on cognitive tests
covering the domains of immediate (p=.015) and delayed verbal memory (p=.002),
attention (

International Cognition and Cancer Task Force Recommendations to
Harmonize Studies of Cognitive Function in Cancer Patients NKI, MDACC,
MSKCC, Sydney Cancer Centre In the past years it has become increasingly
apparent that cytotoxic agents delivered systemically for non Central Nervous System
tumors may have cognitive side-effects, but many fundamental questions require
further elucidation. Answering these questions will benefi t from larger samples
collected over multiple institutions. The International Cognition and Cancer Task
Force brought together experts in the fi eld of cancer and cognition to create research
recommendations and guidelines with the specifi c aim to increase the homogeneity
of study methodology.
Suggestions are formulated by two working groups for a core set of neuropsychological
tests, a common criterion for defi ning cognitive impairment and cognitive
changes, and common methodological approaches that will provide a basis for
combining data across studies. These recommendations will improve research
design and facilitate between study comparisons and meta-analyses, which will help
determine more accurate estimates of incidence, severity, individual risk factors and
causes of cognitive problems associated with chemotherapy for non-CNS tumors.
Information about chemotherapy-associated cognitive problems contributes
to cognitive problems in cancer patients NKI-VU University While increasing
attention is directed at identifying biological mechanisms underlying cognitive
changes observed in cancer patients without CNS disease following chemotherapy,
psychological factors that can contribute to these cognitive changes are much less
studied.
In an online experiment, the infl uence of informing patients about the association
between cognitive problems and chemotherapy on self-reported cognitive
functioning and neuropsychological test performance was investigated. Cancer
patients treated with chemotherapy (n=148) reported higher levels of cognitive
complaints after receiving such information (M = 21.20) than without such
information (M = 19.00; p = .032). No difference was found for chemotherapynaïve
patients (n=88; M = 18.85 vs. 20.08; ns). A similar interaction pattern was
observed on a word learning test. Patients treated with chemotherapy recalled fewer
words after being informed about the association between cognitive problems and
chemotherapy (M = 24.44) than without such information (M = 27.63; p = .010).
No difference was found for chemotherapy-naïve patients (M = 26.35 vs. 25.39; ns).
Patient information may induce a stereotype threat, which affects self-reported
cognitive function and neuropsychological test performance in cancer patients for
whom this information is relevant.
Key publications
95
psychosocial research
Ruiter MB, Reneman L, Boogerd W,
Veltman DJ, van Dam FSAM, Nederveen
AJ, Boven E, Schagen SB. Cerebral
hyporesponsiveness and cognitive
impairment ten years after chemotherapy
for breast cancer. Hum Brain Mapp. 2010
Schilder CM, Seynaeve C, Beex LV,
Boogerd W, Linn SC, Gundy C, Huizenga
HM, Nortier JW, van der Velde CJ,
van Dam FS, Schagen SB. Effects of
Tamoxifen and Exemestane on Cognitive
Functioning of Postmenopausal Patients
With Breast Cancer: Results From the
Neuropsychological Side Study of the
Tamoxifen and Exemestane Adjuvant
Multinational Trial. J Clin Oncol.
2010;28:1294-300
Schilder CM, Seynaeve C, Beex LV,
Boogerd W, Linn SC, Gundy C, Nortier
JW, van der Velde CJ, van Dam FS,
Schagen SB. Cognitive functioning of
postmenopausal breast cancer patients
before adjuvant systemic therapy,
and its association with medical and
psychological factors Critical Reviews in
Oncology/Hematology, Crit Rev Oncol
Hematol. 2010;76:133-41
Schilder CM, Seynaeve C, Linn SC,
Boogerd W, Gundy CM, Beex LV, van
Dam FS, Schagen SB. The impact of
different defi nitions and reference groups
on the prevalence of cognitive impairment:
a study in postmenopausal breast cancer
patients before the start of adjuvant
systemic therapy. Psychooncology.
2010;19:415-22
Seigers R, Timmermans J, van der Horn
HJ, de Vries EF, Dierckx RA, Visser L,
Schagen SB, van Dam FS, Koolhaas
JM, Buwalda B. Methotrexate reduces
hippocampal blood vessel density and
activates microglia in rats but does not
elevate central cytokine release. Behav
Brain Res. 2010;5;207:265-72
Seigers R, Pourtau L, Schagen SB, van
Dam FS, Koolhaas JM, Konsman JP,
Buwalda B. Inhibition of hippocampal
cell proliferation by methotrexate in rats is
not potentiated by the presence of a tumor.
Brain Res Bull. 2010;16;81:472-6

96
psychosocial research
Group leader Eveline Bleiker
Eveline Bleiker PhD Group leader
Annemieke Cats MD Academic staff
Daniela Hahn MSc Academic staff
Irma Kluijt MD Academic staff
Senno Verhoef MD Academic staff
Leonie Woerdeman MD Academic staff
Willem Eijzenga MSc PhD student
Chantal Lammens MSc PhD student
Marijke Wevers MD PhD student
Chad Gundy MSc Senior statistical analyst
Tanja Nagtegaal MSc Junior scientifi c researcher
Grace Sidharta MSc Research assistant
Sophie Van De Velde Genetic counselor
Anja van Rens Genetic counselor
Figure 3: Example of a LFS pedigree
Publications
Ahmed AKJ, Hahn DEE, Hage JJ, Bleiker
EMA, Woerdeman LAE. Temporary
banking of the nipple-areola-complex in
97 skin sparing mastectomies. Plastic and
Reconstructive Surgery (in press)
Douma KF, Bleiker EM, Aaronson NK,
Cats A, Gerritsma MA, Gundy CM, Vasen
HF. Long-term compliance with endoscopic
surveillance for familial adenomatous
polyposis. Colorectal Dis 2010;12:1198-1207
PSYCOSOCIAL ISSUES IN CANCER GENETICS
This research line is being conducted in close collaboration with the NKI-AVL
family cancer clinic. It comprises a number of studies which are focused on two
psychosocial themes in genetic counseling for cancer: 1) the uptake and long-term
psychosocial impact of risk-reducing behavior.; and 2) early detection of psychosocial
problems and the development of psycho-educational interventions.
Psychosocial aspects of genetic testing in families at high risk of multiple
tumors at various sites and ages The aim of this 4 year, multi-center, crosssectional
study is to investigate the uptake of genetic testing for Li-Fraumeni Syndrome
(LFS) and Von Hippel-Lindau Disease (VHL), the psychosocial consequences of (not)
undergoing genetic testing, and compliance with recommended surveillance programs.
Data collection has been completed. In total, 243 individuals (78%) completed a selfreport
questionnaire.
The study on LFS families included 18 families with a p53 germline mutation. Eligible
family members were invited to complete a self-report questionnaire assessing motives
for (not) undergoing genetic testing, LFS-related distress and worries, and healthrelated
quality of life. Uptake of presymptomatic testing was 55% (65/119). Of the total
group, 23% reported clinically relevant levels of LFS-related distress. Carriers were
not signifi cantly more distressed than non-carriers or than those with a 50% risk who
did not undergo genetic testing. Those with a lack of social support were more prone
to report clinically relevant levels of distress (OR 1.3; 95% CI 1.0-1.5). Thus, although
preventive and treatment options for LFS are limited, more than half of the family
members from known LFS families choose to undergo pre-symptomatic testing.
An unfavorable genetic test result, in general, does not cause adverse psychological
effects. Nonetheless, it is important to note that a substantial proportion of individuals,
irrespective of their carrier status, exhibit clinically relevant levels of distress which
potentially warrant psychological support.
In the study on VHL, we assessed compliance with the surveillance program for VHL.
Of the 84 (77%) participants, 78 (93%) indicated having received an advice to undergo
periodic surveillance. Of these, 71 (91%) reported being fully compliant with that advice.
In 64% of the cases, this advice was only in part conform the published guidelines.
Based on the medical fi les, between one-quarter and one-third of individuals did not
undergo surveillance as recommended in the guidelines for central nervous system
(CNS) lesions, and one-half for visceral lesions. Screening delay for CNS lesions was
associated with ‘hospital’ and ‘advice for surveillance that deviates from the guidelines’
(p < .01). These results indicate that the majority of the participants report having
received advice to undergo periodic surveillance. They also report being fully compliant
with that advice. However, for the majority, the advice given is only partially consistent
with published guidelines. Furthermore, delay in surveillance was observed for a
substantial number of individuals. Efforts should be undertaken to stimulate guidelinebased
screening advice, and to minimize screening delay.
Screening for psychosocial problems at the family cancer clinic The aim of this
4 year KWF-study is to develop and evaluate a screening questionnaire as an aid in
identifying individuals experiencing signifi cant psychosocial problems associated
with cancer genetic counseling. In 2009, this multidimensional questionnaire was
developed according to EORTC guidelines for questionnaire module development: 1)
generation of relevant issues, 2) operationalization of these issues into a set of items,
and 3) questionnaire pre-testing. In 2010, this questionnaire has been evaluated for
its reliability, validity, sensitivity, specifi city and positive predictive value for detecting
psychosocial problems and psychosocial support needs. For this validation study, new
counselees who attend the NKI-AVL family cancer clinic for purposes of counseling
and testing are invited to complete the screening questionnaire (by means of a touch
screen computer) just prior to their second visit to the family cancer clinic (thus
after an informative intake session), and at follow-up (three weeks after their fi nal
counseling). At both assessment points, the counselees are also being interviewed by
a trained psychosocial worker who uses a semi-structured interview (‘gold standard’)
to determine the problem areas that warrant further services. In total 130 have
now completed both the screening questionnaire and the clinical interview with a

psychosocial worker. When this screening questionnaire proves to be suffi ciently
valid, a trial will be conducted, to evaluate the effectiveness of the questionnaire
on: decreasing onco-genetic related psychosocial problems, and increasing effective
communication between counsellors and clients, the awareness of problems of
counselees, and appropriate referrals.
Preventive total gastrectomy The aim of this cross-sectional, multi-center study is to
investigate the experiences with, and consequences of gastroscopy screening and
prophylactic total gastrectomy in CDH1 mutation carriers. Mutations in the CDH1 gene
are associated with a 70% lifetime risk for diffuse gastric cancer and an additional 40%
risk for lobular breast cancer in women. The following research questions are being
addressed: (1) What is the impact of prophylactic gastrectomy on quality of life and
future planning? (2) What factors infl uence the decision and timing of prophylactic
gastrectomy? (3) Which sociodemographic, clinical and psychological factors are
associated with quality of life after gastrectomy?, and (4) What can we recommend to
improve the health care in individuals from CDH1 families? Six families were identifi ed
with a CDH1 mutation. All individuals with a CDH1 gene mutation have been invited to
complete a self-report questionnaire and to participate in a semi-structured interview. A
comparison group of individuals who underwent a total gastrectomy because of cancer
are also being invited to complete the self-report questionnaire. In total, 25 of the 31
CDH1 mutation carriers returned the questionnaires (81%). Of these, 20 individuals
had undergone prophylactic total gastrectomy. First results show that ‘the level of
energy’ is the most important factor determining functioning and quality of life after
prophylactic gastrectomy: 65% of the participants experienced reduced excessive
fatigue. Shortly after the surgery, diffi culties in food-intake led to substantial weight
loss. Respondents consider the dumping syndrome and reduced appetite syndrome
as the most impairing functional complaints. About 40% report moderate to severe
impairment in daily activities. Respondents are, in general, satisfi ed with the
multidisciplinary health-care that is offered to them, however, they only stated that
they could have benefi ted from better dietary counseling.
Surveillance of the pancreas in high risk individuals The aim of this study is
to investigate the psychological burden of participating in a pancreatic cancer (PC-)
surveillance program. Since 2006, a multi-center prospective study is investigating
the effectiveness of PC-surveillance (EUS and MRI) in high-risk individuals. Highrisk
individuals are defi ned as (1) fi rst degree relatives (FDR) of patients with familial
pancreatic cancer (FPC) and (2) carriers of a PC-prone gene mutation. PC-prone gene
mutations include CDKN2A (Familial Atypical Multiple Mole Melanoma (FAMMM)syndrome),
LKB1 (Peutz Jegers syndrome), BRCA1 (Hereditary Breast and Ovarian
Cancer (HBOC) syndrome), BRCA2 (HBOC), and p53 (Li-Fraumeni syndrome).
Carriers of a BRCA1/2 mutation or p53 mutation are only eligible when they meet
additional criteria. In 2009, a psychosocial study arm was added to this multicenter
surveillance study. The specifi c research questions of the psychosocial arm, are: 1)
What is the perceived burden of participation in a PC-surveillance program, 2) what
are the motivations to participate in such a program, 3) to what extent are those
participating in the surveillance program worried about developing cancer, and
4) which factors are associated with anxiety experienced after an EUS-MRI-based
surveillance program. Currently, with a cross sectional design, individuals are invited
to complete a questionnaire four weeks after receiving their surveillance results.
Sixty-nine individuals (85%) completed the questionnaire (54% female; mean age 52
yrs). Surveillance was reported as “very to extremely uncomfortable” by 15% for MRI,
and 12% for EUS. Most frequently reported reason to participate was that surveillance
might lead to PC detection in a curable stage. In 27 respondents (39%) EUS and/or
MRI detected an abnormality including cysts and 1 suspicious solid lesion, which was
resected. In total 29% is “often” or “almost always” concerned about developing cancer.
Six respondents (9%) have clinical levels of depression and/or anxiety. Perceived
advantages of surveillance outweighed disadvantages according to 88% of respondents.
The fi nding of ‘abnormal results’, resulting in a shorter screening-interval (n=7) or
surgery (n=1) was not related to a higher level of anxiety. In the total group, levels
of anxiety and depression are comparable to those of the general population. From a
psychosocial point of view PC surveillance in high-risk individuals seems feasible.
97
psychosocial research
Publications (continued)
Douma KF, Bleiker EM, Vasen HF, Gundy
CM, Aaronson NK. Quality of life and
consequences for daily life of familial
adenomatous polyposis (FAP) family
members. Colorectal Dis 2010
Douma KF, Bleiker EM, Vasen HF, Gundy
CM, Gerritsma MA, Aaronson NK.
Psychological distress and quality of life
of partners of individuals with familial
adenomatous polyposis. Psychooncology
2010
Lammens CR, Bleiker EM, Aaronson
NK, Wagner A, Sijmons RH, Ausems
MG, Vriends AH, Ruijs MW, van Os
TA, Spruijt L, Gomez Garcia EB, Cats
A, Nagtegaal T, Verhoef S. Regular
surveillance for Li-fraumeni syndrome:
advice, adherence and perceived benefi ts.
Fam Cancer 2010;9:647-654
Lammens CR, Aaronson NK, Wagner
A, Sijmons RH, Ausems MG, Vriends
AH, Ruijs MW, van Os TA, Spruijt L,
Gomez Garcia EB, Kluijt I, Nagtegaal T,
Verhoef S, Bleiker EM. Genetic testing
in Li-Fraumeni syndrome: uptake and
psychosocial consequences. J Clin Oncol
2010;28:3008-3014
Lammens CR, Bleiker EM, Verhoef S,
Hes FJ, Ausems MG, Majoor-Krakauer
D, Sijmons RH, van der Luijt RB,
van den Ouweland AM, van Os TA,
Hoogerbrugge N, Gomez Garcia EB,
Dommering CJ, Gundy CM, Aaronson
NK. Psychosocial impact of Von Hippel-
Lindau disease: levels and sources of
distress. Clin Genet 2010;77:483-491
Lammens CRM. Living with Li-Fraumeni
Syndrome & Von Hippel-Landau disease.
Thesis 2010
Nieuwenhuis MH, Douma KF, Bleiker
EM, Bemelman WA, Aaronson NK,
Vasen HF. Female fertility after colorectal
surgery for familial a denomatous
polyposis: a nationwide cross-sectional
study. Ann Surg 2010;252:341-344
Smets EMA, Bleiker E, Esch SCM van.
Erfelijkheidsadvisering. In: Kaptein AA,
Prins JB, Colette EH, Hulsman RL.,
editors. Medische Psychologie. Houten:
Bohn, Stafl eu, Van Loghum, 2010:87-99

98
psychosocial research
Group leader Wim van Harten
Wim van Harten MD PhD Group leader
MJM Hummel PhD Academic staff
L Steuten PhD Academic Staff
Peter van Berkel MSc Research staff
Wineke van Lent MSc Research staff
Valesca Retèl MSc Research Staff
Wim Groen PhD Post-doc
Wilma Kuijpers MSc PhD student
Marloes Sanders Research Assistant
Linda Timmer-Arents Research Assistant
Key publications
Bredenhoff E, Van Lent WAM, Van Harten
WH. Exploring types of focused factories in
hospital care: a multiple case study. BMC
Health Services Research. 2010;10:154
Klopper AHJ, Siesling S, Meerdink
N, Wilderom CPM, Van Harten WH.
Quantifying culture gaps between physicians
and managers in Dutch Hospitals: a survey.
BMC Health Services Research, 2010;10;86
Ploem MC, Retèl VP, Linn SC, Schmidt MK,
van Boven HH, de Jong JP, Gevers JMK, Van
Harten WH. Tumour Tissue: who’s in control?
Lancet Oncol. 2010;9-11
Retèl VP, Joore MA, Knauer M, Linn SC,
Hauptmann M, Van Harten WH. Costeffectiveness
of the 70-gene signature
versus St. Gallen guidelines and Adjuvant
Online for early breast cancer. Eur J Cancer
2010;46:1382-1391
Van Berkel PT, Boucherie RJ, Hans EW,
Hurink JL, Van Lent WAM, Van Harten WH.
An exact approach for relating recovering
surgical patient workload to the master
surgical schedule Journal of the Operational
Research Society, 2010
Van Harten WH, Van Bokhorst L, Van
Luenen HGAM. Benchmarking biology
research organizations using a new, dedicated
tool. Mol Oncol 2010;12-8
Van Lent WAM, De Beer RD, Van Harten
WH. International benchmarking of
specialty hospitals. A series of case studies on
comprehensive cancer centres. BMC Health
Services Research, 2010;10:253
EARLY STAGE TECHNOLOGY ASSESSMENT, OPERATIONS
RESEARCH AND CANCER REHABILITATION
Early stage technology assessment From 2003 through 2006, a technology
assessment study was conducted on the introduction of a 70-gene micro array test
as a prognostic tool in the treatment of node negative breast cancer (the RASTERstudy).
This study is being continued as a side study of the European MINDACTstudy.
As the diffusion of this technology is in an early stage and the course of
development is not easy to predict, an evaluation approach has been chosen that
takes the technology dynamics into account, constructive technology assessment
(CTA). The overall CTA has been completed, an internal guideline on patients’
rights concerning banked tissue has been fi nalised and published, and a scenario
session has been conducted together with the international breast group (BIG).
The results of these scenarios are being used for a cost-effectiveness analysis, the
preliminary results of which were submitted to the Dutch Health Insurance Board
in early 2009. In 2010, the CEA modelling has been completed, including real life
scenarios. A comparison between cost-effectiveness of two genomic breast cancer
prognosis tests, involving compliance of prescribers, was conducted and a paper
on the trade off between investments in research versus further investments in
development is being completed. Additionally, in cooperation with the University
of Twente, we have initiated an early stage technology assessment of TIL-transfer
technology in advanced melanoma. A workpackage on this topic in a EU grant
proposal was drafted. In 2011 a PhD student will start to work on early stage
technology assessment in the application of diagnostic/prognostic markers in neoadjuvant
breast cancer treatment.
Operations improvement in oncology Translating operations management and
research (OM/OR) principles into oncologic care is likely to improve both quality
and effi ciency of hospital processes. A series of international benchmarking projects
has been performed comparing performance of 3 Comprehensive Cancer Centers,
3 ambulatory chemotherapy treatment centres (ACT), 6 fundamental research
organisations and 4 radiotherapy departments. Using the experience of earlier OR
simulation techniques (such as a verifi cation of the effi ciency of the operation room
planning and scheduling) we conducted a study on the use of simulation to attain
a high degree of open access in the radiology department, to reduce the length of
the diagnostic track, including CT, from three weeks to one week, and to reduce the
number of hospital visits. This investigation has resulted in several implementation
proposals and papers. In cooperation with the University of Twente, a PhD student
(Peter van Berkel) is working on a mathematical analysis and scheduling of care
pathways within the oncologic hospital setting, and the effect of increased focus
on effi cient capacity use. A study on contingency of OM interventions in hospitals
was performed in 2010. Another study is ongoing, comparing characteristics of
colorectal surgery pathways using structured effi ciency measures and the national
colorectal quality registry. Together with the University of Twente and the Integraal
Kanker Centrum Noord-Oost, a PhD student was employed on a project to evaluate
the added value of accrediting oncology departments in General Hospitals. In 2011
together with the OECI and as part of the Eurocan Platform project, a PhD will
be employed on the development and evaluation of a system of Accreditation &
Designation of (Excellent) European Cancer Centers.
Rehabilitation, physical activity and cancer Survivorship care and rehabilitation
are important elements of a cancer center’s program. In 2009, we have continued
to strengthen and expand athe infrastructure in which studies in this area can be
conducted. A multidisciplinary rehabilitaiton program was started for breast cancer
survivors receiving adjuvant treatment. In addition, a rehabilitation program for
head-and-neck cancer patients has been approved by health insurers and is expected
will be rolled out end of 2010. Finally, a major Alpe d’Huzes KWF grant was
awarded, focusing on patient empowerment, return to work, tele-monitoring and
implementation of relevant fi ndings and programs related to physical exercise and
supported by innovative IT. This program totalling up to 2,8 million Euro, will start
early 2011.

DIVISION OF DIAGNOSTIC ONCOLOGY
DEPARTMENT OF CLINICAL CHEMISTRY
Neuroendocrine tumours
In collaboration with the Division of Medical Oncology (Babs Taal, Paul Baas),
Pathology (Loes van Velthuysen) and Radiology (Warner Prevoo)
In the event of diffuse hepatic metastases, hepatic artery embolization (HAE)
can be a successful treatment option in patients with well differentiated
neuroendocrine tumours (NET). However, embolization causes hypoxia which
stimulates angiogenesis and therefore tumour growth. we investigated angiogenesis
activity following HAE by measuring vascular endothelial growth factor (VEGF),
endothelin-1 (ET-1) and C-terminal pro-endothelin-1 (proET-1) in blood.
Twelve patients with well differentiated NET and liver metastases underwent HAE.
VEGF, ET-1 and proET-1 were measured before embolization and the days following
treatment during hospitalization. Mean (±SE) VEGF level at baseline was 116 (±33)
ng/l which increased after HAE to 313 (±46) ng/l at day 6, followed by a gradual
decrease. ProET-1 showed a similar pattern, with a mean baseline level of 9.2 (±2.0)
pmol/l and a highest level of 40.8 (±5.7) pmol/l at day 6. Some fl uctuations were
observed for ET-1, with maximum levels at day 3 compared to baseline levels.
We conluded that angiogenic growth factors increase temporary. This implies a
need to investigate the effect of antiangiogenic drugs as an adjuvant therapy to
embolization.
Pro-gastrin-releasing peptide (proGRP) is a recently identifi ed biomarker of small
cell lung cancer (SCLC). In well differentiated neuroendocrine tumours (WDNETs)
we investigated the association between proGRP and tumour characteristics, and the
prognostic value of proGRP levels compared with chromogranin A (CgA) levels.
Serum samples were obtained in 282 patients with WDNET. For proGRP the ROC
curve indicated a cut-off level of 90 ng/l (approximately twice the upper reference
value), with a specifi city of 99% and a sensitivity of 43% in distinguishing primary
pulmonary tumours from other sites. In the multivariate Cox model both proGRP
and CgA were strongly associated with survival (p < 0.0001 for both variables).
We concluded that a high-risk proGRP level (more than twice the upper reference
value) in patients with WDNETs is a strong indication for a primary tumour in the
lung. Besides CgA, proGRP is a complementary tumour marker for prognosis and
treatment monitoring in patients with NET.
Ovarian Cancer
In collaboration with the Division of Surgical Oncology (Marc van Beurden)
CA125 is the most widely used biomarker for ovarian cancer, despite its low
sensitivity and specifi city. We investigated whether HE4 in addition to CA125
can improve the sensitivity and specifi city for diagnosis of ovarian cancer versus
healthy subjects, patients with non-malignant tumors and patients with non-ovarian
malignancies. Additionally, we investigated the prognostic value of CA125 and HE4
for overall survival in ovarian cancer patients. Therefore, we measured CA125 and
HE4 in 180 healthy women, 43 women with benign ovarian disease (BENOD), 101
women with non-ovarian cancer (NOC) and 160 women with ovarian cancer (OC).
The upper reference value was established as 26 kU/l for CA125 and 70 pmol/l for
and HE4. CA125 and HE4 were elevated in 42% and 2% in BENOD patients, in 37%
and 38% in NOC patients and in 89% and 84% in OC patients. Sensitivity for CA125
and HE4 at 95% specifi city for the differentiation between the OC and the BENOD
was 64% and 86%, respectively. For the differentiation between the OC and the
NOC, sensitivity was 63% and 48%, respectively. Univariate Cox regression analyses
show signifi cant associations between CA125 (p = 0.001) and HE4 (p < 0.001)
with survival, while in the multivariate analysis HE4 remained the only signifi cant
prognostic variable for overall survival (p < 0.001).
99
diagnostic oncology
Division head Laura van ’t Veer
(till May 2010)
DEPARTMENT OF CLINICAL
CHEMISTRY
Willem Nooijen PhD Academic staff
Hans Bonfrer PhD Academic staff
Tiny Korse MSc Research associate
Dorothé Linders Technical staff
Marian Buning-Kager Technical staff
DEPARTMENT OF NUCLEAR MEDICINE
Cornelis Hoefnagel MD PhD Academic staff
Philippe Baars MD Academic staff
Fijs van Leeuwen PhD Academic staff
Saar Muller PhD Academic staff
Marc Roef MD PhD Academic staff
Michiel Sinaasappel PhD Academic staff
Ferida Sivro-Prndelj MD Academic staff
Marcel Stokkel MD PhD Academic staff
Renato Valdés Olmos MD PhD Academic
staff
Wouter Vogel MD PhD Academic staff
Tjeerd Aukema MD Clinical research fellow
Oscar Brouwer MD Clinical research fellow
Lenka Vermeeren MD Clinical research
fellow
Joeri Kuil PhD Post-doc
Patrick Chin PhD Post-doc
Anton Bunschoten PhD Post-doc
Jasper Emmering MD Registrar
Daphne Rietbergen MD Registrar
Saskia Baank Technical staff
Martine Bakker Technical staff
Carolien Beers-Bauhuis Technical staff
Natascha Bruin Technical staff
Christel Feenstra Technical staff
Rick Muusers Technical staff
Bert Pool Technical staff
Yvonne Pluister Technical staff
Chelvi Mylvaganan Technical staff
Lyandra Rooze Technical staff
Mariska Sonneborn Technical staff
Colinda Vroonland Technical staff
Tessa Buckle Technical staff
Chantal Beekman Technical staff
Martijn Hoogenboom PhD student
DEPARTMENT OF PATHOLOGY
Hester van Boven MD PhD Head
Olga Balague Ponz MD PhD Academic staff
Frans Hogervorst PhD Academic staff
Daphne de Jong MD PhD Academic staff
Petra Nederlof PhD Academic staff
Renée van Pel MD Academic staff

100
diagnostic oncology
Efraim Rosenberg PhD Academic staff
Marc van de Vijver MD PhD Academic staff
Loes van Velthuysen MD PhD Academic
staff
Laura van ’t Veer PhD Academic staff
Jelle Wesseling MD PhD Academic staff
Bart van de Wiel MD PhD Academic staff
Lucie Boerrigter-Barendsen Technical staff
Aafke Wieringa-Ariaens Technical staff
Henrique Ruijter-Schippers Technical staff
Esther Scheerman Technical staff
Carla Schippers-Gillissen Technical staff
Ivon Tielen Technical staff
Miranda van Dongen Technical staff
Suzan van Gerwen Technical staff
THE NETHERLANDS CANCER
INSTITUTE FAMILY CANCER CLINIC
Senno Verhoef MD PhD Head
Frans Hogervorst PhD Academic staff,
head Diagnostic Laboratory PFT
Efraim Rosenberg Academic staff
Laura van ’t Veer PhD Academic staff
Irma Kluijt MD Academic staff
Marielle Ruijs MD PhD Academic staff
Lizet van der Kolk MD PhD Academic staff
Eveline Bleiker PhD Academic staff
Daniela Hahn Academic staff
Petra Nederlof PhD Academic staff
Sophie van der Velden Genetic associate
Anja van Rens Genetic associate
Gea Wigbout Genetic associate
Philip Romer Genetic associate
Daoud Ait Moha Research assistant
Mohamed Achachah Technical staff
Rob Plug Technical staff
Roelof Pruntel Technical staff
Majella Boutmy-de Lange Technical staff
Esther Scheerman Technical staff
Mobien Kasiem Technical staff
Abderrahim Ajouaou Technical staff
DEPARTMENT OF RADIOLOGY
Jelle Teertstra MD Head
Peter Besnard MD Academic staff
Kenneth Gilhuijs PhD Academic staff
Wim Koops MD Academic staff
Charlotte Lange Academic staff
Robert Kröger MD Academic staff
Claudette Loo MD Academic staff
Saar Muller PhD Academic staff
Frank Pameijer MD PhD Academic staff
Warner Prevoo MD Academic staff
Michiel Sinaasappel PhD Academic staff
Fijs van Leeuwen PhD Academic staff
Ingar Seemann PhD student
Patrick Chin PhD Post-doc
Saske Hoving PhD Post-doc
Joeri Kuil PhD Post-doc
Anton Bunschoten PhD Post-doc
Edoardo Pasca PhD Post-doc
In this study HE4 seems to be a helpful tool for the discrimination between benign
or malignant ovarian disease, however CA125 seems to be more helpful when
ovarian cancer is compared with other non-ovarian cancers. CA125 and HE4 have
comparable sensitivities and specifi cities in ovarian cancer patients compared with
healthy women. HE4 might be a more important prognostic marker for survival
than CA125.
Non-small cell lung cancers
In collaboration with the Division of Medical Oncology (Eva Schaake)
Retrospectively, we measured in 55 patients with amphiregulin, soluble EGFR,
TGF- , IGF-1 and IGFBP-3 before and during treatment of erlotinib. The aim was
to investigate if these biomarkers were potential tools to determine objective tumor
responses.
Biobank
In collaboration with the Division of Experimental Therapy (Marjanka Schmidt, Annegien Broeks)
We started with the biobank for storage of serum and full blood. The aim is to
ask an informed consent of every new patient to collect 2 tubes of blood for future
investigations in a broad research area. The approval and logistics are under
responsibility of the Core Facility - Molecular Pathology and Biobank.
Pharmacological studies in mice
Lin Fan, Levi Buil, Mark de Gooijer, Olaf van Tellingen
The main focus of our work is on the development and implementation of in
vivo models for improving therapies against high-grade glioma (GBM). A major
part of our work is dedicated to defi ne the role of the blood-brain barrier (BBB)
in the chemo-resistance of GBM with special emphasis on the role of drug
transporting proteins. Several ABC transporters (e.g. ABCB1, ABCG2, ABCC1-5)
are expressed at the BBB and we are investigating their functional importance
using ABC transporter knockout mouse (KO) models. Because of overlapping
substrate specifi cities, the use of compound knockouts is essential. By using
Abcb1;Abcg2;Abcc4 vs Abcb1;Abcg2 KO mice we have now found that ABCC4
(Mrp4) limits the brain entry of topotecan and irinotecan / SN38 and methotrexate.
This could not be observed in Abcc4 single knockout mice because Abcb1 and Abcg2
are already suffi cient to keep them from penetrating the BBB. We also found that the
BBB penetration of the PARP inhibitor ABT-888 is reduced by 7-fold by Abcb1 and
Abcg2. This fi nding may have important clinical implications as PARP inhibition
Figure 1: High-grade gliomas arising in Pten conditional knockouts (Pten (-/-)) have more intense
staining for Ser473Akt (indicative of an activated PI3K-mTor signaling pathway) and Pten (-/-) tumors
grow much more rapidly than Pten(+/+) tumors (follow up by bioluminescence imaging). This
dependence on Pten makes this model suitable for testing the effi cacy of PI3K-mTor signaling pathway
inhibitors.

as a strategy to enhance the effi cacy of DNA damaging therapies is receiving
considerable interest for treatment of GBM. Clearly, agents that are substrates of
ABC-transporters may not be the best candidates for this disease.
Next to our work with ABC-transporter KO models, we have also developed a set
of spontaneous high-grade glioma mouse models based on Cre-loxP conditional
transgenic mice. Grade III and IV tumors arise in conditional Kras V12 ;Ink4a/
Arf, Kras V12 ;Ink4a/Arf;P53 and Kras V12 ;Ink4a/Arf;Pten mice after stereotactic
intracranial injection of a self-deleting Cre lentivirus. These models may be helpful
to investigate the effi cacy of agents targeting specifi c GBM driving pathways, such as
the PI3K-mTor signaling pathway (fi gure 1). We also found that although all tumors
respond to temozolomide, tumors lacking p53 were most responsive. Concomitant
administration of elacridar, an inhibitor of ABCB1 and ABCG2 increased the
response; in line with our previous fi ndings that temozolomide is a substrate of
these transporters. We are now using this model to demonstrate the impact of ABCtransporters
on treatment of gliomas with temozolomide and ABT-888.
DEPARTMENT OF NUCLEAR MEDICINE
Clinical Nuclear Medicine
P Baas, MJ Baas-Vrancken Peeters, A Bex, J Belderbos, NS van den Berg, JP de Boer, S Burgers, K
Gilhuijs, N Graafl and, J van der Hage, M van den Heuvel, S Horenblas, L Josephson, I Kappers,
H Klomp, C Lange, C Loo, W Meinhardt, OE Nieweg, H Oldenburg, IM van der Ploeg, W. Prevoo,
HG van der Poel, ET Rutgers, EE Schaake, M Verheij, B Taal, J Teertstra, A Velders, M Wouters,
T Aukema, PC Baars, NS van den Berg, O Brouwer, T Buckle, A Bunschoten, CA Hoefnagel,
Hoogenboom M, J Kuil, FWB van Leeuwen, SH Muller, M Roef, F Sivro, M Sinaasappel, M Stokkel,
RA Valdés Olmos, S Vidal Sicart, L Vermeeren, WV Vogel
Both PET-CT and SPECT-CT were evaluated for various clinical applications using
existing and recently introduced tracers. Also the application of new small medicine
devices for radioguided and fl uorescence imaging were further validated as well as
the evaluation of a mini PET ring device for dedicated breast molecular imaging.
At the Annual European Nuclear Medicine Congress held in Vienna the NKI-AVL
received the Eckert & Ziegler Abstract Award 2010 (T Aukema).
SPECT-CT in sentinel node identification In seven patients with clinically stage
I testicular cancer SPECT-CT identifi ed in all patients. When tracer was injected in
the right testicle sentinel nodes were located between aorta and cava, in one patient
together with a sentinel node adjacent to the testicular vessels. In patients with left
testicular cancer SPECT-CT identifi ed para-aortic sentinel nodes in 2 patients and in
one along the testicular vessels. A sentinel node contained metastases in 1 patient.
No recurrences developed in the 6 patients with a negative sentinel node during a
median follow-up of 31 months.
Lymphatic drainage using SPECT-CT was evaluated in 10 treated prostate cancer
patients (4 external beam radiation, 4 brachytherapy, 2 high-intensity-focusedultrasound)
after transrectal ultrasound guided tracer injection into the prostate. In
comparison with 70 untreated patients more sentinels nodes outside the pelvis (80%
vs, 34%, p=0.01) were localized (para-aortic, pre-sacral, inguinal or near the ventral
abdominal wall) in treated patients. In these patients 95% of the sentinel nodes
could be harvested and half of the patients had at least one positive sentinel node on
pathological examination.
Lymphatic mapping in mutifocal/multicentric breast cancer A multicentric
study in patients with multiple invasive tumors in one or more quadrants of the
breast was started. After a fi rst tracer injection in the largest tumor lymphatic
mapping was established using lymphoscintigraphy and SPECT-CT. Subsequently,
a second injection of the tracer followed by the exact same imaging protocol was
performed for the other tumors. In the fi rst evaluation involving 24 patients from
4 centers in the Netherlands additional lymphatic drainage was depicted after the
second and/or third injection in 71%. In 4 patients with a tumour-positive node in
the axilla that was visualized after the fi rst injection, an additionally axillary involved
node was found after subsequent injection. In one patient, isolated tumor cells
101
diagnostic oncology
Wen Qi PhD Post-doc
Christian Siedschlag PhD Post-doc
Lukas Batteau Technical staff
Tessa Buckle Technical staff
Anita Paape Technical staff
Maddalena Rossi Technical staff
Chantal Beekman Technical staff
Wei Chen PhD student
Lotte Elshof PhD student
Lotte Lutkenhaus PhD student
Kenneth Pengel PhD student
Alexander Schmitz PhD student
Annemarie Schmitz PhD student
Kirsten Zuurmond PhD student
Publications
Alderliesten T, Loo C, Paape A, Muller
S, Rutgers E, Peeters MJ, Gilhuijs K. On
the feasibility of MRI-guided navigation to
demarcate breast cancer for breast-conserving
surgery. Med Phys. 2010;37:2617-26
Alderliesten T, Loo CE, Pengel KE,
Oldenburg HSA, Rutgers EJTh, Gilhuijs KGA,
Vrancken Peeters MTFD. Radioactive Seed
Localization of Breast Lesions: an Adequate
Localization Method without Seed Migration.
The Breast Journal 2010 (in press)
Antoniou AC, Wang X, Fredericksen ZS and
other authors. A locus on 19p13 modifi es
risk of breast cancer in BRCA1 mutation
carriers and is associated with hormone
receptor-negative breast cancer in the general
population. Nat Genet. 2010;42:885-92
Antoniou AC, Sinilnikova OM, McGuffog
L and other authors. Common variants in
LSP1, 2q35 and 8q24 and breast cancer risk for
BRCA1 and BRCA2 mutation carriers. Hum
Mol Genet. 2009;15;18:4442-56
Aukema TS, Kappers I, Olmos RA,
Codrington HE, Van Tinteren H, van Pel
R, Klomp HM. Is 18F-FDG PET/CT useful
for the early prediction of histopathologic
response to neoadjuvant erlotinib in patients
with non-small cell lung cancer? J Nucl Med
2010;51:1344-8
Aukema TS, Kappers I, Valdés Olmos RA,
Codrington HE, van Tinteren H, van Pel R,
Klomp HM; NEL Study Group. Is 18F-FDG
PET/CT useful for the early prediction of
histopathologic response to neoadjuvant
erlotinib in patients with non-small cell lung
cancer? J Nucl Med. 2010;51:1344-8

102
diagnostic oncology
Publications (continued)
Aukema TS, Olmos RA, Korse CM, Kroon
BB, Wouters MW, Vogel WV, Bonfrer JM,
Nieweg OE. Utility of FDG PET/CT and
brain MRI in melanoma patients with
increased serum S-100B level during follow-up.
Ann Surg Oncol 2010;17:1657-61
Aukema TS, Rutgers EJ, Vogel WV, Teertstra
HJ, Oldenburg HS, Vrancken Peeters MT,
Wesseling J, Russell NS, Valdés Olmos
RA. The role of FDG PET/CT in patients
with locoregional breast cancer recurrence:
a comparison to conventional imaging
techniques.Eur J Surg Oncol. 2010;36:387-92
Aukema TS, Straver ME, Peeters MJ, Russell
NS, Gilhuijs KG, Vogel WV, Rutgers EJ,
Olmos RA. Detection of extra-axillary lymph
node involvement with FDG PET/CT in
patients with stage II-III breast cancer. Eur J
Cancer. 2010
Aukema TS, Valdés Olmos RA, Korse CM,
Kroon BB, Wouters MW, Vogel WV, Bonfrer
JM, Nieweg OE. Utility of FDG PET/CT
and brain MRI in melanoma patients with
increased serum S-100B level during follow-up.
Ann Surg Oncol. 2010;17:1657-61
Aukema TS, Valdés Olmos RA, Wouters
MW, Klop WM, Kroon BB, Vogel WV, Nieweg
OE. Utility of preoperative 18F-FDG PET/CT
and brain MRI in melanoma patients with
palpable lymph node metastases. Ann Surg
Oncol. 2010;17:2773-8
Aukema TS, Vogel WV, Hoefnagel CA, Valdés
Olmos RA. Prevention of brown adipose tissue
activation in 18F-FDG PET/CT of breast
cancer patients receiving neoadjuvant systemic
therapy. J Nucl Med Technol. 2010;38:24-7
Axwijk P, Kluijt I, de Jong D, Gille JJP,
Teertstra HJ, Horenblas S. Hereditary
causes of kidney tumours. Eur J Clin Invest
2010;40:433-9
Beekman C, Buckle T, van Leeuwen AC,
Rottenberg S, Verheij M, van Leeuwen FWB,
“The value of 99mTc-HYNIC-annexin V
based response monitoring after docetaxel
treatment”, Applied Radiation and isotopes
2010 (accepted)
Beumer JH, Franke NE, Tolboom R, Buckle
T, Rosing H, Lopez-Lazaro L, Schellens JH,
Beijnen JH, van Tellingen O. Disposition and
toxicity of trabectedin (ET-743) in wild-type
and mdr1 gene (P-gp) knock-out mice. Invest
New Drugs. 2010;28:145-55
were found in both an axillary and an additionally found internal mammary chain
sentinel node.
Sentinel node detection using portable imaging devices The use of a portable
gamma camera for sentinel node identifi cation during laparoscopy was evaluated
in 55 patients with prostate cancer. The detector of the camera was placed above
the pelvis and localization of sentinel nodes was guided by a 125 I seed, put on the
collimator of a laparoscopic probe, used as a pointer. Out of 178 sentinel nodes
visualized on SPECT-CT 16 nodes (9%) could not be reproduced with the portable
gamma camera because of a weak signal and 13 (7%) because of their location in
close proximity with the site of the tracer injection in the prostate. In 15 patients the
125 I seed pointer was useful to directly localize nodes (principally along the aorta
and common iliac artery cranial from ureter crossing) and in the other patients the
portable gammacamera was used to verify adequate excision after removal of the
sentinel node leading to the identifi cation of 17 additional sentinel nodes. In two of
these cases sentinel nodes were tumor-positive.
The portable gamma camera was also evaluated for preoperative sentinel node
detection in breast cancer patients in a study performed in cooperation with the
Hospital Clinic of the University of Barcelona. When a lead shield was used to
mask the injection area sentinel node visualization rate was 88% (vs. 95% for the
conventional gamma camera) in a group of 43 patients. This device may enable to
perform preoperative sentinel node imaging and lymphatic mapping in centers
without conventional nuclear medicine equipment.
More recently a freehand SPECT probe was introduced to assist radioguided
excision of breast tumors (ROLL), in some cases combined with sentinel node
lymphadenectomy. The principal advantage of the freehand SPECT probe in
comparison with the standard probe is the generation of 3D images in addition to
the acoustic signals. Up to far 5 patients have been successfully evaluated.
Surgical fl uorescence and radioactive guidance towards the sentinel lymph node
One clinical precedent for the use of nano-sized imaging agents is the localization
of the tumor draining sentinel lymph nodes. In this application, radiocolloids
such as 99m Tc-nanocolloid are currently used to plan the surgical procedure and
to provide acoustic guidance during the intervention. Additional injections of
dyes are common to provide optical surgical guidance. Bimodal imaging agents,
which are both radioactive and fl uorescent, have the potential to be used for both
surgical planning and intraoperative fl uorescence guidance towards the sentinel
lymph nodes. A bimodal ICG- 99m Tc-nanocolloid complex was generated and its
Figure 2: Intraoperative sentinel node detection using a bimodal ICG-99mTc-nanocolloid tracer.
The portable gamma camera indicates the site of the sentinel node in the right side of the neck (A, B) for
incision planning. After skin incision the sentinel node is visualized using a fl uorescence camera (C, D).

effi cacy was evaluated in preclinical breast and prostate related SLN procedures.
Subsequently the particle stability was validated in patients and the value of this
approach in surgical fl uorescence guidance towards sentinel nodes of the prostate is
currently being evaluated. Optical guidance for sentinel node detection potentially
improves accuracy and reduces damage to non-target tissue surrounding the nodes.
Eventually, this research line should provide a rapid translation from bench to ORbedside.
A reproducibility study with this novel tracer has also been started. After a fi rst
injection of 99m Tc-nanocolloid both lymphoscintigraphy and SPECT were acquired
in order to localize sentinel nodes. Subsequently, a second injection of bimodal ICG-
99m Tc-nanocolloid complex followed and the exact imaging protocol was performed.
In 5 patients with head and neck melanoma a similar pattern of lymphatic drainage
was observed without additional sentinel nodes after the second injection. The
bimodal tracer enabled intraoperative fl uorescence imaging of all radioactive
sentinel nodes (fi gure 2). A similar correlation was observed when the excided
sentinel nodes were measured. The study aims to include patients with melanoma,
penile cancer, and breast cancer.
Imaging agents for tumor specific surgical guidance The use of monolabeled
tumor-targeting peptides for molecular imaging is wide-spread. However, it is often
desirable to use the same compound for different clinical applications, e.g. combined
pre- and intraoperative tumor detection. Based on their detection sensitivity, the
combination of radioactivity and fl uorescence is probably the most valuable in
multimodal molecular imaging. Next to generating multimodal imaging agents for
non-tumor specifi c procedures such as the detection of the SLN (see above), we have
developed a number of multimodal imaging agents that specifi cally target tumor
tissue via e.g. the CXCR4 or a vb 3 integrin over expression. These imaging agents are
validated in vitro and in the preclinical setting can be used for in vivo imaging and
surgical guidance toward the tumor.
Figure 3: Schematic representation of targeted
multimodal imaging agents and their application
in surgical guidance. Figure is published in
Bioconjugate Chem. 2010, 21, 1709.
Breast cancer imaging with a small PET ring camera The experience with the
fi rst prototype of a small PET ring camera for dedicated breast imaging was further
extended in stage II-III breast cancer patients (European project MAMMI EU-
037555). The results of this dedicated high resolution system were compared to the
PET-CT images in relation to tumor detectability and location in the breast. FDGavide
primary tumors were visualized in 19 of 20 consecutive patients (95%). In one
patient no primary tumor was visible on FDG PET-CT nor on the dedicated breast
PET (nor on MRI). On the basis of the hanging breast position the small PET ring
camera was able to visualize tumors close to the thoracic wall. The median distance
from the primary tumor to the pectoral muscle was 18 mm (range 10-52 mm).
Evaluation of the results for therapy response is ongoing.
PET-CT in breast cancer To establish the added value of prone position in
30 consecutive patients with breast cancer scheduled to receive neoadjuvant
103
diagnostic oncology
Publications (continued)
Bex A, Vermeeren L, de Windt G, Prevoo
W, Horenblas S, Olmos RA. Feasibility of
sentinel node detection in renal cell carcinoma:
a pilot study.Eur J Nucl Med Mol Imaging.
2010;37:1117-23
Blows FM, Driver KE, Schmidt MK, Broeks
A, van Leeuwen FE, Wesseling J, Cheang
MC, Gelmon K, Nielsen TO, Blomqvist
C, Heikkila P, Heikkinen T, Nevanlinna H,
Akslen LA, Begin LR, Foulkes WD, Couch
FJ, Wang X, Cafourek V, Olson JE, Baglietto
L, Giles GG, Severi G, McLean CA, Southey
MC, Rakha E, Green AR, Ellis IO, Sherman
ME, Lissowska J, Anderson WF, Cox A, Cross
SS, Reed MW, Provenzano E, Dawson SJ,
Dunning AM, Humphreys M, Easton DF,
Garcia-Closas M, Caldas C, Pharoah PD,
Huntsman D. Subtyping of breast cancer
by immunohistochemistry to investigate a
relationship between subtype and short and
long term survival: a collaborative analysis
of data for 10,159 cases from 12 studies. PLoS
Med 2010;7
Bruin SC, Verwaal VJ, Vincent A, van’t Veer
LJ, van Velthuysen ML. A clinicopathologic
analysis of peritoneal metastases of colorectal
and appendiceal origin. Ann Surg Oncol
2010;17:2330-40
Buckle T, Chin PT, van den Berg NS, Loo
CE, Koops W, Gilhuijs KG, van Leeuwen
FW. Tumor bracketing and safety margin
estimation using multimodal marker seeds: a
proof of concept. J Biomed Opt. 2010;15:056021
Buckle T, Chin PT, van Leeuwen FW. (Nontargeted)
radioactive/fl uorescent nanoparticles
and their potential in combined pre- and
intraoperative imaging during sentinel
lymph node resection. Nanotechnology.
2010;21:482001
Buckle T, van Leeuwen AC, Chin PT, Janssen
H, Muller SH, Jonkers J, van Leeuwen FW.
A self-assembled multimodal complex for
combined pre- and intraoperative imaging
of the sentinel lymph node. Nanotechnology.
2010;21:355101
Buckle T, van Leeuwen FW. Validation of
intratracheal instillation of lung tumour cells
in mice using single photon emission computed
tomography/computed tomography imaging.
Lab Anim. 2010;44:40-5

104
diagnostic oncology
Publications (continued)
Bueno-de-Mesquita JM, Nuyten DS,
Wesseling J, Van Tinteren H, Linn SC, van
de Vijver MJ. The impact of inter-observer
variation in pathological assessment of
node-negative breast cancer on clinical risk
assessment and patient selection for adjuvant
systemic treatment. Ann Oncol 2010;21:40-7
Chin PTK, Buckle T, Aguirre de Miguel A,
Meskers SCJ, Janssen RAJ, van Leeuwen
FWB. Dual-emissive quantum dots for
multispectral intraoperative fl uorescence
imaging. Biomaterials. 2010;31:6823
Courrech Staal EF, Aleman BM, Boot H, van
Velthuysen ML, Van Tinteren H, van Sandick
JW. Systematic review of the benefi ts and risks
of neoadjuvant chemoradiation for oesophageal
cancer. Br J Surg 2010;97:1482-96
Courrech Staal EF, Aleman BM, van
Velthuysen ML, Cats A, Boot H, Jansen
EP, van Coevorden F, van Sandick JW.
Chemoradiation for Esophageal Cancer:
Institutional Experience With Three Different
Regimens. Am J Clin Oncol 2010
Courrech Staal EF, Smit VT, van Velthuysen
ML, Spitzer-Naaykens JM, Wouters MW,
Mesker WE, Tollenaar RA, van Sandick
JW. Reproducibility and validation of
tumour stroma ratio scoring on oesophageal
adenocarcinoma biopsies. Eur J Cancer 2010
de Jong D, Janz S. Anaplastic plasmacytoma
of mouse--establishing parallels between
subtypes of mouse and human plasma cell
neoplasia. J Pathol 2010;221:242-7
de Jong MC, Pramana J, van der Wal JE,
Lacko M, Peutz-Kootstra CJ, de Jong JM,
Takes RP, Kaanders JH, van der Laan BF,
Wachters J, Jansen JC, Rasch CR, van
Velthuysen ML, Grenman R, Hoebers FJ,
Schuuring E, van den Brekel MW, Begg AC.
CD44 expression predicts local recurrence after
radiotherapy in larynx cancer. Clin Cancer
Res 2010;16:5329-38
De Jong D, Balagué Ponz O The molecular
background of aggressive B-cell lymphomas
as a basis for targeted therapy. J Pathol 2010
(in press)
De Jong, D, Fest T. Best practice and research
in clinical haematology 2010: Follicular
non-Hodgkin lymphoma. Chapter 4: the
microenvironment in follicular lymphoma.
2010 (in press)
chemotherapy 18 F-FDG PET-CT images obtained in prone position (hanging breast
technique) were compared with those obtained in supine position. In 24 patients
the same number of FDG-avide lesions was observed. In 6 patients more FDG-avide
lymphatic nodes were seen on prone images (in 5 in the axilla and in 1 in the breast).
Based on the comparison of the areas of FDG uptake the prone position technique
achieved higher lesion visualization as well as higher SUVmax values.
PET-CT in lung cancer After the evaluation of the role of FDG PET-CT for early
identifi cation of response to neoadjuvant erlotinib, a second study evaluated
PET-CT and CT in 26 patients with NSCLC eligible for surgical resection. The
patients received preoperatively 150 mg erlotinib once daily for 3 weeks. There was
no apparent relation between the relative difference on CT and the percentage
of necrosis in the excided specimen. By contrast in 8 patients showing a partial
metabolic response on FDG PET-CT the mean percentage of necrosis was 57%
whereas in 18 patients without metabolic response (15 stable and 3 progressive) the
mean percentage was 28%.
FDG PET-CT was involved for assessment of response in a feasibility study
combining cetuximab with concurrent chemoradiotherapy in patients with NSCLC.
Early response monitoring showed a partial metabolic response in 8 out of 10
patients.
New PET tracers The novel proliferation tracer Fluor-18-choline was introduced as
a tool for restaging of prostate cancer with PET, to select patients for local aggressive
salvage treatment or systemic palliative treatment, with. As a novel imaging
modality, the biological parameter infuencing the image quality and detectability
of tumor locations needed to be elucidated. It was determined that physical muscle
exercise had detrimental effects and must be avoided prior to imaging. In addition, it
was determined that therapeutic pharmaceuticals infl uencing the choline pathways,
including colchicine but potentially also taxanes, can disrupt choline metabolism
and should be avoided prior to imaging.
In collaboration with the pharmacy department, the new radiopharmaceutical
Gallium-68-citrate was developed for bloodpool imaging. Ga-citrate binds to
transferrine after intravenous administration and then largely remains in the
circulation, and allows for imaging and quantifi cation of the blood pool in vivo. This
new detection tool has been applied succesfully for 3-dimensional quantifi cation
of left ventricular ejection fraction, with a good image quality and correlation with
phantom measurements. Subsequently, the technique was applied to quantify
the total blood volume in tumor tissue as a parameter of tumor perfusion, and to
quantify extravasation as a parameter of local vascular permeability for transferrine
in tumor tissue. The parameters of tumor bloodpool and vascular permeability are
now available for tumor characterization, and for specifi c response monitoring of
treatment targeted at (neo)vascularization in clinical trials.
SPECT-CT in neuroendocrine tumors In 97 patients somatostatin receptor
scintigraphy (SRS) was retrolectively evaluated in staging and restaging patients
with a carcinoid tumor. The results were compared with Chromogranin-A (CgA)
measurements, demonstrating a higher sensitivity for SRS, but a lower specifi city
than CgA. In about a quarter of the patients, SRS revealed tumor activity in the
absence of elevated CgA levels. The number of lesions scored on the SRS correlated
with the CgA level, but it is not possible to use the CgA levels to subdivide patients
into limited or extensive disease due to the overlap over values. In this study, a new
scorings system was introduced in which the number of lesions and the intensity of
uptake was combined in a score ranging from 0 (=no uptake) to 4 (= intense uptake
in multiple lesions). This scorings system is not of value in staging or restaging
settings, but it might be of value in prognostic stratifi cation, which is part of a new
study.
DEPARTMENT OF PATHOLOGY
(Part of the research of the Pathology Department is carried out within Division of
Experimental Therapy and is described there)

MOLECULAR PATHOLOGY
EGFR mutation in lung carcinomas and response to Iressa therapy
Daphne de Jong, Petra Nederlof, Erik Thunissen, Michel van den Heuvel, Marieke Vollebergh,
Henrique Ruijter, Ivon Tielen, Lucie Boerrigter, Aafke Wieringa-Ariaens
Gefi tinib (Iressa, ZD1839), an inhibitor of epidermal growth factor receptor-tyrosine
kinase (EGFR-TK), has shown potent anti-tumor effects and improved symptom
and quality-of-life of a subset of patients with advanced NSCLC and a mutation in
exon 18, 19, 20 or 21 of the EGFR gene (Tyrosine kinase domain). We have evaluated
specimens of 300 iressa treated patients of a large series of NSCLC from our
institute and the VU medical centre (Dr Erik Thunissen). We correlated response
status with tumour characteristics such as EGFR and KRAS mutation status and
EGFR gene amplifi cation (CISH).
Molecularly supported differentiation models in liposarcoma with
consequences for diagnostic and clinical management
Ronald de Vreeze, Henrique Ruijter, Ivon Tielen, Lucie Boerrigter, Aafke Ariaens,
Petra Nederlof, Rick Haas, Frits van Coevorden, Daphne de Jong
Liposarcoma can be distinguished in well-defi ned disease entities, characterized
by morphology and in part by genetic alterations as well-differentiated (WDLS),
de-differentiated (DDLS), myxoid, round cell (MRLS), and pleomorphic (PLS). WLS
and DDLS are characterized by amplifi cation of the 12q13-15 region including MDM2
and CDK4 genes. MRLS is characterized by a t(12;16) or t(12;22) translocation.
These classifying markers have been used to explore several clinical and biological
aspects of these diseases and their interrelations. In rare instances, mixed-type
liposarcomas with components of MRLS and WDL are seen. On the basis of MRI
features, these cases were collected from the fi les of the NKI-AVL. Molecular
analysis showed that these mixed type liposarcomas should not be regarded as
collision tumors, but as an extreme variant of the morphological spectrum within
a single biological entity. These fi ndings support a stem cell model with different
levels of maturation within specifi c disease entities and expands the morphological
spectrum of MRLS. This stem cell model with tumor progression from intermediate
stages of development was further studied in a large series covering the spectrum
from lipoma to high grade pleiomorphic liposarcoma using immunohistochemistry
and MLPA. Amplifi cation of 12q and in low-grade cases also expression of the target
genes CDK4 and MDM2 in a subset of PLS as well as in in WDLSand DDLS further
supports this model.
Tumor microenvironment in malignant lymphoma
Jan Paul de Boer, Martijn Kerst, Joke Baars, Vanessa Geldhof, Olga Balagué Ponz,
John Raemaekers, Lunenburg Lymphoma Biomarker Consortium, Daphne de Jong
Over the past few years, it has become clear that the biological and clinical behavior
of malignant lymphoma is not only determined by the properties of the tumor
cells themselves, but are also largely by the interaction of the tumor cells with their
non-malignant microenvironment. The composition and functional status of the
tumor microenvironment is highly variable between different classes of malignant
lymphoma and may provide both growth-supportive and growth suppressive signals
via components of the adaptive and innate immune response. We have been mostly
involved in studies in this fi eld in follicular lymphoma (FL) and gastric marginal
zone lymphoma, MALT-type. The precise nature of the cell populations in FL is still
unclear and published data on their prognostic signifi cance are highly confl icting.
In part, this si due to the very poor reproducibility of immunohistochemical
methods to score as were able to show in an international validation study. On the
other hand, varying reported results may be due to heterogeneous composition
of both patient series and treatments. Therefore, we explored this feature in an
immunohistochemical study in patients treated in an EORTC/BNLI trial comparing
fl udarabine to CVP chemotherapy. Most importantly, some T-cell and accessory
cell populations showed an homogeneous prognostic impact, while others had a
105
diagnostic oncology
Publications (continued)
de Ronde J, Wessels L, Wesseling J.
Molecular subtyping of breast cancer: ready to
use? Lancet Oncol 2010;11:306-7
de Ronde JJ, Hannemann J, Halfwerk H,
Mulder L, Straver ME, Vrancken Peeters
MJ, Wesseling J, Van De Vijver M, Wessels
LF, Rodenhuis S. Concordance of clinical
and molecular breast cancer subtyping in the
context of preoperative chemotherapy response.
Breast Cancer Res Treat 2010;119:119-26
de Vreeze R, de Jong D, Nederlof P, Ruijter
HJ, Boerrigter L, Haas R, van Coevorden F.
Multifocal myxoid liposarcoma-metastasis or
second primary tumor?: a molecular biological
analysis. J Mol Diagn. 2010;12:238-43
de Vreeze RS, de Jong D, Koops W, Nederlof
PM, Ariaens A, Haas RL, van Coevorden F.
Oncogenesis and classifi cation of mixed-type
liposarcoma: A radiological, histopathological
and molecular biological analysis. Int J
Cancer. 2010
de Vreeze RS, de Jong D, Nederlof PM,
Ariaens A, Tielen IH, Frenken L, Haas RL,
van Coevorden F. Added Value of Molecular
Biological Analysis in Diagnosis and Clinical
Management of Liposarcoma: A 30-Year
Single-Institution Experience. Ann Surg
Oncol. 2010;17:686-93
de Vreeze RSA. Liposarcoma: from biology
to therapy. Amsterdam: University of
Amsterdam, 2010
de Vries NA, Bruggeman SW, Hulsman D, de
Vries HI, Zevenhoven J, Buckle T, Hamans
BC, Leenders WP, Beijnen JH, van Lohuizen
M, Berns AJ, van Tellingen O. Rapid and
robust transgenic high-grade glioma mouse
models for therapy intervention studies. Clin
Cancer Res. 2010;16:3431-41
de Vries NA, Buckle T, Zhao J, Beijnen JH,
Schellens JH and van Tellingen O. Restricted
brain penetration of the tyrosine kinase
inhibitor erlotinib due to the drug transporters
P-gp and BCRP. Invest New Drugs, 2010
de Zwart IM, de Roos A. MRI for the
evaluation of gastric physiology. Eur Radiol.
2010 Nov;20(11):2609-16. Erratum in: Eur
Radiol. 2010;20:2617
Dik VK, van de Wiel BA, Vasmel WL.
Kimura’s disease of the parotid glands and
multiple cervical lymph nodes. Neth J Med
2010;68:175-7

106
diagnostic oncology
Publications (continued)
Douma KFL, Aaronson NK, Vasen HFA,
Gerritsma MA, Gundy CM, Janssen EPA,
Vriends AHJT, Cats A, Verhoef S, Bleiker
EMA. Psychological distress and use of
psychosocial support in familial adenomatous
polyposis. Psychooncology 2010;19:289-98
Douma KF, Aaronson NK, Vasen HF, Verhoef
S, Gundy CM, Bleiker EM. Attitudes toward
genetic testing in childhood and reproductive
decision-making for familial adenomatous
polyposis. Eur J Hum Genet. 2010;18:186-93.
Elshof LE, Rutgers EJ, Deurloo EE, Loo
CE, Wesseling J, Pengel KE, Gilhuijs KG.
A practical approach to manage additional
lesions at preoperative breast MRI in patients
eligible for breast conserving therapy: results.
Breast Cancer Res Treat. 2010;124:707-15
Engel C, Versmold B, Wappenschmidt B and
other authors. Association of the variants
CASP8 D302H and CASP10 V410I with
breast and ovarian cancer risk in BRCA1 and
BRCA2 mutation carriers. Cancer Epidemiol
Biomarkers Prev. 2010;19:2859-68
Femme Harinck, Canto MI, Schulick R,
Goggins M, Poley JW, Fockens P, Kluijt I,
Marco Bruno MJ. Surveillance in individuals
at hig risk for pancreatic cancer: too early to
tell? Gut 2010;59:1005
Fles R, Hoogendoorn WE, Platteel I,
Scheerman CE, de Leeuw-Mantel G, Mourits
MJE, Hollema H, van Leeuwen FE, van
Boven HH, Nederlof PM. Genomic profi le of
endometrial tumors depends on morphological
subtype, not on tamoxifen exposure Genes,
Chromosomes and Cancer 2010;49;8:699-710
Gaudet MM, Kirchhoff T, Green T and
other authors. Common genetic variants
and modifi cation of penetrance of BRCA2associated
breast cancer. PLoS Genet.
2010;6:e1001183
Graafl and NM, Valdes Olmos RA, Meinhardt
W, Bex A, van der Poel HG, van Boven HH,
Nieweg OE, Horenblas S. Nodal Staging in
Penile Carcinoma by Dynamic Sentinel Node
Biopsy After Previous Therapeutic Primary
Tumour Resection. Eur Urol 2010
Graafl and NM, Valdés Olmos RA, Teertstra
HJ, Kerst JM, Bergman AM, Horenblas S.
18F-FDG PET/CT for monitoring induction
chemotherapy in patients with primary
inoperable penile carcinoma: fi rst clinical
results. Eur J Nucl Med Mol Imaging.
2010;37:1474-80
different and sometimes opposite effect in the treatment arms. Within the NKI-AVL,
this feature is now further explored by FACS analysis on pre- and post treatment
cytological aspirates in FL patients (TAMIL study) and the protocol is currently
activated in various other centers in the Amsterdam region.
Gastric marginal zone lymphoma, MALT-type is a prime example of an
immunologically driven disease in which growth support of the immune
microenvironment is essential in the earlier phases of the disease. This notion has
been the rationale of a clinical study protocol to treat patients with the nucleoside
analog fl udarabine that is directed towards both the tumor B-cells, but also to the
T-cell compartment. Alterations in T-cell subsets were studied in peripheral blood
samples and in sequential gastric biopsies before and after treatment. Treatment
with oral fl udarabine was effective in patients with gastric MALT lymphoma (RR
86%) and resulted in the expected T-cell depletion in the peripheral blood. By
contrast an increase in different T-cell subsets, specifi cally in Tregs, at the tumor
microenvironment was documented that was not seen in control patients treated
with H.pylori eradication only and thus suggesting a direct fl udarabine-related
immune modulation at the tumor site that may contribute to tumor response. These
studies will be further explored in an international collaborative study (EGILS).
Our department is the pathology coordinator of a large international consortium of
lymphoma trial organizations (including a.o. EORTC, HOVON, BNLI, GELA, ECOG)
who jointly validate and study the prognostic impact of (immunohistochemical)
biomarkers in large cohorts of uniformly treated lymphoma patients, concentrating
on FL and diffuse large B-cell lymphoma (DLBCL). Validation studies for DLBCL
were concluded and a series of over 1200 cases of DLBCL has been evaluated for
8 potential prognostic markers. These show that biological parameters with the
exception of BCL2 are of less relevance than previously suggested on smaller series
and mostly overruled by the strong prognostic impact of clinical factors, precluding
the development of a clinically applicable biological prognostic index. After a
successful validation study and subsequent prognostic study in over 1200 cases of
DLBCL, a similar study in FL is now initiated.
Molecular characteristics of low-stage follicular lymphoma
Olga Balagué Ponz, Patricia Gonzales, Marit Roemer, Stefanie Slot, Annegien Broeks,
Linde Braaf, Freek Bot, Max Beijert, Daphne de Jong
Follicular lymphoma (FL) rarely presents as localized disease. The structure of the
translocation t(14;18) suggests a primary oncogenetic origin in precursor B-cells
possibly in the bone marrow, suggesting that nodal FL should be regarded as
disseminated disease per defi nition and contradicting the possibility for localized
disease, which indeed is a rare situaltion. Review of the EORTC 20971 clinical trial
for limited stage nodal low-grade B-cell lymphoma enabled to collect an unique
series of such tumors, in which we explored the morphological, immunological
and molecular characteristics to better characterize low-stage nodal FL and to
delineate this group from the thus far very poorly defi ned group of nodal marginal
zone lymphoma. The far majority of patients treated within the 20971 trial
showed FL with a very high incidence of highly unusual morphological features,
including partial involvement, FL in situ and a “moth eaten” pattern of neoplastic
GC. Low stage FL are characterized by a signifi cantly lower incidence of BCL2
rearrangements (62% versus 98% for stage III/IV disease), both in morphologically
unremarkable cases and among cases with special features. These results suggest
that at least part of low-stage FL may follow a separate oncogenetic pathway with
local development and intranodal recirculation and stress the need for more defi ned
criteria in the differential diagnosis with nodal marginal zone lymphoma.
This notion was further stressed by preliminary results with arrayCGH (Nimblegen)
that showed major differences in genetic make-up of translocation positive and
negative FL, both in complexity of karyotypes and specifi c alterations. Moreover,
translocation negative FL proved to be more alike nodal marginal zone lymphoma
than to translocation positive FL..

Validation of the 70-gene prognosis-signature (MammaPrint)
in breast cancer subpopulations
Stella Mook, Michael Knauer, Marjanka Schmidt, Inge Eekhout, Jelle Wesseling,
Sabine Linn, Marc van de Vijver, Emiel Rutgers, Laura van ’t Veer
The 70-gene MammaPrint prognosis profi le has been shown to be a powerful
prognostic tool in early breast cancer patients. Several validation studies were
conducted, including those for postmenopausal and elderly (>70) patients, as well as
those with 4-9 positive nodes. All studies conformed MammaPrint’s independent
power.
In total for about 1700 NKI diagnosed patients a retrospective 70-gene prognosis
signature was assessed and published in various publications. We compiled all in
one database and have evaluated risk by the prognosis signature in her2postive,
endocrine responsiveness and by tumor diameter.
Most importantly, we have assessed in a series of 541 adjuvantly treated patients, the
additional predictive value of the 70-gene MammaPrint-signature for chemotherapy
(CT) benefi t in addition to endocrine therapy (ET). In the 70-gene high risk group,
BCSS was 81% (ET group) and 94% (ET+CT group) at 5 years with a signifi cant
HR of 0.21(95%CI 0.07-0.59;p

108
diagnostic oncology
Publications (continued)
Josting A, Müller H, Borchmann P, Baars JW,
Metzner B, Döhner H, Aurer I, Smardova L,
Fischer T, Niederwieser D, Schäfer-Eckart K,
Schmitz N, Sureda A, Glossmann J, Diehl V,
De Jong D, Hansmann ML, Raemaekers J,
Engert A. Dose Intensity of Chemotherapy in
Patients With Relapsed Hodgkin’s Lymphoma.
J Clin Oncol. 2010
Kaas R, Verhoef S, Wesseling J, Rookus
MA, Oldenburg HS, Peeters MJ, Rutgers
EJ. Prophylactic Mastectomy in BRCA1 and
BRCA2 Mutation Carriers: Very Low Risk
for Subsequent Breast Cancer. Ann Surg
2010;251:488-92
Kluijt I, Siemerink E, Ausems MGEM,
van Os TA, de Jong D, van Krieken H,
Ligtenberg M, van Riel E, Sijmons R, Plukker
J, van Hillegersberg R, Dekker E, Cats A,
Hoogerbrugge N. CDH1-related hereditary
diffuse gastric cancer syndrome: clinical
variations and implication for counseling.
Submitted to Am J Gastroenterol, 2010
Kluijt I, Sijmons RH, Hoogerbrugge N,
Vasen HFA, Cats A; Namens de Nederlandse
werkgroep erfelijke maagkanker. Familiaire
maagkanker: diagnostiek, behandeling en
periodieke controles. Ned Tijdschr Geneesk.
2010 (accepted)
Kuil J, Velders AH, van Leeuwen FWB,
“Multimodal Tumor-Targeting Peptides
Functionalized with both a Radio-and a
Fluorescent-Label” (review; including cover
feature), Bioconjugate Chemistry 2010, 21,
1709
Kuiper P, Verspaget HW, Biemond I, Jonge-
Muller ES, van Eeden S, van Velthuysen ML,
Taal BG, Lamers CB. Expression and ligand
binding of bombesin receptors in pulmonary
and intestinal carcinoids The role of bombesin
in carcinoids. J Endocrinol Invest 2010
Lammens C, Aaronson N, Wagner A,
Sijmons RH, Ausems MGEM, Vriends
AHJT, Ruijs MWG, van Os TAM, Spruijt
L, Gomez Garcia E, Kluijt I, Nagtegaal T,
Verhoef S, Bleiker E. Genetic testing In Li-
Fraumeni syndrome: uptake and psychosocial
consequences. J Clin Oncol. 2010;28:3008-14
Lammens CR, Bleiker EM, Aaronson NK,
Wagner A, Sijmons RH, Ausems MG,
Vriends AH, Ruijs MW, van Os TA, Spruijt
L, Gomez Garcia EB, Cats A, Nagtegaal
T, Verhoef S. Regular surveillance for Lifraumeni
syndrome: advice, adherence and
perceived benefi ts. Fam Cancer 2010;9:647-54
screened more than 3000 families for germ line mutations in the BRCA1/2 genes
since the start in 1995. These families are obtained through our Family Cancer
Clinic and the Oncogenetic section of the Department of Clinical Genetics of the
Academic Medical Center.
The laboratory has been testing new techniques in order to shorten the through
put time of samples. Last year tested the high resolution melting curve analysis of
BRCA1 which proves to be less complex in handling and this year we implemented
the technique in routine analysis for BRCA1. Furthermore, we introduced an 8
channel pipetting robot to facilitate rapid and controlled pipetting.
Using Array CGH for hereditary breast/ovarian cancer we study whether the
genomic aberrations in breast tumors are similar to the specifi c aberrations found
in BRCA1 and BRCA2 tumors (in collaboration with Petra Nederlof, Division of
Experimental Therapy and Diagnostic Oncology). More than 250 a-CGH analyses
have been completed, for high risk families and unclassifi ed variant analysis mostly.
A BRCA2 classifi er has been validated and implemented in the routine diagnostics
together with the assessment of methylation of the BRCA1 promotor.
For families with hereditary non polyposis colorectal cancer we have mutation
scanning tests for mismatch repair genes hMLH1, hMSH2 and hMSH6 and the
MutY (MYH) gene. Microsatellite instability tests and immunohistochemistry
for the mismatch repair genes is carried out in collaboration with the pathologist
Daphne de Jong. Furthermore methylation of the hMLH1 promoter and the presence
of a specifi c somatic mutation in the BRAF gene (V600E) are being assessed. About
50% of the micro satellite instable(MSI-high) tumors with absent staining of hMLH1
have a methylated promoter. This result has direct consequences for the clinical
interpretation of the family history data.
The Family Cancer Clinic contributes data to several multi-center national and
international research projects, e.g. GEO-HEBON (gene environment interactions
in hereditary breast and ovarian cancer, see Division Psychosocial Research and
Epidemiology), DNA-profi ling by classic cGH and array cGH of breast cancer
patients (see Division of Experimental Therapy), the BCAC and CIMBA consortiums
which focus on the contribution of SNPs to cancer risk (HEBON resource, Matti
Rookus, dept of Psychosodial Research and Epidemiology), studies into the
biological signifi cance of so called unclassifi ed variants (DNA changes of which it is
uncertain whether they be pathogenic mutations or polymorphisms) in collaboration
with the dept Molecular Biology groups of Jos Jonkers (BRCA1 UVs) and Hein te
Riele (MMR UVs), in national and international collaborations with other DNAdiagnostic
and research labs, eg ENIGMA for BRA1/2 UVs (Frans Hogervorst),
psychosocial studies, in collaboration with the department of Psychosocial Research
and Epidemiology and clinical and genetic research in families with gastrointestinal
cancer, including stomach cancer and pancreatic cancer (Annemieke Cats, Division
of Medical Oncology).
The guidelines for hereditary breast cancer management and hereditary colon
carcinoma have been formulated and are being implemented. In collaboration with
the Academic Medical Centre families are included in studies of familial stomach
cancer and pancreatic cancer, for whom periodic screening programs are being
developed, tried out and evaluated.
In 2008 a PhD student started a project on the implementation of genetic
counseling and where necessary rapid DNA-testing in recently diagnosed breast
cancer patients: the TIME- study. In this multi-centre study in collaboration with
the Division of Medical Genetics of the Utrecht Medical Centre, patients from 14
different hospitals will be enrolled for a period of 18 months. The recruitment of
patients has been as planned and inclusion will end on 31 st of December 2010.

DEPARTMENT OF RADIOLOGY
Diagnostic Image Research
KGA Gilhuijs, C Siedschlag, I Dmitriev, W Chen, K Pengel, L Batteau, A Paape,
C Loo, W Koops, S Muller, J Teertstra, W Vogel; FWB van Leeuwen, J Kuil,
PTK Chin, A Bunschoten; S Hovink, I Seemann, T Buckle, C Beekman, H vd Poel,
R Valdes-Olmos, M Sinaasappel, O Brouwer
The diagnostic-imaging laboratory at the department of Radiology pursues new
imaging techniques, image processing, multi-modality registration, pattern
recognition and molecular imaging to improve the sensitivity and specifi city of
cancer diagnosis, pre-treatment assessment of tumor extent, image-guided therapy
and response monitoring.
MRI-model to guide the surgical treatment in breast cancer patients after
neoadjuvant chemotherapy
Although MRI is the most reliable method to assess tumor size after NAC, criteria
for the correct selection of surgery remain unclear. The aim of this study was to
establish an MRI-based interpretation model to facilitate the selection of breastconserving
surgery (BCS) after neoadjuvant chemotherapy (NAC).
In 208 patients, dynamic contrast-enhanced MRI was performed before and after
NAC. Imaging was correlated with pathology. Differences < 20 mm in tumor extent
were considered to accurately indicate disease extent. Multivariate analysis with
cross-validation was performed to analyze features affecting the potential of MRI to
correctly indicate BCS (i.e. residual tumor size < 30 mm on pathology).
The accuracy of MRI to detect residual disease was 76% (158/208). The positive and
negative predictive value of MRI were 90% (130/144) and 44% (28/64), respectively.
In 35 patients (17%) MRI underestimated the tumor size by > 20 mm and in 27
patients (13%) this would have lead to an unjustifi ed selection of BCS. The features
most predictive of indicating feasibility of BCS in tumors < 30 mm on preoperative
MRI were the largest diameter at the baseline MRI, the reduction in diameter and
the tumor subtype based on hormone-, and HER2-status. (AUC=0.78).
We conclude that optimal selection of patients for BCS after NAC based on MRI
should take into account (1) the tumor size at baseline (2) the reduction in tumor
size, and (3) the subtype based on hormone-, and HER2 status.
MRI Response Monitoring of Breast Cancer during Neoadjuvant
Chemotherapy: Relevance of Breast Cancer Subtype
The purpose of this study was to evaluate the relevance of breast cancer subtypes for
MRI markers of therapy response during neoadjuvant chemotherapy (NAC).
MRI examinations were performed in 188 women before and during NAC. MRI
interpretation included lesion morphology at baseline, changes in morphology,
size, and contrast uptake kinetics (initial and late enhancement). Using
immunohistochemistry, tumors were divided into three subtypes: triple-negative,
HER2-positive, and ER-positive/HER2-negative. Tumor response was assessed
dichotomously (presence or absence of residual tumor in the surgical specimen),
and on a continuous scale (using the breast response index (BRI), a measure of the
relative change in tumor stage due to NAC). Multivariate regression analysis and
ROC analysis were employed to establish signifi cant associations.
Residual tumor at pathology was present in 31/47 (66%) of triple-negative tumors,
23/38 (61%) of HER2-positives, and 96/103 (93%) of ER-positives/HER2-negatives.
The BRIs of triple-negative tumors (Pearson’s=0.605, p

110
diagnostic oncology
Publications (continued)
Mook S, Knauer M, Bueno-de-Mesquita
JM, Retel VP, Wesseling J, Linn SC, van ‘t
Veer LJ, Rutgers EJ. Metastatic potential of T1
breast cancer can be predicted by the 70-gene
MammaPrint signature. Ann Surg Oncol
2010;17:1406-13
Mook S, Schmidt MK, Weigelt B, Kreike B,
Eekhout I, van de Vijver MJ, Glas AM, Floore
A, Rutgers EJ, van ‘t Veer LJ. The 70-gene
prognosis signature predicts early metastasis in
breast cancer patients between 55 and 70 years
of age. Ann Oncol 2010;21:717-22
Nuyten DSA. Hypothesis driven gene
expression profi ling in breast cancer.
Amsterdam: University of Amsterdam, 2010
Obdeijn IM, Loo CE, Rijnsburger AJ, Wasser
MN, Bergers E, Kok T, Klijn JG, Boetes C.
Assessment of false-negative cases of breast
MR imaging in women with a familial or
genetic predisposition. Breast Cancer Res Treat
2010;119:399-407
Ockeloen CW, Kempers MJE, Kuiper RP
and other authors. High colorectal and low
endometrial cancer risk in EPCAM deletionpositive
Lynch syndrome: a cohort study.
Accepted. Lancet Oncol, 2010
Oostendorp RL, Buckle T, Lambert G,
Garrigue JS, Beijnen JH, Schellens JH and
van Tellingen O. Paclitaxel in self-micro
emulsifying formulations: oral bioavailability
study in mice. Invest New Drugs, 2010
Osorio A, Milne RL, Pita G and other autors.
Evaluation of a candidate breast cancer
associated SNP in ERCC4 as a risk modifi er
in BRCA1 and BRCA2 mutation carriers.
Results from the Consortium of Investigators of
Modifi ers of BRCA1/BRCA2 (CIMBA). Br J
Cancer. 2009;101:2048-54
Out AA, Tops CM, Nielsen M, Weiss MM,
van Minderhout IJ, Fokkema IF, Buisine
MP, Claes K, Colas C, Fodde R, Fostira F,
Franken PF, Gaustadnes M, Heinimann K,
Hodgson SV, Hogervorst FB, Holinski-Feder
E, Lagerstedt-Robinson K, Olschwang S,
van den Ouweland AM, Redeker EJ, Scott
RJ, Vankeirsbilck B, Grønlund RV, Wijnen JT,
Wikman FP, Aretz S, Sampson JR, Devilee P,
den Dunnen JT, Hes FJ. Leiden open variation
database of the MUTYH gene. Hum Mutat.
2010;31:1205-15
effective in triple-negative or HER2-positive disease but is inaccurate in ER-positive/
HER2-negative breast cancer.
Microscopic disease extension in 3D for Non Small Cell Lung Cancer:
development of a prediction model using pathology validated PET and CT
features
One major uncertainty in radiotherapy planning of non-small cell lung cancer
(NSCLC) concerns the defi nition of the clinical target volume (CTV), meant to cover
potential microscopic disease extension (MDE) around the macroscopically visible
tumor. The primary aim of this study was to establish pre-treatment risk factors for
the presence of MDE. The secondary aim was to establish the impact of these factors
on the accuracy of PET and CT to assess the total tumor-bearing region at pathology
(CTV path).
Thirty-four NSCLC patients, who underwent CT and PET prior to lobectomy,
were included. Specimens were examined microscopically for MDE. The gross
tumor volume (GTV) on CT and PET was compared with the GTV and the CTV
at pathology, taking tissue deformations into account. Using multivariate logistic
regression, image based risk factors for the presence of MDE were identifi ed and a
prediction model was developed based on these factors.
MDE was found in 17 of 34 patients (50%). MDE did not exceed 26 mm in 90%
of patients. In multivariate analysis, two parameters (mean CT tumor density and
GTV CT) were signifi cantly associated with MDE. The AUC of the 2 parameter
prediction model was 0.86. 13 tumors (38%, 95% CI: 24-55%) were identifi ed as
low risk for MDE, being potential candidates for reduced intensity therapy around
the GTV. In the low risk group, the effective diameter of the GTV CT/PET accurately
represented the CTV path. In the high risk group, GTV CT/PET underestimated the
CTV path with on average 19.2 and 26.7 mm, respectively.
We conclude that CT features have potential to predict the presence of MDE. Tumors
identifi ed as low risk of MDE show lower rates of disease around the GTV than high
risk tumors. Both CT and PET accurately visualize the CTV path in low risk tumors,
but underestimate it in high risk tumors.
On the Feasibility of MRI-Guided Navigation to Demarcate Breast Cancer for
Breast-Conserving Surgery
The aim of this study was to investigate the feasibility of image-guided navigation
approaches to demarcate breast cancer on the basis of pre-acquired magnetic
resonance imaging (MRI) in supine patient orientation.
Strategies were examined to minimize the uncertainty in the instrument-tip
position, based on the hypothesis that release of instrument pressure returns
the breast tissue to its pre-deformed state. For this purpose, four sources of
uncertainty were taken into account: 1) U ligaments: uncertainty in the reproducibility
of the internal mammary-gland geometry during repeat patient setup in supine
orientation; 2) U r_breathing: residual uncertainty in registration of the breast after
compensation for breathing motion using an external marker; 3) U reconstruction:
uncertainty in the reconstructed location of the tip of the needle using an optical
image-navigation system (phantom experiments, n=50); 4) U deformation: uncertainty
in displacement of breast tumors due to needle-induced tissue deformations
(patients, n=21). A Monte Carlo study was performed to establish the 95%
confi dence interval (CI) of the combined uncertainties. This region of uncertainty
was subsequently visualized around the reconstructed needle tip as an additional
navigational aid in the pre-acquired MR images.
Validation of the system was performed in fi ve healthy volunteers (localization of
skin markers only), and in two patients. In the patients, the navigation system was
used to monitor ultrasound-guided radioactive seed localization of breast cancer.

Nearest distances between the needle tip and the tumor boundary in the ultrasound
images were compared with those in the concurrently reconstructed MR images.
Combined impact resulted in 3.9 mm uncertainty in the position of the needle tip
(95% CI) after release of pressure. The volunteer study showed a targeting accuracy
comparable to that in the phantom experiments: 2.9 mm ± 1.3 mm versus 2.7 mm
± 1.1 mm respectively. In the patient feasibility study, the deviations were within the
3.9 mm CI.
We conclude that Image-guided navigation to demarcate breast cancer on the
basis of pre-acquired MR images in supine orientation appears feasible if patient
breathing is tracked during the navigation procedure, positional uncertainty
is visualized and pressure on the localization instrument is released prior to
verifi cation of its position.
3D bracketing of tumor margins with multimodal marker seeds
Surgeons often rely on guide wires or radioactive markers for guidance towards the
lesion. Further improvement in surgical guidance may be obtained by adding optical
guidance to currently used methods, in the form of intraoperative fl uorescence
imaging. To achieve such a multimodal approach we have generated markers
which can be used in a pre-, intra-, and post-operative setting, based on a cocktail
of our previously developed dual-emissive InP/ZnS QDs, lipids and pertechnetate.
Phantom experiments demonstrate that these seeds can be placed accurately around
a surrogate tumor using ultrasound. Combined with the multimodal nature, this
provides the opportunity to predetermine the resection margins by validating the
placement accuracy using multiple imaging modalities (namely X-ray, MRI, SPECT/
CT, and US). The dual-emissive fl uorescent properties of the InP/ZnS QDs provide
the unique opportunity to intra-operatively estimate the depth of the seed and assess
the safety margins, in the tissue; emission green max = 520nm < 5mm penetration
vs. emission red max = 660 nm < 12mm penetration. We have now even extended
this approach with a third color, yielding a so-called traffi c light approach for
approaching the lesion. By using particles with different colors, the original
geographic orientation of the excised tissue can also be determined.
Figure 4: Schematic representation of the combined pre-, intra-, and postoperative imaging paradigm
that can be achieved with multimodal marker seeds. A) Tumor diagnosing imaging procedure.
B) Image guided placement of bracketing tumor marker seeds. C) Preoperative validation of the marker
placement accuracy. D) Optical intraoperative detection of the tumor markers. E) Geographical
(re)orientation of the excised tissue section at pathology. Figure is published in J Biomed Opt. 2010,
15, 056021
111
diagnostic oncology
Publications (continued)
Pijpe A, Manders P, Brohet RM, Collee JM,
Verhoef S, Vasen HFA, Hoogerbrugge N,
van Asperen CJ, Dommering C, Ausems
MGEM, Aalfs CM, Gomez-Garcia EB, van’t
Veer LJ, van Leeuwen FE, Rookus MA.
Physical activity and the risk of breast cancer
in BRCA1/2 mutation carriers. Breast Cancer
Res Treat 2010;120:235-44
Pirker R, Herth FJ, Kerr KM, Filipits M, Taron
M, Gandara D, Hirsch FR, Grunenwald D,
Popper H, Smit E, Dietel M, Marchetti A,
Manegold C, Schirmacher P, Thomas M,
Rosell R, Cappuzzo F, Stahel R; European
EGFR Workshop Group. Consensus for EGFR
mutation testing in non-small cell lung cancer:
results from a European workshop. J Thorac
Oncol. 2010;5:1706-13
Ploem MC, Retèl VP, Linn SC, van Boven H,
Schmidt M.K., De Jong J.Ph., Gevers J.K.M.,
Van Harten WH. Tumour tissue: who is in
control? Lancet Oncol 2010;11:9-11
Prevoo W, van den Munckhof MP, Meinhardt
W, Horenblas S, van den Bosch MA.
Radiofrequency ablation of kidney tumours in
patients with a solitary kidney. Clin Radiol.
2010;65:230-6
Rafael M, Kluijt I, Koot B, Smets A, Tilanus
M, Bras H, van de Wetering M. Gastric
adenocarcinoma in a 13-year old boy, a
diagnosis not often seen in this age group.
Pediatric Hematology-Oncology 2010
(accepted)
Rasch CRN, Hauptmann M, Schornagel J,
Wijers O, Buter J, Gregor T, Wiggenraad
R, de Boer JP, Ackerstaff AH, Kroger R,
Hoebers FJP, Balm AJM. Intra-arterial versus
intravenous chemoradiation for advanced
head and neck cancer: Results of a randomized
phase 3 trial. Cancer 2010;116:2159-65
Rayavarapu RG, Petersen W, Chin P,
Janssen H, van Leeuwen FWB, Manohar
S, van Leeuwen TG. In vitro toxicity and
cellular uptake studies of PEGylated and
PSS-coated gold nanorods. NanoTechnology.
2010;21:145101
Reese E, Sluzevich J, Kluijt I, Teertstra HJ,
de Jong D, Horenblas S, Ryu J. Birt-Hogg-
Dubé Syndrome. In: Riegert-Johnson DL,
Boardman LA, Hefferon T, Roberts M (ed).
Familial Cancer Syndromes. National Center
for Biotechnology Information: Bethesda, US,
2009

112
diagnostic oncology
Publications (continued) Publications (continued)
Rietbergen DD, Scholte AJ, Al Younis
I, Stokkel MP. Myocardial perfusion
scintigraphy before and after cardioversion for
atrial fi brillation: Recovery of quantitative
parameters. J Nucl Cardiol. 2010
Rijnsburger AJ, Obdeijn IM, Kaas R, Tilanus-
Linthorst MM, Boetes C, Loo CE, Wasser
MN, Bergers E, Kok T, Muller SH, Peterse
H, Tollenaar RA, Hoogerbrugge N, Meijer S,
Bartels CC, Seynaeve C, Hooning MJ, Kriege
M, Schmitz PI, Oosterwijk JC, de Koning HJ,
Rutgers EJ, Klijn JG. BRCA1-Associated Breast
Cancers Present Differently From BRCA2-
Associated and Familial Cases: Long-Term
Follow-Up of the Dutch MRISC Screening
Study. J Clin Oncol. 2010
Rodenhuis S, Mandjes IA, Wesseling J, van
de Vijver MJ, Peeters MJ, Sonke GS, Linn
SC. A simple system for grading the response
of breast cancer to neoadjuvant chemotherapy.
Ann Oncol 2010;21:481-7
Roef M, Vogel WV. The effects of muscle
exercise and bed rest on [(18)F] methylcholine
PET/CT. Eur J Nucl Med Mol Imaging. 2010
Ruggi A, Beekman C, Wasserberg D,
Subramaniam V, Reinhoudt DN, van
Leeuwen FWB, Velders AH. Dendritic
Ruthenium(II)-based dyes tunable for
diagnostic or therapeutic applications. Chem.
Eur. J. 2010 (accepted)
Ruijs MWG, Verhoef S, Rookus MA, Pruntel
R, van der Hout AH, Hogervorst FBL, Kluijt
I, Sijmons RH, Aalfs CM, Wagner A, Ausems
MGEM, Hoogerbrugge N, van Asperen
C, Gomez Garcia EB, Meijers-Heijboer H,
ten Kate LP, Menko FH, van ’t Veer. TP53
germline mutation testing in 180 families
suspected of Li-Fraumeni syndrome: mutation
detection rate and relative frequency of cancers
in different familial phenotypes. J Med Genet
2010;47421-8
Ruijs MWG. Li-Fraumeni syndrome, clinical
and molecular genetics. Amsterdam: Free
University 2010
Scheenstra RJ, Muller SH, Hilgers FJ.
Endotracheal temperature and humidity in
laryngectomized patients in a warm and
dry environment and the effect of a heat and
moisture exchanger. Head Neck. 2010;27
Scheenstra RJ, Muller SH, Vincent A, Ackerstaff
AH, Jacobi I, Hilgers FJ. Short-term endotracheal
climate changes and clinical effects of a heat
and moisture exchanger with an integrated
electrostatic virus and bacterial fi lter developed for
laryngectomized individuals.Acta Otolaryngol.
2010;130:739-46
Scheenstra RJ, Muller SH, Vincent A, Hilgers FJ.
Heat and moisture exchange capacity of the upper
respiratory tract and the effect of tracheotomy
breathing on endotracheal climate. Head Neck
2010;29
Scheenstra RJ, Muller SH, Vincent A, Sinaasappel
M, Hilgers FJ. Infl uence of breathing resistance
of heat and moisture exchangers on tracheal
climate and breathing pattern in laryngectomized
individuals. Head Neck. 2010;32:1069-78
Schmitz AC, van den Bosch MA, Loo CE, Mali WP,
Bartelink H, Gertenbach M, Holland R, Peterse JL,
Rutgers EJ, Gilhuijs KG. Precise correlation between
MRI and histopathology - Exploring treatment
margins for MRI-guided localized breast cancer
therapy. Radiother Oncol. 2010;97:225-32
Stokkel MP, Handkiewicz Junak D, Lassmann M,
Dietlein M, Luster M. EANM procedure guidelines
for therapy of benign thyroid disease. Eur J Nucl
Med Mol Imaging. 2010;37:2218-28
Straver ME, Aukema TS, Valdés Olmos RA,
Rutgers EJ, Gilhuijs KG, Schot ME, Vogel
WV, Peeters MJ. Feasibility of FDG PET/CT
to monitor the response of axillary lymph node
metastases to neoadjuvant chemotherapy in breast
cancer patients. Eur J Nucl Med Mol Imaging.
2010;37:1069-76
Straver ME, Glas AM, Hannemann J, Wesseling
J, van de Vijver MJ, Rutgers EJ, Vrancken Peeters
MJ, Van Tinteren H, van’t Veer LJ, Rodenhuis S.
The 70-gene signature as a response predictor for
neoadjuvant chemotherapy in breast cancer. Breast
Cancer Res Treat 2010;119:551-8
Straver ME, Loo CE, Rutgers EJ, Oldenburg HS,
Wesseling J, Vrancken Peeters MJ, Gilhuijs KG.
MRI-model to guide the surgical treatment in breast
cancer patients after neoadjuvant chemotherapy.
Ann Surg 2010;251:701-7
Straver ME, Rutgers EJ, Rodenhuis S, Linn SC,
Loo CE, Wesseling J, Russell NS, Oldenburg
HS, Antonini N, Vrancken Peeters MT. The
relevance of breast cancer subtypes in the outcome
of neoadjuvant chemotherapy. Ann Surg Oncol
2010;17:2411-8
Teertstra HJ, Loo CE, van den Bosch MA, van
Tinteren H, Rutgers EJ, Muller SH, Gilhuijs KG.
Breast tomosynthesis in clinical practice: initial
results. Eur Radiol. 2010;20:16-24
Teertstra HJ, Muller SH. De cytotron, peperduur en
gevaarlijk. Med Contact 2010;65:26-7
Thunnissen E, Smit EF, Nederlof PM, Dingemans
AC. Doelgerichte behandeling van het nietkleincellig
longcarcinoom: het belang van de EGFR
mutatie analyse. NTvG 2010 (accepted)
Valdés Olmos RA, Vermeeren L, Hoefnagel CA,
Aukema TS, Vogel WV. New dimensions of nuclear
medicine in oncology. Almenara (in press)
Valdés Olmos RA, Vidal-Sicart S, Nieweg OE.
Technological innovation in the sentinel node
procedure: towards 3-D intraoperative imaging.
Eur J Nucl Med Mol Imaging. 2010;37:1449-51
Van den Bosch MAAJ, Prevoo W, Van der Linden
E, Meijerink MR, Van Delden OM, Mali WPTM,
Reekers JA. Oncologische interventieradiologie.
Gamma Prof 2010;60:3-7
van der Ploeg IMC, Oldenburg HSA, Rutgers EJT,
Baas-Vrancken Peeters MJ, Kroon BBR, Valdés
Olmos RA, Nieweg OE. Lymphatic Drainage
Patterns from the Treated Breast. Ann Surg Oncol
2010;17:1069-75
van der Vegt B, Wesseling J, Pijnappel RM,
Dorrius MD, den Heeten GJ, de Roos MA, de
Bock GH. Aggressiveness of ‘true’ interval invasive
ductal carcinomas of the breast in postmenopausal
women. Mod Pathol 2010;23:629-36
van Deurzen CH, Cserni G, Bianchi S, Vezzosi
V, Arisio R, Wesseling J, Asslaber M, Foschini
MP, Sapino A, Castellano I, Callagy G, Faverly D,
Martin-Martinez MD, Quinn C, Amendoeira I,
Kulka J, Reiner-Concin A, Cordoba A, Seldenrijk
CA, van Diest PJ. Nodal-stage classifi cation in
invasive lobular breast carcinoma: infl uence of
different interpretations of the pTNM classifi cation.
J Clin Oncol 2010;28:999-1004
van Leeuwen AC, Buckle T, Vermeeren L,
Valdes-Olmos R, van der Poel HG, van Leeuwen
FWB. “Cocktail” injections for combined pre- and
intraoperative multimodal imaging of the tumor
draining lymph nodes in a spontaneous mouse
prostate tumor model. J. Biomedical Optics 2010
(accepted)
van Leeuwen FW, Buckle T, Batteau L, Pool B,
Sinaasappel M, Jonkers J, Gilhuijs KG. Potential
value of color-coded dynamic breast-specifi c gammaimaging;
comparing (99m)Tc-(V)-DMSA, (99m)
Tc-MIBI, and (99m)Tc-HDP in a mouse mammary
tumor model. Appl Radiat Isot. 2010;68:2117-24

Publications (continued)
van Leeuwen FWB. Evaluation of multimodal
imaging approaches for combined pre-and
intraoperative imaging of the SLN and tumor
margins. IEEE: Proceedings international
symposium on biomedical imaging: from nano to
macro (ISBI 2010; peer reviewed manuscript)
van Loon J, Siedschlag C, Stroom J, Blauwgeers
H, van Suylen RJ, Knegjens J, Rossi M, van
Baardwijk A, Boersma L, Klomp H, Vogel W,
Burgers S, Gilhuijs K. Microscopic Disease
Extension in Three Dimensions for Non-Small-Cell
Lung Cancer: Development of a Prediction Model
Using Pathology-Validated Positron Emission
Tomography and Computed Tomography Features.
Int J Radiat Oncol Biol Phys. 2010;23
van Munster BC, Korevaar JC, Korse CM, Bonfrer
JM, Zwinderman AH, de Rooij SE. Serum S100B
in elderly patients with and without delirium. Int J
Geriatr Psychiatry 2010;25:234-9.
van Oosten M, Crane LMA, Bart J, van Leeuwen
FWB, van Dam GM. Selecting potential targetable
biomarkers for imaging purposes in colorectal
cancer using TArget Selection Criteria (TASC):
a novel target identifi cation tool. Translational
Oncology 2010 (accepted)
Van Riel E, Ausems MGEM, Hogervorst FBL,
Kluijt I, van Gijn ME, van Echtelt AJ, Scheidel-
Jacobse K, Hennekam FAM, Stulp RP, Sijmons
RH, te Riele H, Offerhaus GJA, Menko FH, Gille
JJP. A novel missense mutation of the MLH1 gene,
c.112A>C, p.Asn38His, in six families with features
of Lynch syndrome. Hereditary Cancer in Clinical
Practice 2010;8:7
Van Riel E, Warlam-Rodenhuis CC, Verhoef
S, Rutgers EJ, Ausems MG. BRCA testing of
breast cancer patients: medical specialists’ referral
patterns, knowledge and attitudes to genetic testing.
Eur J Cancer Care (Engl) 2009;19:369-76
Vanneste BG, Haas RL, Bard MP, Rijna H,
Valdes Olmos RA, Belderbos JS. Involved fi eld
radiotherapy for locally advanced non-small cell
lung cancer: isolated mediastinal nodal relapse.
Lung Cancer 2010;70:218-20
Verloop J, van Leeuwen FE, Helmerhorst TJ,
van Boven HH, Rookus MA. Cancer risk in DES
daughters. Cancer Causes Control 2010;21:999-
1007
Vermaat JS, van der Tweel I, Mehra N, Sleijfer S,
Haanen JB, Roodhart JM, Engwegen JY, Korse
CM, Langenberg MH, Kruit W, Groenewegen
G, Giles RH, Schellens JH, Beijnen JH, Voest
EE. Two-protein signature of novel serological
markers apolipoprotein-A2 and serum amyloid
alpha predicts prognosis in patients with metastatic
renal cell cancer and improves the currently
used prognostic survival models. Ann Oncol
2010;21:1472-81
Vermeeren L, Meinhardt W, Bex A, van der Poel
HG, Vogel WV, Hoefnagel CA, Horenblas S,
Valdés Olmos RA. Paraaortic sentinel lymph
nodes: toward optimal detection and intraoperative
localization using SPECT/CT and intraoperative
real-time imaging. J Nucl Med. 2010;51:376-82
Vermeeren L, Meinhardt W, Valdes Olmos RA.
Prostatic lymphatic drainage with sentinel nodes at
the ventral abdominal wall visualized with SPECT/
CT: a case series. Clin Nucl Med. 2010;35:71-3
Vermeeren L, Meinhardt W, van der Poel HG,
Valdés Olmos RA. Lymphatic drainage from the
treated versus untreated prostate: feasibility of
sentinel node biopsy in recurrent cancer. Eur J Nucl
Med Mol Imaging. 2010;37:2021-6
Vermeeren L, Muller SH, Meinhardt W, Valdés
Olmos RA. Optimizing the colloid particle
concentration for improved preoperative and
intraoperative image-guided detection of sentinel
nodes in prostate cancer. Eur J Nucl Med Mol
Imaging. 2010;37:1328-34
Vermeeren L, Tanis PJ, Nieweg OE, Valdes Olmos
RA. Lymphoscintigraphy of a breast tumor showing
focal tracer accumulation along the falciform
ligament of the liver. Clin Nucl Med 2010;35:168-9
Vermeeren L, Valdés Olmos RA, Klop WM, Balm
AJ, van den Brekel MW. A portable gamma-camera
for intraoperative detection of sentinel nodes in the
head and neck region.J Nucl Med. 2010;51:700-3
Vermeeren L, Valdés Olmos RA, Klop WM, van
der Ploeg IM, Nieweg OE, Balm AJ, van den
Brekel MW. SPECT/CT for sentinel lymph node
mapping in head and neck melanoma. Head Neck.
2010;31
Vidal-Sicart S, Aukema TS, Vogel WV, Hoefnagel
CA, Valdés Olmos RA. Added value of prone
position technique for PET-TAC in breast cancer
patients. Rev Esp Med Nucl. 2010;29:230-5
113
diagnostic oncology
Publications (continued)
Vollebergh MA, Lips E, Nederlof PM,
Wessels LFA, Schmidt MK, van Beers EH,
Cornelissen S, Holtkamp M, Froklage FE,
de Vries EGE, Schrama JG, Wesseling J, van
de Vijver MJ, van Tinteren H, de Bruin M,
Hauptmann M, Rodenhuis S, Linn SC. A
comparative genomic hybridization classifi er
derived from BRCA1-mutated breast cancer
and benefi t of high-dose, platinum-containing,
chemotherapy in breast cancer patients.
Annals of Oncol (accepted)
Vyth-Dreese FA, Sein J, Van De Kasteele W,
Dellemijn TAM, van den Bogaard C, Nooijen
WJ, de Gast GC, Haanen JBAG, Bex A. Lack
of anti-tumour reactivity despite enhanced
numbers of circulating natural killer T cells
in two patients with metastatic renal cell
carcinoma. Clin Exp Immunol 2010 (in press)
Waddell N, Arnold J, Cocciardi S, da Silva L,
Marsh A, Riley J, Johnstone CN, Orloff M,
Assie G, Eng C, Reid L, Keith P, Yan M, Fox
S, Devilee P, Godwin AK, Hogervorst FBL,
Couch F, Grimmond S, Flanagan JM, Khanna
K, Simpson PT, Lakhani SR, Chenevix-Trench
G. Subtypes of familial breast tumours revealed
by expression and copy number profi ling.
Breast Cancer Res Treat 2009;4
Wang X, Pankratz VS, Fredericksen Z and
other authors. Common variants associated
with breast cancer in genome-wide association
studies are modifi ers of breast cancer risk in
BRCA1 and BRCA2 mutation carriers. Hum
Mol Genet. 2010;19:2886-97
Waddell N, Arnold J, Cocciardi S, da Silva L,
Marsh A, Riley J, Johnstone CN, Orloff M,
Assie G, Eng C, Reid L, Keith P, Yan M, Fox
S, Devilee P, Godwin AK, Hogervorst FB,
Couch F; kConFab Investigators, Grimmond
S, Flanagan JM, Khanna K, Simpson PT,
Lakhani SR, Chenevix-Trench G. Subtypes of
familial breast tumours revealed by expression
and copy number profi ling. Breast Cancer Res
Treat. 2010;123:661-77
Wesseling J. Relevantie van amplifi catie van
het en coderend voor de oestrogeen-receptoralfa
(ESR1) in mammacarcinoom. Ned
Tijdschr Oncol 2010;7:49-55
Yang TJ, Aukema TS, van Tinteren H, Burgers
S, Valdés Olmos R, Verheij M. Predicting
early chemotherapy response with technetium-
99m methoxyisobutylisonitrile SPECT/CT
in advanced non-small cell lung cancer. Mol
Imaging Biol. 2010;12:174-80

114
medical oncology
Division head John Haanen
John Haanen MD PhD Head
Joke Baars MD PhD Academic staff
Paul Baas MD PhD Academic staff
André Bergman MD PhD Academic staff
Jos Beijnen PhD Academic staff
Christian Blank MD PhD Academic staff
Willem Boogerd MD PhD Academic staff
Henk Boot MD PhD Academic staff
Wieneke Buikhuisen MD PhD Academic staff
Sjaak Burgers MD PhD Academic staff
Annemieke Cats MD PhD Academic staff
Jan Paul De Boer MD PhD Academic staff
Dieta Brandsma MD PhD Academic staff
Elke Brouwers PhD Academic staff
Alwin Huitema PhD Academic staff
Martijn Kerst MD PhD Academic staff
Marjolein Klous PhD Academic staff
Sabine Linn MD PhD Academic staff
Anne Lukas MD PhD Academic staff
Serena Marchetti MD Academic staff
Bastiaan Nuijen PhD Academic staff
Sjoerd Rodenhuis MD PhD Head
Hilde Rosing PhD Academic staff
Jan Schellens MD PhD Academic staff
Gabe Sonke MD Academic staff
Neeltje Steeghs MD PhD Academic staff
Babs Taal MD PhD Academic staff
Margot Tesselaar MD Academic staff
Michel Van Den Heuvel MD PhD Academic
staff
Marchien Van Der Weide MD PhD Academic
staff
Roel Van Gijn PhD Academic staff
Marije Appels Temporary staff
Sandra Bakker Temporary staff
Marja Bonarius Temporary staff
Melanie Bos Temporary staff
Ilja Bot Temporary staff
Rogier Boshuizen Temporary staff
Annebeth Haringhuizen Temporary staff
Emile Kerver MD Temporary staff
Peter Kunst MD PhD Temporary staff
Alet Mager Temporary staff
Boelo Poppema Temporary staff
Josine Quispel Temporary staff
Quirine van Rossum-Schornagel Temporary
staff
Dirkje Sommeijer Temporary staff
Teun van Strien Temporary staff
Suzan Vrijaldenhoven Temporary staff
Ralph de Backer PhD student
Karin Beelen PhD student
David Boss PhD student
DIVISION OF MEDICAL ONCOLOGY
CLINICAL PHARMACOLOGY OF ANTICANCER DRUGS
Jan Schellens, Henk Boot, Annemieke Cats, Bastiaan Nuijen, Hilde Rosing, Serena Marchetti,
Neeltje Steeghs, Alwin Huitema, Jos Beijnen
BACKGROUND AND OBJECTIVES
The division of pharmacology is involved in research in different phases of
anticancer drug development. This concerns: I) Pharmaceutical formulation, II)
Bioanalytical method development and implementation in clinical pharmacological
studies, III) Early clinical phase I/II studies and IV) supportive care studies in
patients with breast cancer.
Research activities of the division of Clinical Pharmacology of the department
of Medical Oncology, the department of Pharmacy & Pharmacology and the
department of Experimental Therapy (group Schellens) are closely integrated.
In 2010 we continued clinical research fully compliant with ICH-GCP guidelines,
previously certifi ed by the Dutch Ministry of Health. The department of Pharmacy
& Pharmacology successfully continued its offi cial governmental GLP (in the fi eld
of analytical chemistry), GMP (formulation and manufacturing of investigational
cytotoxic drugs) and GDP (ISO9002 for worldwide distribution of clinical supplies)
licenses.
I PHARMACEUTICAL FORMULATION
Clinical manufacturing (Thalidomide tablets) of oral formulations of anticancer
agents was continued. A pharmaceutical development program of oral taxanes
resulted in a fi rst formulation composed of a solid dispersion of docetaxel
that entered the clinic (ModraDoc001). A similar formulation of paclitaxel
(ModraPac001) was developed and is clinically tested.
A fi rst formulation project aimed at the GMP-manufacture of ready-to-label
molecular imaging agents is started with bevacizumab, which is now available as
an off-the-shelf product for imaging studies. A second project with DOTATATE was
initiated.
The Amsterdam BioTherapeutics Unit (AmBTU) is fully operational for the manufacture
of GMP-grade DNA-plasmid. A phase I clinical study with the in-house
manufactured DNA-plasmid pDERMATT (plasmid DNA Encoding Recombinant
Mart-1 and Tetanus Toxin fragment-c) is ongoing in our institute. Currently,
pharmaceutical products of HPV E6 and E7 pDNA vaccines are developed and
manufactured. It is expected that these pDNA products will enter the clinic fi rst
half of 2011. Clean room facilities are in place to enable GMP-preparation of
T-lymphocytes for TIL and T-cell receptor gene therapy. Clinical trials are expected
to start beginning of 2011. The research program in collaboration with Utrecht
University aiming at the pharmaceutical development of non-viral vectors (e.g.
lipoplexes and polyplexes) for improvement of DNA-plasmid transfection is ongoing.
An ex vivo human skin screening model is in place and has helped to identify
important aspects of intradermal pDNA administration and non-viral vector composition.
Ultimate aim of this program is translation of promising vectors to the clinic.
II BIOANALYTICAL METHOD DEVELOPMENT + IMPLEMENTATION IN
PK STUDIES
The bioanalytical support of three mass balance studies (E7389, E7080 and
bendamustine) has been completed this year. E7389 is a synthetic analog of
Halichondrin B (HalB), acting by interfering with normal mitotic spindle formation,
resulting in blockage of cells in mitosis, leading to cell death via apoptosis. Results
showed that E7389 was slowly metabolised and only minor monooxygenated
metabolites were detected using our LTQ XL MS instrument. Radioactive E7080,
an orally active multi–tyrosine kinase inhibitor, was orally administered to 6
patients, followed by collection of blood and plasma samples and collection of all
excreta for a period of at least 8 days. E7080 was quantifi ed in plasma, urine and

faeces using LC-MS/MS. Furthermore, the collected samples were used for total
radioactivity determination and metabolic profi ling. The average total recovery of
the radioactive dose in excreta was ~89%, of which ~25% was excreted in urine and
~64% in faeces. Comparison of total radioactivity results with LC-MS/MS results
indicated large amounts of metabolites in urine and faeces. The metabolite profi ling
revealed products of E7080 oxidation and demethylation in both urine and faeces
and products of demethylation and glucuronidation in urine and, in small amounts,
in plasma. Bendamustine is an alkylating agent for the treatment of chronic
lymphocytic leukemia (CLL), Hodgkin’s lymphoma, non-Hodgkin’s lymphoma
(NHL) and with activity in small-cell lung cancers. We investigated bendamustine’s
disposition in a mass balance study. Mean total recovery of the radioactivity
in excreta was ~76% of the radiochemical dose, with approximately half of the
radiochemical dose recovered in the urine. Results of urinary metabolite profi ling
using LC-LTQ ion trap MS revealed that bendamustine is extensively metabolized
via oxidative (N-dealkylation and hydroxylation), hydrolytic (chloride displacement
and possibly hydrolysis of alkylated products), and conjugative (possibly glutathione
conjugation) pathways.
A method for the quantifi cation of tamoxifen and its phase I metabolites in plasma
has been developed and validated to support a study in premenopausal women
treated with adjuvant tamoxifen versus continued treatment.
New validated methods for the determination of capecitabine and its metabolite were
developed and validated.
The following sample analysis were performed for clinical trials within and outside
the Institute concerning paclitaxel, docetaxel, topotecan, irrinotecan and its active
metabolite SN-38, trabectedin, AS703026 (an inhibitor of MEK 1 and MEK2),
lonafarnib, gemcitabine and dFdU, gemcitabine triphosphate, 5-hydroxy methyl
uracil, cyclophosphamide (in combination with fl udarabine) and its metabolite
4-hydroxy cyclophosphamide, and platinum (originating from cisplatin, carboplatin
and oxaliplatin).
III EARLY CLINICAL PHASE I/II STUDIES
III-1 Novel approaches to improve oral bioavailability
The ‘boosting’ concept of oral docetaxel plus ritonavir, an effi cacious inhibitor of
gut wall and hepatic CYP3A4, has been further developed this year. The major step
forward is that the oral capsule formulation of docetaxel, denoted ModraDoc001,
turns out to have benefi cial pharmaceutical properties in patients. The ongoing
phase I study is approaching maximum tolerated dose (MTD) and now recruits
patients at the level of 80 mg ModraDoc001 plus 200 mg ritonavir. Main toxicities
are diarrhea and general malaise. The concentration-time profi les of docetaxel,
administered as ModraDoc001, at the tested dose-levels administered together
with ritonavir are shown in fi gure 1. The novel oral paclitaxel capsule formulation
(ModraPac001) showed excellent systemic exposure to paclitaxel and has therefore
been taken further into the clinic. The phase I study with daily low-dose or
metronomic ModraPac001 plus booster drug ritonavir is being executed.
Figure 1: Plasma concentration-time
profi les of patients who were treated
with oral docetaxel formulated
as capsules ModraDoc001 on all
occasions taken together with one
single dose of 100 mg or 200 mg of
the boosting agent ritonavir.
115
medical oncology
Maarten Deenen PhD student
Lot Devriese PhD student
Thomas Dorlo PhD student
Anne Charlotte Dubbelman PhD student
Corinne Ekhart PhD student
Geert Frederikx PhD student
Andrew Goey PhD student
Claudia Heijens PhD student
Helgi Helgason PhD student
Tine Janssens PhD student
Eefje Jong PhD student
Ron Keizer PhD student
Heinz Josef Klümpen PhD student
Stijn Koolen PhD student
Nienke Lankheet PhD student
Suzanne Leijen PhD student
Jelte Meulenaar PhD student
Johannes Moes PhD student
Ruud van de Noll PhD student
Philip Schouten PhD student
Rik Stuurman PhD student
Maurits Swellengrebel PhD student
Bas Teunissen PhD student
Linda Tulner PhD student
Joost Van Den Berg PhD student
Jolanda Van Den Hoven PhD student
Coen Van Hasselt PhD student
Evita Van Der Steeg PhD student
Mariska Van Vliet PhD student
Annemieke Van Winden PhD student
Carolien Alderden Technical staff
Abadi Gebretensae Technical staff
Michel Hillebrand Technical staff
Ciska Koopman-Kroon Technical staff
Luc Lucas Technical staff
Lianda Nan-Offeringa Technical staff
Joke Schol Technical staff
Bas Thijssen Technical staff
Matthijs Tibben Technical staff
Marja Merqui-Roelvink Clinical trial manager
Sandra Adriaansz Nurse practitioner
Roel Blanken Nurse practitioner
Annelies Boekhout Nurse practitioner
Karina Bucholtz Nurse practitioner
Ria Dubbelman Nurse practitioner
Joke Foekema Nurse practitioner
Emmy Harms Nurse practitioner
Marjo Holtkamp Nurse practitioner
Marianne Keessen Nurse practitioner
Annemieke Koenen Nurse practitioner
Maria Kuiper Nurse practitioner
Lisette Saveur Nurse practitioner
Henk Mallo Nurse practitioner
Annemarie Nol Nurse practitioner
Suzanne Onderwater Nurse practitioner
Margaret Schot Nurse practitioner
Wilma Uyterlinde Nurse practitioner
Jana Van der Sar Nurse practitioner
Dick Visser Nurse practitioner
Marion Zimmerman Nurse practitioner

116
medical oncology
Publications
Aleman BM, de Bruin ML, Dorresteijn
LD, Krol AD, van ’t Veer, Boogerd W, van
Leeuwen FE. Late effects of radiation therapy:
the hits just keep on coming. J Natl Cancer
Inst 2010;102:576-577
Annema JT, Bohoslavsky R, Burgers S,
Smits M, Taal B, Venmans B, Nabers H,
van de Borne B, van Balkom R, Haitjema
T, Welling A, Staaks G, Dekkers OM, van
Tinteren H, Rabe KF. Implementation of
endoscopic ultrasound for lung cancer staging.
Gastrointest Endosc. 2010;71:64-70,70.e1
Baas P, Burgers JA. Longkanker. In: van
der Hoeven JJM, Wagener DTT, Dalmeijer
JP, editors. Het oncologie formularium, een
praktische leidraad. Houten: Bohn Stafl eu
van Loghum, 2010:39-52
Bendle GM, Linnemann C, Hooijkaas AI,
Bies L, de Witte MA, Jorritsma A, Kaiser AD,
Pouw N, Debets R, Kieback E, Uckert W,
Song JY, Haanen JB, Schumacher TN. Lethal
graft-versus-host disease in mouse models
of T cell receptor gene therapy.Nat Med.
2010;16:565-70
Van den Berg JH, Nuijen B, Schumacher TN,
Haanen JB, Storm G, Beijnen JH, Hennink
WE. Synthetic vehicles for DNA vaccination. J
Drug Target. 2010;18:1-14
Van den Berg JH, Oosterhuis K, Hennink
WE, Storm G, van der Aa LJ, Engbersen
JF, Haanen JB, Beijnen JH, Schumacher
TN, Nuijen B. Shielding the cationic charge
of nanoparticle-formulated dermal DNA
vaccines is essential for antigen expression
and immunogenicity. J Control Release.
2010;141:234-40
Van den Berg JH, Oosterhuis K, Beijnen
JH, Nuijen B, Haanen JB. DNA vaccination
in oncology: current status, opportunities
and perspectives. Curr Clin Pharmacol.
2010;5:218-25
Bergman AM, Kerst JM. Rol van, en
ontwikkelingen in, chemotherapie bij
prostaatkanker. Nederlands Tijdschrift voor
Oncologie (submitted)
Bergman AM, Adema AD, Balzarini J,
Bruheim S, Fichtner I, Noordhuis P, Fodstad
O, Myhren F, Sandvold ML, Hendriks
HR, Peters GJ. Antiproliferative activity,
mechanism of action and oral antitumor
activity of CP-4126, a fatty acid derivative
of gemcitabine, in in vitro and in vivo tumor
models. Invest New Drugs. 2010 (in press)
III-2 Pharmacology (PK/PD, ADME, mass balance) of novel anticancer drugs
Currently, we perform twenty-eight phase I/II, pharmacological and proof of concept
studies, which number is stable compared with last year. We recruited more than
240 new patients in these studies this year. Sixteen of these studies are two- or
multicenter studies. Representative examples are described.
a. Studies with the poly-ADP-ribose polymerase (PARP1) inhibitor olaparib (AZD2281)
The ongoing two-center phase I trial to test the safety, optimal dose and schedule
of AZD2281 when given daily BID in combination with three-weekly carboplatin
and paclitaxel, or in combination with weekly paclitaxel has entered its 22 nd doselevel.
We demonstrated promising anticancer activity in a range of tumor types,
but especially patients with tumors harboring BRCA1/2 mutations benefi tted most.
Prolonged myelosuppression is the main toxicity of the combination.
b. Phase I studies with oral fi broblast growth factor receptor (FGFR) inhibitors
Two trials are being executed, with BGJ398 and AZD4547 respectively. The study
with AZD4547 has reached its MTD, which is hyperphosphatemia, skin and nail
toxicity and general malaise. The hyperphosphatemia seems to indicate that the
FGFR mediates phosphate excretion in the kidneys.
c. Studies with angiogenesis inhibitor pazopanib
We continued the trial with the angiogenesis agent pazopanib given in combination
with topotecan. Of interest, signifi cant antitumor activity is observed in this study in
a range of heavily pretreated patients.
d. Other phase I studies
This year we started a number of fi rst-in-man phase I studies, including the novel
androgen receptor down regulating molecule AZD3514, indicated for hormone
refractory prostate cancer. This is an orally available drug given on a daily
continuous schedule. The fi rst-in-man study with the combination of erlotinib and
the PI3K inhibitor GDC0941 progressed. Intermittent dosing of GDC0941, together
with continuous daily dosing of erlotinib, was implemented as continuous dosing
of both compounds was found unsafe. Main toxicity is skin toxicity. This study is
especially of interest for patients progressing but previously responding to erlotinib.
The fi rst in man study with the MEK inhibitor BO21189 was put on hold as the DLT
had been reached. Main toxicity is EGFR-inhibitor-like skin toxicity. This is also a
daily continuous schedule. We continued the phase I study with the Wee1 inhibitor
in combination with or gemcitabine, cisplatin or carboplatin. Main toxicities are
general malaise, nausea and vomiting, which can be controlled well with intensive
antiemetic medication. This concept is currently been tested in tumors with p53
pathway mutation, especially refractory ovarian cancer and advanced melanoma.
III-3 Pharmacokinetic and pharmacodynamic (PK/PD) modelling and simulation
PK/PD modelling and simulation was used to investigate whether a relevant
therapeutic dose was reached in a fi rst-in-human phase I study of a novel integrin
inhibitor (E7820). For this purpose, preclinical data on PK, tumor growth inhibition
in xenografts and biomarker data were combined with clinical biomarker and PK
data. The required level of biomarker inhibition for tumor stasis in the preclinical
experiments was used as target for the phase I study. At the recommended dose level
from the phase I study indeed this level of biomarker inhibition was reached.
A semi-mechanistic PK model for the complex interaction between orally
administered docetaxel and ritonavir has been developed both on human as on
preclinical data. Excellent extrapolation from preclinical experiments to humans
was found using allometric scaling.
In clinical research, often datasets are obtained which contain some degree
of missing data. We investigated several methods to deal with two commonly
encountered types of missing data (data below the limit of quantitation and missing
categorical data) on the outcome of PK/PD analyses and applied these methods to
some real datasets. Appropriate handling of missing data may heavily reduce bias in
PK/PD analyses.

III-4 Pharmacogenomics to identify patients at risk for toxicity or undertreatment
Pharmacogenetic screening of patients treated with 5-FU/Capecitabine.
DYPD*2 genotyping in routine clinical practice was continued. To date a population
of more than 1600 patients has been genotyped prior to start of capecitabine
therapy, amongst which seventeen heterozygotes have been identifi ed. Doseadaptation
could be performed in twelve of these patients. Safety was excellent. No
major toxicities were reported and no toxic deaths occurred. The cost analysis of the
genotyping approach testing demonstrated that the adaptive dosing in DPYD*2A
patients is cost-effective.
IV SUPPORTIVE CARE STUDIES IN PATIENTS WITH BREAST CANCER
The intervention study in female patients with menopausal complaints induced
by treatment for breast cancer with placebo, venlafaxine (Efexor ®) and clonidine
hydrochloride demonstrated that both venlafaxine and clonidine signifi cantly
reduced hot fl ash scores. Side-effects were manageable in most patients
We continued the multicenter cardiac protection trial with the angiotensine II (ATI)
receptor antagonist candesartan in patients with Her 2 positive breast cancer who
received adjuvant treatment with trastuzumab. This is a double blind randomized
trial versus placebo. There are 19 centers actively recruiting and in total 198 of the
planned 200 patients have been registered. The recruitment rate has signifi cantly
increased over the past year.
We started a retrospective analysis to assess the severity of cardiac toxicity induced
by trastuzumab in patients who received trastuzumab in the adjuvant or metastatic
disease setting. Data of more than 130 patients are available for this analysis. Left
ventricular ejection fraction data illustrate that about 20% of the patients who
started with normal LVEF (>50%) drop to values < 50% and 15 % reduction in LVEF.
This confi rms that trastuzumab induces signifi cant cardiac toxicity.
V COLLABORATION WITH THE DUTCH MEDICINES EVALUATION
BOARD
We performed the following dossiers for the CBG/EMA this year: Co-rapporteur
report about erlotinib (Tarceva) for fi rst line treatment of patients with NSCLC with
EGFR activating mutations (type II variation), Rapporteur report about mifamurtide
(Mepact) for osteosarcoma (type II variation) and Co-rapporteur report about
vandetanib (Zactima) for metastasized or locally advanced medullary thyroid cancer.
In addition Scientifi c advices were given about PF00299804 (HER1,2,4 TKI) in
NSCLC, about MGN901 (maytansinoid immunoconjugate) in Merkel cell carcinoma,
about AMG479 (anti IGF-1R antibody) in pancreas cancer, about pazopanib
(Votrient) in NSCLC and ovarian cancer, about RO5185426 (Braf inhibitor) for
metastasized or locally advanced melanoma and about tivantinib (ARQ197; C-Met
inhibitor) for NSCLC
We continued the scientifi c advices to the minister of Health and the Dutch Health
Insurance Authority (NZA) as chairperson of the “Committee for Pharmaceutical
Aid”.
CLINICAL IMMUNOTHERAPY AND TARGETED THERAPY
John Haanen, Christian Blank, Sandra Adriaansz, Henk Mallo, Loes Pronk
BACKGROUND AND OBJECTIVES
The clinical immuno- and targeted therapy group is responsible for the treatment
of melanoma and renal cell carcinoma patients. Translational immunotherapy
research focuses on adoptive cellular therapies, such as T-cell receptor gene therapy
and treatment with tumor-infi ltrating lymphocytes (TIL) for melanoma and DNA
and peptide vaccination studies for HPV-related squamous cell cancers. For renal
cell cancer our group is leading in the development of investigator-initiated phase
II/III trials to improve the treatment with small molecule receptor tyrosine kinase
inhibitors (RTKI), mTOR kinase inhibitors and combinations of cytokines and antiangiogenesis
drugs.
Publications (continued)
117
medical oncology
Bex A, van der Veldt AA, Boven E, Haanen
JB. Re: Surgical resection of renal cell
carcinoma after targeted therapy. Thomas
AA, Rini BI, Stephenson AJ, Garcia JA,
Fergany A, Krishnamurthi V, Novick AC,
Gill IS, Klein EA, Zhou M and Campbell
SC. J Urol 2009;182:881-886. J Urol.
2010;183:1646-7;author reply 1647
Bex A, Sonke GS, Pos FJ, Brandsma D,
Kerst JM, Horenblas S. Symptomatic brain
metatastases from small-cell carcinoma of
the urinary bladder: The Netherlands Cancer
Institute experience and literature review.
Ann Oncol. 2010
Bex A, Van der Veldt AA, Blank C, Meijerink
MR, Boven E, Haanen JB. Progression of
a caval vein thrombus in two patients with
primary renal cell carcinoma on pretreatment
with sunitinib. Acta Oncol. 2010;49:520-3
Borkner L, Kaiser A, van de Kasteele W,
Andreesen R, Mackensen A, Haanen JB,
Schumacher TN, Blank C. RNA interference
targeting programmed death receptor-1
improves immune functions of tumor-specifi c
T cells.Cancer Immunol Immunother.
2010;59:1173-83
Brandsma D, Dorlo TP, Haanen JH, Beijnen
JH, Boogerd W. Severe encephalopathy and
polyneuropathy induced by dichloroacetate J
Neurol. 2010
Buikhuisen WA,Burgers JA, Vincent AD,
Schellens JHM, Beijnen JH, Smit EF,
Joerger M. Pemetrexed Pathway–Associated
Germline Polymorphisms: A Useful Tool for
Treatment Individualization? JCO 2010
Cats A, Verheij M, van Grieken NCT, van de
Velde CJH. Maagcarcinoom. In: Oncologie.
van de Velde, van der Graaf, van Krieken,
Marijnen, Vermorken, eds. Bohn Stafl eu,
2010:353-359 (in press)
Courrech Staal EF, Aleman BM, van
Velthuysen ML, Cats A, Boot H, Jansen
EP, van Coevorden F, van Sandick JW.
Chemoradiation for Esophageal Cancer:
Institutional Experience With Three Different
Regimens. Am J Clin Oncol. 2010
Courrech Staal EF, van Sandick JW, van
Tinteren H, Cats A, Aaronson NK. Healthrelated
quality of life in long-term esophageal
cancer survivors after potentially curative
treatment. J Thorac Cardiovasc Surg.
2010;140:777-83

118
medical oncology
Publications (continued)
Deenen MJ, Terpstra WE, Cats A, Boot H,
Schellens JHM. Standard dose UFT is not a
safe treatment alternative after severe toxicity
to capecitabine or 5-FU in DPD-defi cient
patients. Ann Int Med. 2010 (in press)
Dikken JL, Jansen EP, Cats A, Bakker B,
Hartgrink HH, Kranenbarg EM, Boot H,
Putter H, Peeters KC, van de Velde CJ,
Verheij M. Impact of the extent of surgery
and postoperative chemoradiotherapy on
recurrence patterns in gastric cancer. J Clin
Oncol. 2010;28:2430-6
Douma KFL, Aaronson NK, Vasen HFA,
Gerritsma MA, Gundy CM, Janssen E,
Vriends AHJT, Cats A, Verhoef S, Bleiker
EMA. Psychological distress and use of
professional psychosocial support in families
at high risk for familial adenomatous
polyposis Psycho-Oncology 2010;19:289-298
Douma KF, Bleiker EM, Aaronson NK,
Cats A, Gerritsma MA, Gundy CM,
Vasen HF.Long-term compliance with
endoscopic surveillance advice for familial
adenomatous polyposis (FAP).Colorectal Dis
2010;12(12):1198-207.
Van Erp NP, Mathijssen RH, van der Veldt
AA, Haanen JB, Reyners AK, Eechoute
K, Boven E, Wessels JA, Guchelaar HJ,
Gelderblom H. Myelosuppression by
sunitinib is fl t-3 genotype dependent. Br J
Cancer. 2010;103:757-8
Gadiot J, Hooijkaas AI, Kaiser ADM,
Tinteren H, van Boven H, Blank C.
Overall survival and PD-L1 expression in
metastasized malignant melanoma. Cancer
(in press)
Geurts TW, Klomp HM, Burgers JA,
van Tinteren H, Roukema BY, Balm
AJ. Resection of secondary pulmonary
malignancies in head and neck cancer
patients. J Laryngol Otol. 2010:1-6
Giovannetti E, Zucali PA, Peters GJ, Cortesi
F, D’Incecco A, Smit EF, Falcone A, Burgers
JA, Santoro A, Danesi R, Giaccone G,
Tibaldi C. Association of polymorphisms in
AKT1 and EGFR with clinical outcome and
toxicity in non-small cell lung cancer patients
treated with gefi tinib. Mol Cancer Ther.
2010;9:581-93
MELANOMA
Translational research with DNA vaccination in melanoma patients
In January 2009 we started a fi rst in man study with DNA tattoo vaccination.
In this dose-escalating phase I clinical study advanced-stage HLA-A*0201posiitve
melanoma patients are treated with an in-house manufactured DNA
vaccine, pDERMATT, encoding Tetanus Toxin Fragment C (TTFC) fused to an
immunodominant epitope of the melanocyte differentiation antigen MART-1. The
DNA vaccine (5 mg/ml) is being tattoed into the epidermis of patients on days 0, 3
and 6 and a booster vaccination is administered at days 28, 31 and 34. The starting
dose is 1 mg of DNA applied to a total skin surface of 2 x 2 cm. Dose escalation is
performed by increasing the skin area that is being tattooed (8 cm 2 , 16 cm 2 and 32
cm 2 ). In 2010 the 2nd dose cohort (8 cm 2 ) with three patients was fi nished. The
study is now also open for enrolment at the Leiden University Medical Center. So
far, only local vaccination site toxicity (grade 1 and 2) was observed. In biopsies taken
one week after the last tattoo (both priming and booster) from the vaccination site
showed the presence of CD8+ MART-1-specifi c T cells, indicating that a cellular
immune response had been induced. We are awaiting the fi nal results from
peripheral blood samples (both MART-specifi c and TTFC-specifi c responses).
Translational research with tumor-infiltrating lymphocytes in melanoma patients
We are preparing for a clinical phase II study using tumor-infi ltrating lymphocytes
for patients with metastatic melanoma. This is based on results from a single arm,
single institution trial performed at the Surgery Branch of the NIH, Bethesda,
USA, which showed a 50% objective response rate in heavily pre-treated stage IV
melanoma patients. Recently, these impressive response rates were confi rmed in
a trial performed in Israel, using the exact same protocol as was used at the NIH.
Again a 50% objective response rate was found in heavily pre-treated stage IV
melanoma patients. Ours will be the fi rst trial in Europe with TIL and the fi rst trial
comparing TIL treatment to standard dacarbazine chemotherapy. As of September
2008 the Division of Immunology, Medical Oncology and Pharmacy is working
closely together to prepare for this high
impact trial. A new clean room for the
culturing of TIL according GMP guidelines
has been added to the AM-BTU and all steps
in the production of the TIL product are
being validated (fi gure 2). The full protocol
including IMPD is under review of the
Central Committee on Research involving
Human Subjects (CCMO). We expect to
enrol patients in this study in early 2011.
Anti-CTLA4 treatment
In 2006 we participated (European top includer) in the randomized double-blind
placebo-controlled phase III trial comparing the combination of Ipilimumab, a
fully human anti-CTLA4 monoclonal antibody, and gp100 peptide vaccine, to
Ipilimumab or gp100 vaccine alone. In June 2010, the results were presented
at ASCO, showing a statistical signifi cant and meaningful overall survival
improvement of both Ipilimumab arms compared to gp100 vaccine arm. As of
June 2010, Ipilimumab is available at our institute in a Named Patient Program for
treatment of patients with metastatic melanoma, who have failed 1 st line treatment.
In 2010 over 50 patients have been enrolled in this program and a novel research
project (see section IV) is being developed to analyze the immune response changes
upon Ipilimumab treatment.
BRAF V600E mutated melanoma
In 2010 we participated in the multicenter randomized controlled phase III trial,
BRIM3, comparing standard dacarbazine with an oral BRAF V600E inhibitor
in metastatic melanoma patients with tumors harbouring this BRAF mutation.
Close to 80 patients were screened and in about 40% this mutation was present. 23
patients were randomized. Impressive objective responses were seen, some of which
were unfortunately short-lived. Enrolment was closed in November 2010.

RENAL CELL CARCINOMA
In the renal cell carcinoma program we closely collaborate with Dr Axel Bex from
the urology-oncology group.
In 2005 we started participation in a treatment-use program of the small molecule
sunitinib, a multiple receptor tyrosine kinase inhibitor with high affi nity for
VEGF-R, PDGF-R, c-KIT and FLT3. Since VEGF and PDGF play prominent roles
in the pathogenesis of sporadic clear cell renal cell cancer, inhibition of kinase
activity of their receptors appeared to be a rational therapy in this patient population.
In 2010, still 2 out of 53 patients that were treated in this study were on study
medication, indicating that in a minority of patients sunitinib can induce long-term
disease stabilization. Serum samples from amongst others these 53 patients were
used in a collaborative study to perform a genome wide association study, analyzing
single nucleotide polymorphisms that are correlated with toxicity of sunitinib
treatment.
An investigator-initiated study to defi ne the optimal time point for tumor
nephrectomy in primary metastatic renal cell carcinoma patients is ongoing. A
total of 20 patients have been treated in this small proof of principle trial. The idea
of defi ning the optimal timing for nephrectomy in primary metastatic RCC was
adopted by the EORTC GU group and has been approved as a randomized controlled
multicenter phase III trial (NCT01099423) that has started enrolment in 2010.
Patients will be randomized to receive either 3 cycles of sunitinib treatment prior to
tumor nephrectomy or immediate nephrectomy. Post surgery both groups will (re)
start sunitinib treatment until disease progression. Study endpoint: Progression-free
and overall survival.
In addition, we participated in a phase I study: sorafenib + IL-2 as second line
treatment for metastatic clear cell renal cell cancer and in a randomized controlled
multicenter phase III trial in metastatic clear cell renal cell cancer comparing two
receptor tyrosine kinase inhibitors, pazopanib and sunitinib, the latter being the
standard of care at the moment.
BREAST CANCER THERAPY AND RESPONSE PREDICTION
Sabine Linn, Sjoerd Rodenhuis, Gabe Sonke, Karin Beelen, Jos Beijnen, Jorma De Ronde, Marjo
Holtkamp, Alwin Huitema, Rutger Koornstra, Esther Lips, Ingrid Mandjes, Lennart Mulder, Paula
Nederlof, Margaret Schot, Philip Schouten, Marieke Vollebergh, Jelle Wesseling, Lodewyk Wessels
BACKGROUND AND OBJECTIVES
The objective of this program is to develop and improve potentially curative therapy
for patients with locoregional or oligometastatic breast cancer. For all studies, close
collaborations are maintained with many other clinical departments and research
divisions. In 2010, about 140 patients were entered in sixteen medical studies in
breast cancer, either focusing on the treatment of early breast cancer or on advanced
disease.
PREOPERATIVE CHEMOTHERAPY
The preoperative chemotherapy program continues to accrue over nearly 100
patients per year and now includes data and tissues of some 500 patients. The
program is the core of a multidisciplinary research effort to optimize response
prediction and to improve response monitoring. A pCR (pathologic complete
remission) is rare in luminal tumors (usually characterized by a positive
estrogen receptor and the absence of a HER2 amplifi cation). The expression of
the Progesterone Receptor and the molecular subtype were shown to be weakly
predictive of response but cannot be used to withhold chemotherapy. In addition to
the Dutch multicenter phase II study of Trastuzumab, Carboplatin and Paclitaxel in
HER2+ disease, a second multicenter study was launched for triple-negative breast
cancers (NCT01057069). In October, it had recruited the fi rst 16 patients while
additional centers were still being added to the list of investigators. It studies the
effi cacy of intensive alkylating agents in tumors that have a defect in the DNA repair
mechanism called ‘Homologous ‘Recombination’ in a phase III setting.
Attempts to develop a chemotherapy sensitivity gene expression signature using
mRNA expression arrays have not yet been successful and the same is true for the
Publications (continued)
119
medical oncology
Graafl and NM, Valdés Olmos RA, Teertstra
HJ, Kerst JM, Bergman AM, Horenblas S.
18F-FDG PET/CT for monitoring induction
chemotherapy in patients with primary
inoperable penile carcinoma: fi rst clinical
results. Eur. J. Nucl. Med Mol Imaging.
2010;37:1474-80
Hamberg P, Steeghs N, Loos WJ, van de
Biessen D, den Hollander M, Tascilar
M, Verweij J, Gelderblom H, Sleijfer S.
Decreased exposure to Sunitinib due to
concomitant administration of ifosfamide:
results of a phase I and pharmacokinetic
study on the combination of Sunitinib and
Ifosfamide in patients with advanced solid
malignancies. Br J Cancer. 2010;102:1699-
706
Heemskerk B, Jorritsma A, Gomez-Eerland
R, Toebes M, Haanen JB, Schumacher TN.
Microbead-assisted retroviral transduction
for clinical application.Hum Gene Ther.
2010;21:1335-42
Helgason HH, Engwegen JYMN, Zapatka
M, Vincent A, Cats A, Boot H, Beijnen
JH, Schellens JHM. Identifi cation of serum
proteins as prognostic and predictive markers
of colorectal cancer using surface-enhanced
laser desorption ionization – time of fl ight
mass spectrometry (SELDI-TOF MS).
Oncology Reports 2010;24:57-64
Helgason HH, Engwegen JYMN, Zapatka M,
Cats A, Boot H, Beijnen JH, Schellens JHM.
Serum proteomics and disease-specifi c
biomarkers of patients with advanced
gastric cancer. Oncol Letters 2010;1:327-333
Hodi FS, O’Day SJ, McDermott DF, Weber
RW, Sosman JA, Haanen JB, Gonzalez
R, Robert C, Schadendorf D, Hassel JC,
Akerley W, van den Eertwegh AJ, Lutzky
J, Lorigan P, Vaubel JM, Linette GP, Hogg
D, Ottensmeier CH, Lebbé C, Peschel C,
Quirt I, Clark JI, Wolchok JD, Weber JS,
Tian J, Yellin MJ, Nichol GM, Hoos A, Urba
WJ. Improved survival with ipilimumab in
patients with metastatic melanoma.N Engl J
Med. 2010;363:711-23. Erratum in: N Engl J
Med. 2010;363:1290
Jansen EPM, Boot H, Dubbelman R, Verheij
M, Cats A. Postoperative chemoradiotherapy
in gastric cancer. A phase I-II study of
radiotherapy with dose-escalation of
weekly cisplatin and daily capecitabine
chemotherapy. Ann Oncol, 2010;21:530-534

120
medical oncology
Publications (continued)
Kappers I, van Sandick JW, Burgers JA,
Belderbos JS, van Zandwijk N, Klomp HM.
Surgery after induction chemotherapy in
stage IIIA-N2 non-small cell lung cancer: why
pneumonectomy should be avoided. Lung
Cancer. 2010;68:222-7
Kerst JM, Moonen L, Graafl and NM,
Bergman AM, Pos FJ, Horenblas S. The
role of chemotherapy and radiotherapy in the
treatment of penile cancer. Springer Verlag
(in press)
Kluijt I, Sijmons RH, Hoogerbrugge N,
Vasen HFA, Cats A.Familiaire maagkanker:
diagnostiek en periodieke controles Namens de
Nederlandse werkgroep erfelijke maagkanker.
Ned Tijdschr Geneesk, 2010 (in press)
Knauer M, Mook S, Rutgers EJ, Bender RA,
Hauptmann M, van de Vijver MJ, Koornstra
RH, Bueno-de-Mesquita JM, Linn SC, van
‘t Veer LJ. The predictive value of the 70-gene
signature for adjuvant chemotherapy in
early breast cancer. Breast Cancer Res Treat,
2010;120:655-61
Kok M, Zwart W, Holm C, Fles R,
Hauptmann M, Van ‘t Veer LJ, Wessels LF,
Neefjes J, Stål O, Linn SC, Landberg G,
Michalides R. PKA-induced phosphorylation
of ERalpha at serine 305 and high PAK1 levels
is associated with sensitivity to tamoxifen in
ER-positive breast cancer. Breast Cancer Res
Treat 2010
Kroep JR, Linn SC, Boven E, Bloemendal HJ,
Baas J, Mandjes IAM, van den Bosch J, Smit
WM, de Graaf H, Schröder CP, Vermeulen
GJ, Hop WCJ, Nortier JWR. Lapatinib:
clinical benefi t in patients with HER2positive
advanced breast cancer. Neth J Med,
2010;68:371-6
Lammens CR, Bleiker EM, Aaronson NK,
Wagner A, Sijmons RH, Ausems MG,
Vriends AH, Ruijs MW, van Os TA, Spruijt
L, Gómez García EB, Cats A, Nagtegaal
T, Verhoef S.Regular surveillance for Li-
Fraumeni syndrome: advice, adherence and
perceived benefi ts. Fam Cancer. 2010;9:647-54
Lee SM, Baas P, Wakelee H. Antiangiogenesis
drugs in lung cancer. Respirology.
2010;15:387-92
validation of signatures published by other groups. A chemotherapy resistance test
based on gene expression may be a more realistic goal, as resistance may be caused
by abnormal activity of a single gene. First hypothetical results that should be
testable in future patients have been generated.
ADJUVANT SYSTEMIC THERAPY, PROGNOSIS AND PREDICTION
The primary objective of the Matador study is to identify predictive gene expression
profi les for a disease-free survival benefi t of either a docetaxel-containing regimen
(docetaxel-doxorubicin-cyclophosphamide (TAC)) or an accelerated doxorubicincyclophophamide
(ACdd) regimen (ISRCTN61893718). As such, frozen tissue of the
primary tumor is mandatory to be eligible. The study, open since 2004, is running
in over 30 centers in the Netherlands and ~460 patients of the planned 600 patients
have been included.
The preoperative window trial to study responsiveness of hormone-receptor
positive breast cancers to tamoxifen, anastrozole or anastrozole in combination
with fulvestrant in postmenopausal patients has been extended to other centers
(NCT00738777). Blood and tumor tissue is collected pre and post treatment for
translational research, including gene expression profi ling. Changes in Ki67
expression, in combination with changes in TUNEL labeling as a marker for
apoptosis are used as a read-out for hormonal therapy responsiveness. Chromosome
immune precipitation (ChIP-seq) studies on the fi rst paired samples of patients
exposed to the combination of fulvestrant and anastrozole revealed changes in
estrogen receptor binding to the DNA in over 3000 sites.
In collaboration with Leiden University Medical Center and the TEAM study
group, the TEAM II study has been initiated to investigate the optimal duration of
neoadjuvant exemestane therapy (currently ~40 patients included) and to study the
benefi t of three years adjuvant oral ibandronate in addition to standard adjuvant
systemic therapy in postmenopausal patients with hormone-receptor positive early
breast cancer (currently ~ 500 patients included) (ISRCTN17633610).
THORACIC ONCOLOGY
Paul Baas, Sjaak Burgers, Michel van den Heuvel, Wieneke Buikhuisen, Josine Quispel-Janssen,
Houke Klomp, Michel Wouters, Jose Belderbos, Petra Nederlof, Jacques Neefjes
The department’s clinical research focuses on immunological studies, combined
modality approaches and mesothelioma treatment.
NON SMALL CELL LUNG CANCER (NSCLC)
Patients with resectable early stage NSCLC are included in a neo-adjuvant study with
erlotinib given for 3 weeks. This study is led by Dr Klomp and shows that this short
course of targeted agent treatment already induces signifi cant apoptosis. The study
is now under evaluation with 55 patients entered.
Several I and II clinical trials in locally advanced disease focus on concurrent
chemoradiotherapy and explore the usage of both innovating new radiation
strategies and the implementation of agents such as Cetuximab and Olaparib.
A clinical care pathway has been established and several studies aim to improve
supportive care in this group of patients.
In 3 different vaccination trials are ongoing: a maintenance study with Selectikine
after a short course of RT has just ended; two phase 3 randomized trials, one testing
the peptide vaccine Stimuvax after chemoradiotherapy, the other testing a tumor-cell
vaccin Lucanix in an adjuvant setting.
In second line in advanced NSCLC a national randomized study of erlotinib vs.
erlotinib + chemotherapy is ongoing (PI: S Burgers) with pharmaco-kinetic and
genomic studies.
For 2011 we are planning to construct a SOP in order to profi le tumors with regard
to relevant signalling pathways and chemosensitivity. Finally, tumor tissue form
patients is cultured in mice and as cell lines in order to test multiple compound
libraries and. We hope to elucidate molecular pathways involved in thoracic
malignancies and to develop an ex vivo drug sensitivity screening method.

SMALL CELL LUNG CANCER (SCLC)
In patients with Limited Stage disease we join the European phase 3 CONVERT
study which investigates the effects of concurrent twice daily RT with chemotherapy
vs. sequential chemotherapy and RT. For patients with Extensive Stage a window of
opportunity EORTC phase 2 study with Sunitinib induction therapy has just been
fi nished. The results of this study are being analyzed.
PLEURAL DISEASES
In 2007 the pleura bank study was initiated to allow the development of innovative
diagnostic and treatment algorithms. All patients presenting with a pleural effusion
are asked to participate and to allow research in the near future with frozen serum
and pleural fl uid samples. Currently we have patient data and matched samples in
over 600 patients. A national pleurodesis study has been initiated to compare the
primary pleurodesis with the use of indwelling catheters. A total of 120 patients is
planned for this study.
MALIGNANT PLEURAL MESOTHELIOMA (MPM)
The national randomized phase 3 maintenance study of thalidomide after standard
chemotherapy in MPM is now prepared for publication. A subgroup of patients in
both arms will be analyzed for predictive and prognostic factors of serial serum
samples including VEGF, bFGF, IL-6 and Cytokeratin markers. Independent
radiology monitoring is ongoing.
For second and third line therapy we offer patients a international randomized
phase 3 study of Vorinostat vs. placebo. Of the 660 planned patients we are the top
recruiting centre with over 40 patients included.
We have started a randomized phase1-2 study examining the effect of Axitinib (a
potent oral anti vascular drug) to standard chemotherapy in patients with MPM.
Biopsies are taken before and after 3 courses of chemotherapy and will be used
to evaluate the effect of the addition of Axitinib. The randomized phase II part is
currently ongoing. The translational research in this study will focus on vascular
staining, apoptosis and circulating endothelial/tumor cells.
Fresh tumor samples are currently used to prepare cell lines in the lab of A Berns. In
special immuno-compromised mice human tissue samples are implanted in order to
test specifi c promising compounds.
GASTROENEROLOGY
Henk Boot, Annemieke Cats, Babs Taal, Margot Tesselaar, Hans Bonfrer, Maarten Deenen, Luc
Dewit, Tiny Korse, Jan Schellens, Maurits Swellengrebel, Marcel Verheij
GASTRIC CANCER
New treatment regimens are indicated for the treatment of gastro-esophageal cancer.
In a phase I/II dose-fi nding study we explored the safety and preliminary activity
of the combination of docetaxel, oxaliplatin and capecitabine. In 22 patients treated
at the maximal tolerated dose level, toxicity mostly remained grade ≤ 2, with grade
3 toxicity consisting of fatigue, diarrhea and infection (1 patient each). Overall
response rate was 46% (95% CI: 27-66%). Median progression-free and overall
survival were 6.9 months (95% CI: 5.6 – 8.2) and 11.6 months (95% CI: 8.7 – 14.5),
respectively. The polymorphisms GSTP1 313A>G and CDA 79C>A were individually
predictive for hematological toxicity, and three patients homozygous polymorphic for
GSTP1 313A>G experienced a prolonged progression-free survival.
In another study, we evaluated the effect of gastric surgery and radiotherapy on the
systemic exposure to oral capecitabine and its primary metabolites. Patients with
a total or partial resection absorbed capecitabine signifi cantly more rapidly, and
showed signifi cantly higher peak plasma concentrations of capecitabine, 5’-dFCR
and 5’-dFUR compared with non-gastrectomized patients. Esophagocardiac resection
had no signifi cant effect on capecitabine pharmacokinetics.
RECTAL CANCER
According to national guidelines, neoadjuvant (chemo)radiotherapy should be given
to all rectal cancer patients >T1M0. Between 2006-2008 we evaluated the additional
Publications (continued)
121
medical oncology
Lips EH, Mulder L, Hannemann J, Laddach
N, Vrancken Peeters MT, van de Vijver
MJ, Wesseling J, Nederlof PM, Rodenhuis
S. Indicators of homologous recombination
defi ciency in breast cancer and association
with response to neoadjuvant chemotherapy.
Ann Oncol. 2010
Loo C, Straver ME, Rodenhuis S, Muller S,
Wesseling J, Vrancken Peeters MJ, Gilhuijs
K. MRI response monitoring of breast cancer
during neoadjuvant chemotherapy: Relevance
of breast cancer subtype. J Clin Oncol. 2010
(in press)
Mook S, Knauer M, Bueno-de-Mesquita JM,
Retel VP, Wesseling J, Linn SC, Van’t Veer
LJ, Rutgers EJ. Metastatic Potential of T1
Breast Cancer can be Predicted by the 70-gene
MammaPrint Signature. Ann Surg Oncol
2010
Van Meerbeeck JP, Scherpereel A, Surmont
VF, Baas P. Malignant pleural mesothelioma:
The standard of care and challenges for future
management. Crit Rev Oncol Hematol. 2010
Oosterhuis K, van den Berg JH, Schumacher
TN, Haanen JB. DNA Vaccines and
Intradermal Vaccination by DNA Tattooing.
Curr Top Microbiol Immunol. 2010
Van der Poel HG, Zevenhoven J, Bergman
AM. Pim1 regulates androgen-dependent
survival signaling in prostate cancer cells. Urol
Int. 2010;84:212-20
Quaak SG, Haanen JB, Beijnen JH, Nuijen
B. Naked plasmid DNA formulation: effect
of different disaccharides on stability after
lyophilisation. AAPS PharmSciTech.
2010;11:344-50
Rasch, CR, Hauptmann M, Schornagel J,
Wijers O, Buter J., Gregor T, Wiggenraad
R, De Boer JP, Ackerstaff AH, Kroger R,
Hoebers FJ, Balm AJ, Hilgers FJ. Intraarterila
versus intravenous chemoradiation
for advanced head and neck cancer: Results
of a randomized phase 3 trial. Cancer
2010;116:2159
Retèl VP, Joore MA, Knauer M, Linn
SC, Hauptmann M, Harten WH. Costeffectiveness
of the 70-gene signature versus
Sankt Gallen guidelines and Adjuvant Online
for early breast cancer. Eur J Cancer 2010

122
medical oncology
Publications (continued)
Rodenhuis S, Mandjes IA, Wesseling J, van
de Vijver MJ, Peeters MJ, Sonke GS, Linn
SC. A simple system for grading the response
of breast cancer to neoadjuvant chemotherapy.
Ann Oncol. 2010;21:481-7
De Ronde JJ, Hannemann J, Halfwerk H,
Mulder L, Straver ME, Vrancken Peeters
MJ, Wesseling J, van de Vijver M, Wessels
LF, Rodenhuis S. Concordance of clinical
and molecular breast cancer subtyping in the
context of preoperative chemotherapy response.
Breast Cancer Res Treat. 2010;119:119-26
De Ruiter MB, Reneman L, Boogerd W,
Veltman DJ, van Dam FS, Nederveen
AJ, Boven E, Schagen SB. Cerebral
hyporesponsivenessness and cognitive
impairment 10 years after chemotherapy for
breast cancer. Hum Brain Mapp 2010
Scherpereel A, Astoul P, Baas P, Berghmans
T, Clayson H et a. Guidelines of the European
Respiratory Society and the European Society
of Thoracic Surgeons for the management of
malignant pleural mesothelioma. Eur Respir J
2010;35:479-495
Van Schil PE, Baas P, Gaafar R, Maat
AP, van de Pol M, Hasan B, Klomp HM,
Abdelrahman AM, Welch J, Van Meerbeeck
J; on behalf of the EORTC Lung Cancer
Group. Phase II trial of trimodality therapy
for malignant pleural mesothelioma (EORTC
08031). Eur Respir J. 2010
Schilder CM, Seynaeve C, Linn SC, Boogerd
W, Beex LV, Gundy CM, Nortier JW, van
de Velde CJ, van Dam FS, Schagen SB.
Cognitive functioning of postmenopausal
breast cancer patients before adjuvant systemic
therapy, and its association with medical and
psychological factors. Crit Rev Oncol Hematol
2010;76:133-141
Schilder CM, Seynave C, Beex LV, Boogerd
W, Linn SC, Gundy CM, Huizenga HM,
Nortier JW, van de Velde CJ, van Dam
FS, Schagen SB. Effects of tamoxifen and
exemestane on cognitive functioning of
postmenopausal patients with breast cancer:
results from the neuropsychological side
study of the tamoxifen and exemestane
adjuvant multinational trial. J Clin Oncol
2010;28:1294-1300
value of discussing rectal cancer patients in a multidisciplinary team (MDT) in
the greater Amsterdam region. The primary endpoint was the number of positive
circumferential resections margins (CRM ≤ 1 mm). MDT discussion took place in
116 patients (55%). The proportion of patients with advanced disease was higher in
the MDT+ group (88% ≥T3/N+ versus 68%, p=0.001). The overall CRM+ rate was
13% and did not differ between the MDT+ and the MDT- group.
In locally advanced rectal cancer, we test the feasibility of preoperative chemoradiotherapy
with capecitabine and bevacizumab. Currently, 36 patients are included in
the study and toxicity is evaluated by an independent data monitoring committee.
NEUROENDOCRINE TUMORS (NET)
In patients with hypervascular metastases of NET beyond surgical resection
hepatic artery embolization induces tumor regression. However, embolization
causes hypoxia, which may stimulate angiogenesis and therefore tumor growth.
This hypothesis was studied in 12 patients. Multiple blood samples were drawn
before and daily after embolization. The vascular endothelial growth factor (VEGF),
endothelin-1 (ET-1) and C-terminal pro-Endothelin-1 (CTproET-1) showed indeed a
clear increase at day 6.
GENOTYPING OF THE DPYD GENE IN PATIENTS TREATED WITH
CAPECITABINE
DPYD coding region was sequenced in 45 metastasized colorectal cancer cases
with grade ≥3 capecitabine-related toxicity and in 100 randomly selected controls
having been treated with capecitabine, oxaliplatin, bevacizumab ± cetuximab. DPYD
IVS14+1G>A and 2846A>T showed to be predictive markers for severe toxicity to
capecitabine, for which patients require dose reductions of up to 50%.
Figure 3: Dose modifi cations of capecitabine by genotype. Mean cumulative doses of capecitabine (plus
standard deviations) expressed as a percentage of the planned dose according to the protocol for wild
type and mutant patients for IVS14+1G>A and 2846A>T.

Publications (continued)
Sigmond J, Bergman AM, Leon LG, Loves WJ,
HOebe EK, Peters GJ. Staurosporine increases
toxicity of gemcitabine in non-small cell lung
cancer cells: role of protein kinase C, deoxycytidine
kinase and ribonucleotide reductase. Anticancer
Drugs. 2010;21:591-9
Sleijfer S, Ouali M, van Glabbeke M, Krarup-
Hansen A, Rodenhuis S, Le Cesne A, Hogendoorn
PC, Verweij J, Blay JY. Prognostic and predictive
factors for outcome to fi rst-line ifosfamidecontaining
chemotherapy for adult patients with
advanced soft tissue sarcomas: an exploratory,
retrospective analysis on large series from the European
Organization for Research and Treatment of
Cancer-Soft Tissue and Bone Sarcoma Group
(EORTC-STBSG). Eur J Cancer. 2010;46:72-83
Snoeren N, Voest EE, Bergman AM, Dalesio
O, Verheul HM, Tollenaar RA, van der Sijp JR,
Schouten SB, Borel Rinkes IH, van Hilligersberg
R. A randomized two arm phase III study in
patients post radical resection of liver metastases
of colorectal cancer tot investigate bevacizumab
in combination with capecitabine plus oxaliplatin
(CAPOX) vs CAPOX alone as adjuvant treatment.
BMC Cancer. 2010;10:545
Sonke GS, Wesseling J. Moeten we mammacarcinoom
patiënten met (sub) micrometastasen in de
schildwachtklier adjuvant systemisch behandelen?
Ned Tijdschr Onccol 2010;7:19-20
Sonke GS, Rottenberg S, Linn SC, Jonkers J.
PARP remmers bij de behandeling van BRCA1/2
mutatiedraagsters. Kanker Breed 2010;2:3-8
Stahel R, Baas P, Faivre-Finn C, Dooms C,
Passlick B, Mazières J, Cappuzzo F, Früh M,
Sorensen JB, Blackhall F, Taron M, Gridelli C,
O’Byrne K, Rosell R. Meeting report: 2nd meeting
of the European Thoracic Oncology Platform
(ETOP). Lung Cancer. 2010;68:121-4
Steeghs N, Mathijssen, RH, Wessels JA, de Graan
AJ, van der Straaten T, Mariani M, Laffranchi B,
Comis S, de Jonge MJ, Gelderblom H, Guchelaar
HJ. Infl uence of pharmacogenetic variability on the
pharmacokinetics and toxicity of the aurora kinase
inhibitor danusertib. Invest New Drugs. 2010
Steeghs N, Rabelink Tj, op ‘t Roodt J, Batman E,
Cluitmans FH, Weijl NI, de Koning E, Gelderblom
H. Reversibility of capillary density after
discontinuation of bevacizumab treatment. Ann
Oncol. 2010;21:1100-5
Steeghs N, van Coevorden F, Luykx SA, Cats
A. Complicaties van bevacizumab behandeling.
Nederl Tijdschr Oncol 2010;7:27-36
Straver ME, Glas AM, Hannemann J, Wesseling
J, van de Vijver MJ, Rutgers EJ, Vrancken Peeters
MJ, van Tinteren H, van ‘t Veer LJ, Rodenhuis S.
The 70-gene signature as a response predictor for
neoadjuvant chemotherapy in breast cancer. Breast
Cancer Res Treat. 2010;119:551-8
Straver ME, Rutgers EJ, Rodenhuis S, Linn SC,
Loo CE, Wesseling J, Russell NS, Oldenburg
HS, Antonini N, Vrancken Peeters MT. The
relevance of breast cancer subtypes in the outcome
of neoadjuvant chemotherapy. Ann Surg Oncol.
2010;17:2411-8
Swellengrebe HAM, Marijnen CAM, Vincent A,
Cats A. Evaluating long-term attachment of two
endoclips in the human gastrointestinal tract.
World J Gastroenterol. 2010 (in press)
Swellengrebel HA, Marijnen CA, Verwaal VJ,
Vincent A, Heuff G, Gerhards MF, van Geloven
AA, van Tets WF, Verheij M, Cats A. Toxicity and
complications of preoperative chemoradiotherapy
for locally advanced rectal cancer. Br J Surg. 2010
Tol J, Koopman M, Miller MC, Tibbe A, Cats
A, Creemers GJM, Vos AH, Nagtegaal ID,
Terstappen LWMM, Punt CJA. Circulating tumour
cells early predict progression-free and overall
survival in advanced colorectal cancer patients
treated with chemotherapy and targeted agents.
Ann Oncol 2010;21:1006-12
Vasen HF, Abdirahman M, Brohet R, Langers
AM, Kleibeuker JH, van Kouwen M, Koornstra JJ,
Boot H, Cats A, Dekker E, Sanduleanu S, Poley
JW, Hardwick JC, de Vos Tot Nederveen Cappel
WH, van der Meulen-de Jong AE, Tan TG, Jacobs
MA, Mohamed FL, de Boer SY, van de Meeberg
PC, Verhulst ML, Salemans JM, van Bentem
N, Westerveld BD, Vecht J, Nagengast FM.One
to 2-year surveillance intervals reduce risk of
colorectal cancer in families with Lynch syndrome.
Gastroenterology. 2010;138:2300-6
Van der Veldt AA, Meijerink MR, van den
Eertwegh AJ, Haanen JB, Boven E. Choi response
criteria for early prediction of clinical outcome in
patients with metastatic renal cell cancer treated
with sunitinib. Br J Cancer. 2010;102:803-9
Van der Veldt AA, Eechoute K, Gelderblom H,
Gietema JA, Guchelaar HJ, van Erp N, Van den
Eertwegh AJ, Haanen JB, Mathijssen RH, Wessels
JA. Genetic Polymorphisms Associated With a
Prolonged Progression-Free Survival in Patients
With Metastatic Renal Cell Cancer Treated With
Sunitinib. Clin Cancer Res. 2010
Publications (continued)
123
medical oncology
Vermaat JS, van der Tweel I, Mehra N,
Sleijfer S, Haanen JB, Roodhart JM,
Engwegen JY, Korse CM, Langenberg
MH, Kruit W, Groenewegen G, Giles RH,
Schellens JH, Beijnen JH, Voest EE. Twoprotein
signature of novel serological markers
apolipoprotein-A2 and serum amyloid alpha
predicts prognosis in patients with metastatic
renal cell cancer and improves the currently
used prognostic survival models. Ann Oncol.
2010;21:1472-81
Vollebergh MA, Lips EH, Nederlof PM,
Wessels LFA, Schmidt MK, van Beers EH,
Cornelissen S, Holtkamp M, Froklage FE,
de Vries EGE, Schrama JG, Wesseling J,
van de Vijver MJ, van Tinteren H, de Bruin
M, Hauptmann M, Rodenhuis S, Linn SC.
An aCGH classifi er derived from BRCA1mutated
breast cancer and benefi t of high-dose
platinum-based chemotherapy in HER2negative
breast cancer patients. Ann of Oncol.
2010 (in press)
Vollebergh MA, Kappers I, Klomp HM,
Buning-Kager JC, Korse CM, Hauptmann M,
de Visser KE, van den Heuvel MM,
and Linn SC. Ligands of EGFR and the
insulin-like growth factor family as serum
biomarkers for response to EGFR-inhibitors in
patients with advanced NSCLC. J Thorac Onc
2010 (in press)
Vyth-Dreese FA, Sein J, Van De Kasteele W,
Dellemijn TA, Van Den Bogaard C,
Nooijen WJ, De Gast GC, Haanen JB,
Bex A. Lack of anti-tumour reactivity despite
enhanced numbers of circulating natural
killer T cells in two patients with metastatic
renal cell carcinoma. Clin Exp Immunol.
2010;162:447-59
Van Winden A, van den Broek I, Gast M-C,
Engwegen J, Sparidans R, van Dulken E,
Depla, A, Cats A, Schellens J, Peeters PHM,
Beijnen J, van Gils C. Serum degradome
markers for the detection of breast cancer.
Journal of Proteome Research 2010;9:3781-
3788
Yang TJ, Aukema TS, van Tinteren H,
Burgers S, Valdés Olmos R, Verheij M.
Predicting early chemotherapy response with
technetium-99m methoxyisobutylisonitrile
SPECT/CT in advanced non-small cell lung
cancer. Mol Imaging Biol. 2010;12:174-80

124
radiotherapy
Division head Marcel Verheij
Marcel Verheij MD PhD Head
Berthe Aleman MD PhD Academic staff
Harry Bartelink MD PhD Academic staff
José Belderbos MD PhD Academic staff
Monique Bloemers MD Academic staff
Eugène Damen PhD Academic staff
Roel de Boer PhD Academic staff
Luc Dewit MD PhD Academic staff
Paula Elkhuizen MD PhD Academic staff
Rick Haas MD PhD Academic staff
Olga Hamming-Vrieze MD Academic staff
Wilma Heemsbergen PhD Academic staff
Frank Hoebers MD PhD Academic staff
Edwin Jansen MD PhD Academic staff
Joost Knegjens MD Academic staff
Han Krewinkel MSc Academic staff
Joos Lebesque MD PhD Academic staff
Ben Mijnheer PhD Academic staff
Luc Moonen MD PhD Academic staff
Arash Navran MD Academic staff
Heike Peulen MD Academic staff
Floris Pos MD PhD Academic staff
Coen Rasch MD PhD Academic staff
Babs Reichgelt MD Academic staff
Peter Remeijer PhD Academic staff
Nicola Russell MD PhD Academic staff
Govert Salverda MD Academic staff
Christoph Schneider PhD Academic staff
Jan-Jakob Sonke PhD Academic staff
Joep Stroom PhD Academic staff
Marcel van Herk PhD Academic staff
Baukelien van Triest MD PhD Academic staff
Karijn Verschueren MD Academic staff
Corine van Vliet-Vroegindeweij PhD Academic
staff
Thelma Witteveen MD PhD Academic staff
Frits Wittkämper PhD Academic staff
Gerben Borst MD PhD Temporary staff
Mieke Harrick MD Temporary staff
Birgit Hollmann MD Temporary staff
Monique de Jong MD Temporary staff
Philip Meijnen MD PhD Temporary staff
Stella Mook MD Temporary staff
Marlies Nowee MD PhD Temporary staff
Brenda Tomasoa MD Temporary staff
Femke van der Leij MD Temporary staff
Wouter Vogel MD PhD Temporary staff
Ben Vanneste MD Temporary staff
Brian Vendel MD Temporary staff
Francine Voncken MD Temporary staff
Tanja Alderliesten PhD Post-doc
Chun Chen PhD Post-doc
Alessia Gasparini PhD Post-doc
DIVISION OF RADIOTHERAPY
In 2010, our research activities continued to focus on: (1) improving treatment
accuracy by optimal dose distribution, geometrical precision and dosimetric
treatment verifi cation, (2) enhancing radiation response by altered fractionation
schedules, chemoradiation and biological response modifi ers, and (3) predicting
treatment response and prognosis by genetic profi ling, functional imaging and
biomarkers.
Adaptive strategies (ART) and volumetric rotational radiation delivery (VMAT)
have become standard of care in an increasing number of indications. The in-house
developed 3-dimensional EPID dosimetry is now available for all curatively and
radically treated patients and ensures the safe delivery of the radiation dose. This
innovation ranked third in the National Patient Safety Contest. As a direct result
of these technological advances, extremely hypofractionated regimens such as
stereotactic body radiotherapy (SBRT) have become a valuable alternative to surgery
for early stage disease and oligometastases. To increase patient comfort during
irradiation, one of our treatment suites was equipped with a technology that provides
a personalized visual and acoustic ambience, which is expected to have a positive
impact on patient convenience and stability. An increasing number of targeted
agents fi nd their way to the clinic as potential radiosensitizers. Recently established
collaborations with pharmaceutical companies have resulted in new combined
modality initiatives. To aid the preclinical identifi cation of promising agents and
their optimal scheduling, we have acquired an image-guided microbeam irradiator
for small animals, integrated in the mouse cancer clinic facility. Functional imaging
like FDG-PET and fMRI is increasingly used in our treatment planning protocols
and allows a better prediction of response. New prognostic/predictive genetic
profi les are being explored in early stage laryngeal and breast cancer, contributing to
a more customized therapeutic approach.
In 2010 the division had its quinquennial scientifi c site visit. An international panel
of experts qualifi ed the division as an international leader in the fi eld of radiotherapy
with a very impressive scientifi c program. Useful recommendations will be worked
out in the coming years.
In September 2010, the third scientifi c meeting of the Descartes Cancer Consortium
was organized by the Karolinska Institutet, Sweden. This collaboration of three
European cancer centers (Institut Gustave Roussy, Karolinska Institutet and NKI-
AVL) was founded in 2008 to provide a platform of complementary expertise for
conducting early clinical trials with an emphasis on novel targeted agents. Three
trials are now running simultaneously at the three institutes.
The Radiotherapy department remains an active proponent of introducing proton
therapy in The Netherlands and collaborates with Erasmus MC, Leiden University
MC and TU Delft within the Holland Therapy Center (HollandPTC) consortium.
Four co-workers of the division successfully defended their theses: Edwin Jansen
(chemoradiotherapy in gastric cancer), Dimitry Nuyten (gene expression profi ling in
breast cancer), Monique Smitsmans (IGRT of prostate cancer) and Marcel Steggerda
(prostate brachytherapy).

IMAGE ACQUISITION AND PROCESSING
Tanja Alderliesten Suzanne van Beek, Anja Betgen, Johan de Boer, Roman Bohoslavsky, Kristy
Brock, Niel Burnett, Richard Clarkson1 , Hermine Dees-Ribbers, Andre Dekker, Joop Duppen,
Alessia Gasparini, Rick Haas, Rutger Heddes, Maarten Hulshof, Rianne de Jong, Vincent Khoo1 ,
Simon van Kranen, Philippe Lambin2 , Joos Lebesque, Patricia Lindsay1 , Corrie Marijnen, Angelo
Mencarelli, Cathryn Nagel, Thao-Nguyen Nguyen, Jasper Nijkamp, Steven Petit, Lennert Ploeger,
Floris Pos, Coen Rasch, Theo de Reijke3 , Peter Remeijer, Simon Rit, Peter de Ruiter, Sarah Swift,
Rajko Topolnjak, Corine van Vliet-Vroegindeweij, Marnix Witte, Lambert Zijp, Jan-Jakob Sonke,
Marcel van Herk
1 The Royal Marsden NHS, London, UK
2 MAASTRO clinic, Maastricht, The Netherlands
3 Free University of Amsterdam, The Netherlands
TARGET VOLUME DEFINITION
Microscopic disease extension in 3D for Non Small Cell Lung Cancer:
development of a prediction model using pathology validated PET/CT
features In radiotherapy planning of non-small cell lung cancer (NSCLC),
uncertainties exist concerning the clinical target volume (CTV), meant to cover
potential microscopic disease extension (MDE) around the visible tumor. The aim
of the current study was to establish pre-treatment risk factors for the presence of
MDE, The total tumor-bearing region at pathology is called CTVpath. 34 NSCLC
patients, who underwent CT and PET prior to lobectomy, were included. The
specimen was examined microscopically for MDE and image based prediction
model was developed for the presence of MDE. MDE was found in 17 of the 34
patients (50%). After deformation correction, MDE was less than 26 mm in 90%
of patients. A two-parameter model (mean tumor density and tumor volume on
CT) predicted the presence of MDE (AUC: 0.86). 13 patients (38%, 95% CI: 24-55%)
were identifi ed as low risk for MDE, being potential candidates for reduced intensity
therapy around the GTV. In the low risk group, the effective diameter of the
GTVCT/PET accurately represented the diameter of the CTVpath. In the high-risk
group, CT and PET underestimated the CTVpath by 2-3 cm. In conclusion, CT and
PET accurately visualize the CTVpath in low risk patients, but underestimate the
CTVpath in high-risk patients.
Validation of a mid-position PET scan for treatment planning in radiotherapy
Recently, respiratory correlated PET/CT (4D-PET) became commercially available.
While 4D-PET reduces respiratory induced blurring, noise is increased since
each phase uses fewer counts. We previously developed mid-position (MidP) CT
reconstruction technique (motion compensated CT) for 3D radiotherapy planning
using deformable image registration. To improve the quality of our PET data
we developed a similar technique for 4D PET. 4D PET/CT scans of a dynamic
respiration phantom were taken on a Philips Gemini 16. The phantom contained
four spheres of different sizes (20 ml, 5 ml, 2 ml and 1 ml). The phantom moved
with amplitudes ranging from 0 to 2 cm. To compensate for motion in the PET, we
determined the motion from the 4D CT. Subsequently all voxels in the PET scan
are relocated to the local mean position of the CT motion trajectory. After this,
the 4D PET dataset is averaged, resulting in a 3D MidP PET dataset. To validate
this method, we compared the uncompensated and the motion compensated PET
data for maximum and mean intensity in the spheres (Imax and Imean) and the
apparent sphere size. Compared to a static situation, a motion amplitude of 2 cm
reduced Imax largely (ranging 6% for the 20 ml to 56% for the 1 ml sphere). Motion
compensation reduced the loss of intensity by about 50%. Motion also increased
the apparent size of the spheres. For instance, an amplitude of 2 cm increased the
estimated size of the 5 ml sphere by 37%. After motion compensation, the size
restored to within 2%. The MidP PET is expected to improve PET based radiotherapy
planning (fi gure 1).
Publications (continued)
125
radiotherapy
Marnix Maas PhD Post-doc
Anton Mans PhD Post-doc
Vanessa Mexner PhD Post-doc
Anke van Mourik PhD Post-doc
Raul Pecharroman Gallego PhD Post-doc
Simon Rit PhD Post-doc
Roel Rozendaal PhD Post-doc
Alize Scheenstra PhD Post-doc
Hanno Spreeuw PhD Post-doc
Rajko Topolnjak PhD Post-doc
Markus Wendling PhD Post-doc
Marnix Witte PhD Post-doc
Barry Doodeman Physician assistant
Marcel Jonker Physician assistant
Robin Kalisvaart Physician assistant
Margriet Kwint Physician assistant
Sandra Vreeswijk Physician assistant
Anja Betgen MSc Technical staff
Josien de Bois Technical staff
Joop Duppen Technical staff
Joeri Honnef Technical staff
Rianne de Jong Technical staff
Angelo Mencarelli Technical staff
Tom Minderhoud Technical staff
Danny Minkema Technical staff
Agnieszka Olszewska MSc Technical staff
Carmen Panneman MSc Technical staff
Carolien Peters Technical staff
Kenneth Pengel MSc Technical staff
Lennert Ploeger Technical staff
Maddalena Rossi MSc Technical staff
René Tielenburg Technical staff
Lambert Zijp MSc Technical staff
Figure 1: Depictions of the sphere with
PET-based delineations (based on 40% of
Imax). The spheres were moving vertically.
It can be seen that the shape of the object
in the PET image is improved after motion
compensation. (CT = CT-data, Static
= PET with no amplitude, 2 cm = PET
with 2 cm amplitude. Comp = Motion
compensated PET)

126
radiotherapy
Publications
Alderliesten T, Loo C, Paape A, Muller
S, Rutgers E, Peeters MJ, Gilhuijs K. On
the feasibility of MRI-guided navigation to
demarcate breast cancer for breast-conserving
surgery. Med Phys 2010;37:2617-26
Aleman BM, Haas RL, van der Maazen
RW. Role of radiotherapy in the treatment of
lymphomas of the gastrointestinal tract. Best
Pract Res Clin Gastroenterol. 2010;24:27-34
Aleman BM, De Bruin ML, Dorresteijn
LD, Krol AD, ’t Veer MB, Boogerd W, van
Leeuwen FE. Re: Late effects from radiation
therapy: the hits just keep on coming. J Natl
Cancer Inst 2010;102:576-577
Al-Mamgani, Heemsbergen WD, Levendag
PC, Lebesque JV. Subgroup analysis of
patients with localized prostate cancer treated
within the Dutch-randomized dose escalation
trial. Radiother Oncol 2010;96:13-8
Al-Mamgani, Lebesque JV, Heemsbergen
WD, Tans L, Kirkels WJ, Levendag PC,
Incrocci L. Controversies in the treatment
of high-risk prostate cancer – what is the
optimal combination of hormonal therapy
and radiotherapy: a review of literature.
Prostate 2010;70:701-09
Annema JT, Bohoslavsky R, Burgers S,
Smits M, Taal B, Venmans B, Nabers H,
van de Borne B, van Balkom R, Haitjema
T, Welling A, Staaks G, Dekkers OM, van
Tinteren H, RAbe KF. Implementation of
endoscopic ultrasound for lung cancer staging.
Gastrointest Enosc. 2010;71:64-70
Aukema TS, Rutgers EJ, Vogel WV, Teerstra
HJ, Oldenburg HS, Vrancken Peeters MT,
Wesseling J, Russell NS, Valdés Olmos
RA. The role of FDG PET/CT in patients
with locoregional breast cancer recurrence:
A comparison to conventional imaging
techniques. Eur J Surg Oncol 2010;36:387-392
Aukema TS, Straver ME, Vrancken Peeters
MT, Russell NS, Gilhuijs KG, Vogel WV,
Rutgers EJ, Valdés Olmos RA. Detection of
extra-axillary lymph node involvement with
FDG PET/CT in patients with stage II-III
breast cancer. Eur J Can 2010;46:3205-10
Bartelink H, Meijnen P, Straver ME, Rutgers
EJ. The EORTC experience: from DCIS to
locally advanced breast cancer. IN: Kuerer’s
Breast Surgical Oncology. Chapter 45. Kuerer
HM, Ed. McGraw-Hill, New York 2010
TREATMENT PLANNING
Including rotations in probabilistic IMRT planning for prostate cancer
Probabilistic planning directly includes geometrical uncertainties during
optimization without safety margins, and generally leads to better plans when the
PTV overlaps OARs. Our aim was to develop probabilistic planning for markerbased
prostate IGRT (where rotations are the main residual uncertainties). Using the
prostate’s center of mass as rotation point, the effect of random errors is included
by blurring the dose distribution for every voxel of the CTV. Next, similar to the
Multiple Instance Geometry Approximation, probable CTV instances are calculated
for various systematic rotation errors, and then combined with translational errors.
Optimization based on a confi dence level for a given dose constraint (e.g. DVH
point) is achieved by summation over a predefi ned percentage of lowest cost cases
from all systematic errors (typically 90%).
Clinical prostate treatment plans were replanned with probabilistic objectives. Based
on a marker-match protocol, uncertainties are largest for systematic rotations around
the LR axis (5° SD) and small for translations. Treatment plans from 8 prostate
cancer patients were replanned aiming for the same target coverage. Including
rotations increased optimization time somewhat (3-5 min). The dose distributions
achieved were similar but showed a steeper dose gradient in the rectum region,
compensated by an increase in the anterior section of the prostate. Inclusion of
rotations led to a dose increase in the seminal vesicle area adjacent to the bladder
wall (fi gure 2). Maintaining the same target coverage, dose to the rectum was
reduced by 5.1±1.4Gy (mean dose). This is the fi rst planning method that correctly
includes rotational uncertainties, and inclusion of rotations takes margin-less
planning for prostate RT one step closer to clinical implementation.
Figure 2: Isodose contours
of clinical plan dose (black)
and probabilistic plan dose
(white) for one of the analyzed
patients. Arrows indicate the
effect of including rotational
uncertainties in optimization
Sliding eggoid: an accurate algorithm for anisotropic expansion of organs
A recent survey of seven planning systems found a 5% variation of expanded
volumes. A sliding ellipsoid does not deal correctly with unequal expansion sizes.
The purpose of this work is to develop a fast and accurate expansion algorithm.
We propose a sliding ‘eggoid’: a shape defi ned by an arbitrary ellipsoid in each
octant, with the restriction that the fi rst order derivative of the shape is continuous
at the octant borders. The organs are defi ned as triangulated contours with caps
at both ends at half a slice distance. To accelerate the algorithm, only the triangleedges
are sampled, while the original triangles are displaced according to tangent
of the triangle to the eggoid. On a normal PC (Intel dual core 3.16 GHz) a 10 mm
expansion of a prostate takes 2.1 seconds using a 1.0 mm grid. The accuracy was
verifi ed by calculating the volume of an expanded cube and was for all the above
grids within 0.5% when a cube of 1.0 cm 3 is anisotropically expanded with eggoids
in the range of 10 mm. The novel sliding eggoid algorithm correctly handles
anisotropic expansions.
IMAGE GUIDANCE
Evaluation of ghosting correction in cone beam CT Ghosting artifacts reduce
image quality and Hounsfi eld unit accuracy of cone beam CT (CBCT) scans.
Typically, ghosting shows up in transversal planes of CBCT scans as a partial
ring with high intensity, and is therefore also referred to as a radar artifact. The

magnitude of ghosting depends on patient and scan geometry and is most apparent
when part of the beam is temporarily not attenuated by the patient during gantry
rotation. In this study, the image quality improvements of a previously developed
correction algorithm were evaluated on CBCT scans of different treatment sites. The
parameters for exponential decay were fi tted to the falling edge response of a series
of cone-beam projection images. To evaluate clinical applicability we reconstructed
scans with and without ghosting correction for the treatment sites breast, prostate
and liver, manifesting strong, medium and mild ghosting artifacts. Image quality
was indeed improved for all reconstructions and the radar artifact was almost
completely removed.
In-vivo analysis of interplay effects due to respiration and respiration
irregularities during free-breathing VMAT delivery of SBRT In free-breathing
delivery of lung SBRT with IGRT, the effect of respiratory motion is considered
to be a blurring of the dose distribution that can be addressed by small margins.
VMAT speeds up dose delivery of SBRT and changes the delivery patterns leading
to unknown interplay effects, potentially resulting in unexpected tumor dose
heterogeneity. The purpose of this study was to quantify the interplay effect in-vivo
in individual VMAT patients. We analyzed 5 patients (peak-peak tumor motion
0.2-3 cm, PTV diameter 2.5-6 cm). The analysis uses 3 existing components: 1)
The second to second delivered 3D dose is reconstructed using EPID dosimetry
software. 2) A motion model of the lung is derived from the 4D planning CT by
deformable registration using a phase-based optical fl ow method. 3) A recording
of the respiratory pattern during delivery is obtained by automatic tracking of the
diaphragm position in X-rays acquired for 4D CBCT on the linac. When CBCT
data is acquired simultaneously with treatment, the interplay effect can be totally
quantifi ed in-vivo for individual patients. For this analysis, motion traces acquired
during separate CBCT acquisitions were used as surrogates. The 4DCT motion
model was updated using the diaphragm data as a “navigator channel” to include
respiration irregularities. Next, dose reconstructions were deformed according to
the instantaneous motion model and accumulated to calculate the actual delivered
dose including interplay effects and breathing irregularities. Here we compared
the accumulated dose (with interplay effect) with the original 3D EPID dose, and
with a simple blurred dose (without interplay effects) according to the motion in
the 4D planning CT. With 3 cm motion, the 80% isodose line shifted as expected
by about 0.5 cm in the cumulative dose compared to the 3D dose, while there was
limited interplay effect: a maximum distance to agreement of 0.3 cm. In other cases,
the interplay effect was smaller. In conclusion, we have developed a methodology
to analyze interplay effects in-vivo in individual patients. The tested VMAT
treatments were remarkably robust to the interplay effect and observed respiration
irregularities. Therefore, a simple blurring of the 3D dose distribution based on the
planning PDF accurately predicts the delivered dose.
ADAPTIVE RADIOTHERAPY
Plan adaptation for systematic deformations in head & neck cancer
radiotherapy based on an average patient model A new CT is commonly
acquired to adapt to progressive anatomy changes (e.g. weight loss) during
radiotherapy for H&N cancer patients. However, both the planning and the
repeat CT are snapshots of a variable patient pose and thus introduce systematic
deformations. The purpose of this study was to validate the application of an average
patient model obtained by the modifi cation of the planning CT. Displacement
Vector fi elds (DVF) for 10 patients were obtained by B-spline deformable registration
between the planning CT and daily CBCTs (± 31 fractions per patient). We compared
the following protocols: online couch shifts (as reference), single plan adaptation
with one after N scans, and weekly adaptation based on an from
the previous week. We calculated the average vector length (over all patients) of
the average displacement vector (over all fractions) of bony (B) and soft tissue
(ST) landmarks. Effectiveness of single plan adaptation depended on the number
of measurements, with an optimum at 12, though the overall effectiveness was
modest (B: 1.5 mm, ST: 2.9 mm), indicating progressive changes. Weekly plan
adaptation showed a higher accuracy of (B: 1.2 mm, ST: 2.1 mm) as it performs
Publications (continued)
127
radiotherapy
Beekman CA, Buckle T, van Leeuwen AC,
Valdés Olmos RA, Verheij M, Rottenberg S,
van Leeuwen FW. Questioning the value of
(99m)Tc-HYNIC-annexin V based response
monitoring after docetaxel treatment in a
mouse model for hereditary breast cancer.
Appl Radiat Isot. 2010 (in press)
Bex A, Sonke GS, Pos FJ, Brandsma D,
Kerst JM, Horenblas S. Symptomatic brain
metastases from small-cell carcinoma of the
urinary bladder: The Netherlands Cancer
Institute experience and literature review.
Ann Oncol 2010;11:2240-5
Borst GR, Sonke JJ, Belderbos JS, Lebesque
JV. Normal tissue complication probability
after hypofractionation increased due to
the high dose per fraction or the high total
biological equivalent dose? Radiother Oncol
2010;94:388
Borst GR, Ishikawa M, Nijkamp J,
Hauptmann M, Shirato H, Bengua G,
Onimaru R, Josien Bois AD, Lebesque
JV, Sonke JJ. Radiation pneumonitis after
hypofractionated radiotherapy; evaluation
of the LQ (L) model and different dose
parameters. Int J Radiat Oncol Biol Phys
2010;77:1596-603
Borst GR, Sonke JJ, Hollander SD, Betgen
A, Remeijer P, van Giersbergen A, Russell
NS, Elkhuizen PH, Bartelink H, van Vliet-
Vroegindeweij C. Clinical results of Image-
Guided Deep Inspiration Breath Hold Breast
Irradiation. Int J Radiat Oncol Biol Phys
2010;78:1346-51
Broeks A, Braaf LM, Wessels LF, van de
Vijver M, de Bruin ML, Stovall M, Russell
NS, van Leeuwen FE, van ’t Veer LJ.
Radiation-associated breast tumors display a
distinct gene expression profi le. Int J Radiat
Oncol Biol Phys 2010;76:504-7
Burke SM, van de Giessen E, de Win M,
Schilt T, van Herk M, van den Brink W, Booij
J. Serotonin and dopamine transporters in
relation to neuropsychological functioning,
personality traits and mood in young adult
healthy subjects. Psychol Med 2010;6:1-11
Case RB, Moseley DJ, Sonke JJ, Eccles CL,
Dinniwell RE, Kim J, Bezjak A,Milosevic
M, Brock KK, Dawson LA. Interfraction
and intrafraction changes in amplitude
of breathing motion in stereotactic liver
radiotherapy. Int J Radiat Oncol Biol Phys
2010;77:918-925

128
radiotherapy
Publications (continued)
Chai X, van Herk M, van de Kamer JB,
Remeijer P, Bex A, Betgen A, De Reijke
TM, Hulshof MC, Pos FJ, Bel A. Behavior
of Lipiodol Markers During Image Guided
Radiotherapy of Bladder Cancer. Int J Radiat
Oncol Biol Phys 2010;77:309-14
Courrech Staal EF, Aleman BM, Boot H,
van Velthuysen MF, van Tinteren H, van
Sandick JW. Systematic review of the benefi ts
and risks of neoadjuvant chemoradiation
for oesophageal cancer. British J of Surg
2010;97:1482-96
Courrech Staal EF, Aleman BM, van
Velthuysen ML, Cats A, Boot H, Jansen
EP, van Coevorden F, van Sandick JW.
Chemoradiation for esophageal cancer:
Institutional experience with three different
regimens. Am J Clin Oncol 2010 (in press)
de Jong MC, Pramana J, Knegjens JL, Balm
AJ, van den Brekel MW, Hauptmann M,
Begg AC, Rasch CR. HPV and high-risk
gene expression profi les predict response to
chemoradiotherapy in head and neck cancer,
independent of clinical factors. Radiother
Oncol 2010;95:365-70
de Jong MC, Pramana J, van der Wal JE,
Lacko M, Peutz-Kootstra CJ, de Jong JM,
Takes RP, Kaanders JH, van der Laan BF,
Wachters J, Jansen JC, Rasch CR, van
Velthuysen ML, Grenman RA, Hoebers FJ,
Schuuring E, van den Brekel MW, Begg AC.
CD44 expression predicts local recurrence
after radiotherapy in larynx cancer. Clin
Cancer Res 2010;16:5329-38
de Vreeze R, de Jong D, Nederlof P, Ruijter
HJ, Boerrigter L, Haas R, van Coevorden F.
Multifocal myxoid liposarcoma-metastasis
or second primary tumor?: a molecular
biological analysis. J Mol Diagn.
2010;12:238-43
de Vreeze RS, de Jong D, Nederlof PM,
Ariaens A, Tielen IH, Frenken L, Haas RL,
van Coevorden F. Added value of molecular
biological analysis in diagnosis and clinical
management of liposarcoma: A 30-Year
single-institution experience. Ann. Surg
Oncol 2010;17:683-93
de Vreeze RS, de Jong D, Koops W, Nederlof
PM, Ariaens, Haas RL, van Coevorden R.
Oncogenesis and classifi cation of mixed-type
liposarcoma: a radiological, histopathological
and molecular biological analysis. Int J
Cancer 2010 (in press)
better on progressive changes. For patients with overall systematic deformations
≥ 3mm, weekly adaptation reduced systematic deformations by more than 45%.
Inability of the deformable registration method to register sliding tissue (tip of uvula
and epiglottis occasionally led to large systematic ST errors. We conclude that the
benefi ts of this method are potentially high for patients with large deformations.
FOLLOW-UP RESEARCH
Post chemoradiation FDG-PET derived local radiation dose effect relation for
oesophageal toxicity in NSCLC patients Non Small Cell Lung Cancer (NSCLC)
patients undergoing concurrent chemoradiation (CRT) using IMRT in our institute,
often develop severe acute radiation oesophagitis. The purpose of this work was to
correlate the planned dose distribution with the post-CRT local oesophageal FDG-
PET uptake as a surrogate for oesophageal toxicity to improve our understanding of
the dose effect relationship. Ten NSCLC patients (2 groups: 5 patients with grade 1-2,
and 5 patients with grade 3 oesophageal toxicity) partaking in a phase II combined
modality treatment study (concurrent chemoradiation with cetuximab) were selected
from a total of 43 study patients. Patients were selected where the oesophagus was
within the planning target volume (PTV) and an FDG-PET/CT scan was performed
approximately 30 days post-CRT. IMRT was combined with daily cisplatin and
weekly cetuximab. The planned dose was sampled on the oesophagus surface. The
post-CRT-PET scan was recalculated to standard uptake values (SUV) and sampled
on the oesophagus surface to establish the oesophageal dose to FDG-PET SUV
relationship. The mean PET SUV for the oesophagus surface was signifi cantly
higher in the grade 3 oesophageal toxicity group compared to the grade 1-2 group (2.7
vs. 2.1, p=0.04 1-sided t-test). A local dose-effect relationship was observed with a
sharp increase in average oesophageal FDG-PET activity at dose levels above 60 Gy.
At these high dose levels signifi cant differences in planned oesophageal dose were
present between patients with grade 1-2 and grade 3 toxicity. The results indicate
that elevated SUV post-CRT in the oesophagus is correlated with severe acute
oesophageal toxicity and that local FDG-PET uptake values mostly increase at EQD2
levels above 60 Gy. These observations indicate that the previously used predictor
V35 may not be accurate for oesphagus toxicity after IMRT (fi gure 3).
Figure 3: Average relative
oesophagus dose-surface
histogram for patients with
grade 3 toxicity (thick black)
and patients with grade 1-2
toxicity (thick white) and their
95% CI (thin lines).
The line with square markers
denotes the relationship
between planned dose at the
surface of the oesophagus and
the PET Standard Uptake
Value measured at that same
point post-RT, error bars are
95% CI.
Use of principal component analysis to analyze inter-patient dose variation
Dose variation between prostate cancer patients is the result of a limited
number of factors, such as local differences in the shape, position and volume
prostate and seminal vesicles. The purpose of this study is to analyze the interpatient
dose variation using principal component analysis (PCA), in relation to
outcome. A subgroup of 67 patients with localized prostate cancer and a high
risk for extraprostatic disease was selected from the Dutch trial. Failure (n=30)
was biochemical or clinical determined at a 4 year endpoint. For each patient a
3-dimensional dose map was created around the prostate, on a fi xed grid of dose
points. Afterwards, we applied PCA to obtain the uncorrelated principal components
(PCs). Every PC represents a weighted linear combination of dose voxels. Pearson
correlation coeffi cients were then computed between these PCs and several factors.

Finally, the logistic regression model was used to evaluate the predicted value of
the PCs on failure. The fi rst three PCs explain the most variation (37.5%, 15.4% and
7.6%). All three PCs appear to correlate with the length of the delineated seminal
vesicles in LR, CC or AP directions: PC1 mainly correlates with CC direction
(R=0.65, P

130
radiotherapy
Figure 5: Locations of the local recurrences
in the RT (top) and no –RT (bottom)
group colored for NO and CRM – (black
dots and N+ and /or CRM+ (white dots)
Top plane indicates the cranial border of
the used RT fi elds, while the bottom plane
indicates the S2 - S3 inter - space level.
Figure 6: a) PTV DVHs from planned
dose distribution (`A’), EPID reconstructed
dose distributions of fractions 1–3
(`B’–`D’) and total EPID reconstructed
dose distribution (fractions 1–5) (`E’). (b)
Axial plane from a 3D -evaluation of the
fi rst fraction, intersecting the isocenter; the
PTV is indicated.
treated in the Dutch total mesorectal excision (TME) trial, either with a TME alone
(no-RT) or with 5x5 Gy RT followed by a TME (RT), with a local recurrence were
analyzed. A local recurrence was found in 114 of 1417 patients in the trial. For 94
(25 RT; 69 no-RT) recurrences the location was adequately known to be placed in a
3D pelvic model. The infl uence of preoperative RT on patterns of local recurrence
was evaluated. The use of RT mainly reduces anastomotic, lateral and perineal
recurrences. In patients with N0 disease and a negative CRM only one recurrence
was found cranially of the S2-S3 inter-space (fi gure 5). This was for an RT patient
and only 2 lymph nodes were examined during pathology. A reduction of the CTV
border cranially to the S2-S3 inter-space is proposed which would lower the cranial
border by approximately 4 cm while a maximum increase in local recurrence rate
of 0.2% can be expected. With the proposed CTV, a reduction in absolute small
bowel exposure (>15 Gy) of approximately 40% and 60% could be achieved for 3-fi eld
conformal- and IMRT, respectively.
PRECLINICAL IMAGING
Performance characterization of a high-resolution imaging system for small
animal radiation research µIGRT systems bridge the technology gap between
pre-clinical radiation research and clinical radiation therapy by combining a high
accuracy CBCT imaging system and a high dose therapeutic X-ray source on the
same platform. The purpose of this research was to quantify the CBCT image
quality improvements of a new fl at panel imager (FPI).
We evaluated µIGRT systems: Unit 1 contains a FPI with a Gd2O3 scintillator and a
pixel pitch of 400 mm (512x512 pixels), Unit 2 contains a FPI with a CsI scintillator
and a 200 mm pixel pitch (1024x1024 pixels). Image uniformity, system spatial
resolution and contrast to noise ratio (CNR) were studied on a µCT image quality
phantom.
The full width at half maximum of the modulated transfer function (MTF)
increased from 0.7 mm-1 of unit 1 to 2.1 mm-1 for unit 2. The MTF value at 10% of
the maximum indicates that structures of 200 µm can be resolved with Unit 2. CNR
was about 30% lower in unit 2 for the same voxel size. Image uniformity was similar
between the two units with variations of about 4%.
EPID DOSIMETRY
Priscilla Camargo, Anton Mans, Igor Olaciregui-Ruiz, Raul Pecharromán-Gallego,
Jan-Jakob Sonke, Joep Stroom, Marcel van Herk, Ben Mijnheer
The potential for incidents and the ever-increasing complexity of patient treatments
emphasize the need for accurate dosimetric verifi cation in radiotherapy. For this
reason, all curative treatments are verifi ed by EPID (Electronic Portal Imaging
Device) dosimetry. Since the clinical introduction of the method in February 2005
treatment plans of more than 5000 patients have been verifi ed. The results of
these measurements have recently been analyzed. Among these plans, 17 serious
errors were detected that have led to an intervention. Due to their origin, 9 of these
errors would not have been detected with pre-treatment verifi cation, including
a plan transfer error detected in a 5x5 Gy IMRT rectum treatment (see fi gure 6).
Investigation of the plan transfer chain revealed that due to a network transfer error
(“lost delayed write data”) the plan was corrupted. 3D analysis of the acquired EPID
data revealed serious under-dosage of the PTV: on average 11.6%, locally up to 20%.
The information was used by the physician to determine whether and to what extent

compensatory measures were needed in the remaining four fractions. This analysis
shows the importance of in vivo (EPID) dosimetry for all treatment plans as well as
the ability of our method to assess the dosimetric impact of deviations found.
Volumetric-modulated arc therapy (VMAT) of prostate and lung cancer has
been introduced in our department in 2009. VMAT is a complex technique in
which gantry speed, fi eld shape and dose rate are continuously varied during
gantry rotation. Consequently, accurate verifi cation is essential for a safe clinical
implementation of VMAT. EPID images obtained during VMAT delivery using
gantry-angle resolved data acquisition are converted to 3D dose distributions inside
a phantom or patient using our back-projection model. In addition to verifi cation of
the dose at the isocentre, 3D gamma analysis is performed to compare planned and
EPID-reconstructed 3D VMAT dose distributions. Gamma evaluation parameters
are mean and maximum (1%) gamma and % gamma < 1. VMAT plans of 10 prostate
and 10 lung cancer patients, verifi ed by means of 3D EPID dosimetry, have been
analysed. The average difference of the dose at the isocentre was for prostate cancer
VMAT -0.2 ± 1.6 % and +0.4 ± 1.6 %, pre-treatment and in vivo, respectively, and
for the hypo-fractionated lung cancer VMAT plans +2.0 ± 1.2 % and -0.7 ± 2.3 %.
3D gamma evaluation showed almost the same results for pre-treatment and in vivo
verifi cation of the 3D dose distribution for prostate cancer VMAT, while somewhat
larger deviations were observed for lung cancer VMAT verifi cation. The results of
this study show that EPID dosimetry is a valuable tool for 3D verifi cation of VMAT
delivery, both pre-treatment and in vivo.
Currently the transmission of the photon beam through a phantom or patient,
an essential ingredient of the back-projection model, is estimated from the ratio
of EPID measurements with and without a phantom/patient in the beam. Thus,
an additional irradiation to obtain “open images” under the same conditions
as the actual phantom/patient irradiation is required. However, by calculating
the transmission in the direction of the beam instead of using “open images”,
this extra measurement can be avoided. This was achieved by using a model
that includes the effect of beam hardening and off-axis dependence of the EPID
response. The parameters in the model were empirically obtained by performing
EPID measurements using polystyrene slab phantoms of different thickness. A
theoretical analysis to verify the sensitivity of the model with patient thickness
changes was also performed. The new model was applied for EPID dose verifi cation
measurements of various IMRT treatments. The results showed generally good
agreement with the dose determined using the measured transmission. The average
difference between EPID-based in vivo dose at the isocentre determined using either
the new model for transmission or its measured value, was 2.6 ± 3.1, 0.2 ± 3.1 and
2.2 ± 3.9 %, for 47 patients treated with 6, 10 and 18 MV IMRT beams, respectively.
For a sub-group of 11 patients pre-treatment verifi cation was also performed showing
similar dose differences at the isocentre: -1.9 ± 0.9, -1.4 ± 1.2 and -0.4 ± 2.4 %.
It can be concluded that calculating instead of measuring the transmission leads
to differences in dose generally smaller than 2 % and yielded only slightly higher
gamma evaluation parameter values. The new model is now tested clinically for a
large variety of treatment sites.
Our current EPID-based back-projection dose reconstruction model had to be
modifi ed to make it also applicable to wedged beams. For this purpose an energydependent
correction factor, taking into account the changes in beam quality caused
by the wedge and the presence of a phantom or patient, was inserted in the EPID
dose-response function. The resulting reconstructed dose distribution was in good
agreement with the planned dose in a plane at 10 cm depth in a patient but does not
take the depth dependence of the wedge factor into account. A start has been made
to extend the wedge correction model for 3D dose verifi cation of wedged beams.
TREATMENT PLANNING
Corine van Vliet-Vroegindeweij, Andrea Holt, Tomas Janssen, Zdenko van Kesteren, Emmy Lamers,
Annemarie Lakeman, Angela Tijhuis, Suzanne den Hollander, Anton Mans, Anke van Mourik,
Roman Bohoslavsky, Marnix Witte, José Belderbos, Coen Rasch, Joost Knegjens, Jan-Jakob Sonke,
Paula Elkhuizen, Tanja Alderliesten, Jonathan Yang, Eugène Damen
Publications (continued)
131
radiotherapy
Jansen EP, Boot H, Dubbelman R, Verheij
M, Cats A. Postoperative chemoradiotherapy
in gastric cancer –a phase I-II study
of radiotherapy with dose escalation of
weekly cisplatin and daily capecitabine
chemotherapy. Ann Oncol. 2010;21:530-4
Jansen EPM, Nijkamp J, Gubanski M, Lind
PARM, Verheij M. Interobserver variation
of clinical target volume delineation in
gastric cancer. Int J Radiat Oncol Biol Phys
2010;77:1166-70
Kappers I, van Sandick JW, Burgers SA,
Belderbos JS, van ZN, Klomp HM. Surgery
after induction chemotherapy in stage
IIIA-N2 non-small cell lung cancer: Why
pneumonectomy should be avoided. Lung
Cancer 2010;68:222-7
Knauer M, Mook S, Rutgers EJ, Bender RA,
Hauptmann M, van de Vijver MJ, Koornstra
RH, Bueno-de-Mesquita JM, Linn SC, van
’t Veer LJ. The predictive value of the 70-gene
signature for adjuvant chemotherapy in
early breast cancer. Breast Cancer Res Treat
2010;120:655-61
Knegjens JL, Hauptmann M, Pameijer FA,
Balm AJ, Hoebers FJ, de Bois JA, Kaanders
JH et a. Tumor volume as prognostic factor in
chemoradiation for advanced head and neck
cancer. Head & Neck, 2010;1-9
Kreike B, Hart G, Bartelink H, van de Vijver
MJ. Analysis of breast cancer related gene
expression using natural splines and the
Cox proportional hazard model to identify
prognostic associations. Breast Cancer Res
Treat 2010;122:711-20
Le C, van de Weijer EP, Pos FJ, Vogel WV.
Active infl ammation in 18F-methylcholine
PET/CT. Eur J Nucl Med Mol Imaging
2010;37:654-655
Mann RM, Loo CE, Wobbes T, Bult P,
Barentsz JO, Gilhuijs KGA, Boetes C. The
impact of preoperative breast MRI on the
re-excision rate in invasive lobular carcinoma
of the breast. Breast Cancer Res Treat
2010;119:415-422
Mans A, Remeijer P, Olaciregui-Ruiz I,
Wendling M, Sonke JJ, Mijnheer B, van Herk
M, Stroom JC. 3D Dosimetric verifi cation of
volumetric-modulated arc therapy by portal
dosimetry. Radiother Oncol. 2010;94:181-7

132
radiotherapy
Publications (continued)
Mans A, Wendling M, McDermott LN,
Sonke JJ, Tielenburg R, Vijlbrief R, Mijnheer
B, van Herk M, Stroom JC. Catching errors
with in vivo EPID dosimetry. Med Phys
2010;37:2638-44
Matzinger O, Heimsoth I, Poortmans P,
Collette L, Struikmans H, Van Den Bogaert
W, Fourquet A, Bartelink H, Ataman F,
Gulyban A, Pierart M, Van Tienhoven
G; EORTC Radiation Oncology & Breast
Cancer Groups. Toxicity at three years
with and without irradiation of the internal
mammary and medial supraclaviculair
lymph node chain in stage I to III breast
cancer (EORTC trial 22922/10925). Acta
Oncol 2010;49:24-34
Mook S, Knauer M, Bueno-de-Mesquita JM,
Retel VP, Wesselink J, Linn SC, van ’t Veer
LJ, Rutgers EJ. Metastatic Potential of T1
Breast Cancer can be predicted by the 70-gene
MammaPrint Signature. Ann Surg Oncol
2010;17:1406-13
Mook S, Schmidt MK, Weigelt B, Kreike B,
Eekhout I, van de Vijver MJ, Glas AM, Floore
A, Rutgers EK, van ’t Veer LJ. The 70-gene
prognosis signature predicts early metastasis
in breast cancer patients between 55 and 70
years of age. Ann Surg Oncol 2010;21:717-22
Nijkamp J, Kusters M, Beets-Tan RGH,
Martijn H, Beets GL, Van de Velde CJH,
Marijnen CAM. Three-dimensional analysis
of recurrence patterns in rectal cancer:
The cranial border in hypofractionated
preoperative radiotherapy can be lowered. Int
J Radiat Oncol Biol Phys 2010 (in press)
Okines A, Verheij M, Allum W, Cunningham
D & Cervantes A on behalf of the ESMO
guidelines working group. Gastric cancer:
ESMO clinical practice guidelines for
diagnosis, treatment and follow-up. Annals of
Oncol 2010;21;50-54
Oldenborg S, Van Os RM, Van Rij CM,
Crezee J, Van de Kamer JB, Rutgers EJ,
Geijsen ED, Zum vörde sive vörding PJ,
Koning CC, Van Tienhoven G. Elective
re-irradiation and hyperthermia following
resection of persistent locoregional recurrent
breast cancer: A retrospective study. Int J
Hyperthermia 2010;26:136-44
Pos F, Remeijer P. Adaptive management of
bladder cancer radiotherapy. Semin Radiat
Oncol 2010;20:116-120
Biological Target Volume (BTV) boost phase II study on dose-escalation of
the high uptake FDG-PET regions inside the primary tumor for NSCLC
Recurrences in NSCLC occur mainly in the high FDG uptake regions visible on the
pre-treatment CT-PET scan. Dose-escalation of these high FDG uptake regions
might improve local control and overall survival. In cooperation with Maastro clinic,
we therefore designed a randomized phase II trial with 2 treatment arms: doseescalation
up to the normal tissue constraints using a simultaneous integrated boost
(SIB) to the entire primary tumour (Arm A) or to the regions inside the primary
tumour having an FDG-uptake ≥ 50% of maximum SUV (Arm B). A treatment
planning study was performed on 7 patients to investigate the feasibility of this trial
design. Current clinical plans deliver a dose of 66 Gy in 24 fractions to the primary
tumor. Dose-escalation is planned as a SIB up to a maximum fraction dose of 5.5 Gy
based on the normal tissue constraints; e.g. mediastinal structures: max. 94 Gy,
brachial plexus: max. 66 Gy, lungs: MLD 20 Gy, spinal cord: max. 52 Gy, all doses in
EQD2.
In 2 patients the brachial plexus restricted dose-escalation, and for 2 patients the
acceptable lung toxicity was reached already at the conventional dose level of 66 Gy
due to the large volume of the PTV. For 3 patients, the dose-escalation was planned
for both treatment arms. Mean dose-escalation levels for the entire PTV (Arm A)
were up to 73 Gy, 74 Gy and 101 Gy, whereas higher levels of dose-escalation were
obtained in Arm B: 76 Gy, 77 Gy and 134 Gy, respectively. The normal tissues
limiting further dose-escalation were lung (N=2) and the mediastinal structures
(N=3). For one patient in Arm B, the tumour dose reached the maximum allowed
dose-escalation.
Based on these results, we conclude that dose-escalation using a SIB to the high
FDG uptake regions or the entire primary tumour is feasible. Because of the size of
the primary tumour in stage II/III NSCLC, dose-escalation is frequently restricted
by the normal mediastinal structures (blood vessels), mean lung dose and the
brachial plexus.
The fi rst patient entered the phase II randomized trial in May 2010 and 9 patients
have been entered so far.
VMAT for stereotactic body radiation therapy of lung tumors: a comparison
with conventional IMRT techniques For a random selection of 27 patients eligible
for SBRT, we generated treatment plans for three different treatment techniques:
1. a coplanar VMAT technique (using the SmartArc module in Pinnacle 3 ), 2. our
clinically used non-coplanar IMRT technique including typically 12-16 non-coplanar
beams, and 3) a coplanar IMRT technique including 9 equidistant beams. A dose of
3x18 Gy was prescribed to the isodose encompassing 95% of the PTV. The dose to
healthy lung tissue was judged using the biologically equivalent dose in fractions of
2 Gy (EQD 2) using the linear-quadratic model with / = 3 Gy and should not exceed
16 Gy. The maximum dose for OAR’s should be below physical doses of 18 Gy for the
spinal cord, 30 Gy for the heart, 27 Gy for the esophagus, and 24 Gy for the brachial
plexus.
Clinically acceptable VMAT plans could be prepared using two coplanar arcs, where
radiation entering through the contralateral lung was avoided. Treatment delivery
times for VMAT could be reduced to an average of 6.6 minutes as compared to 23.7
minutes for non-coplanar IMRT. The mean dose to healthy lung in EQD 2 was found
to be very similar for VMAT (8.5 Gy) and non-coplanar IMRT (8.4 Gy), and slightly
increased for coplanar IMRT (8.9 Gy). The dose conformity of the 54 Gy isodose
level was 1.13 for VMAT and 1.11 for non-coplanar IMRT and 1.12 for coplanar
IMRT. For the lower isodose levels the conformity was found to be slightly better for
non-coplanar IMRT with 4.8 vs. 5.2 for VMAT at the 27 Gy isodose level. Sparing
of OAR’s including spinal cord, esophagus, heart, brachial plexus and large blood
vessels can be achieved with VMAT at levels comparable to and sometimes even
better than with non-coplanar IMRT.
We concluded that coplanar VMAT for SBRT of early-stage lung cancer achieved a
plan quality and sparing of OAR’s comparable to non-coplanar IMRT and slightly
better to that of coplanar IMRT, while delivery time can be reduced by up to 70%
with VMAT. Based on these results, VMAT for SBRT of lung cancer has been
successfully implemented in our clinic.

Sequentially delivered boost plans are superior to simultaneously delivered
plans in head-and-neck cancer when the boost volume is located further away
from the parotid glands We evaluated 50 recently treated head and neck cancer
patients. Apart from the clinical plan with a sequentially (SEQ) given boost using
an IMRT technique, a simultaneous integrated boost (SIB) technique plan was
constructed with the same beam set-up. The mean dose to the parotid glands was
compared. The elective nodal areas were bilateral in all cases, with a boost on either
one side or both sides of the neck.
When the parotid gland volume and the Planning Target Volume (PTV) for the
boost overlap, there is on average a lower dose to the parotid gland with a SIB
technique (-1.2 Gy) which is however not signifi cant (p=0.08). For all parotid glands
with no boost PTV overlap, there is a benefi t from a SEQ technique compared
to a SIB technique for the gland evaluated (on average a 2.5 Gy lower dose to the
parotid gland, p

134
radiotherapy
Publications (continued)
Straver ME, Loo CE, Rutgers EJ, Oldenburg
HS, Wesseling J, Vrancken Peeters MJ,
Gilhuijs KG. MRI-Model to Guide the
Surgical Treatment in Breast Cancer Patients
After Neoadjuvant Chemotherapy. Ann Surg
2010;251:701-7
Straver ME, Meijnen P, van Tienhoven
G, van de Velde CJ, Mansel RE, Bogaerts
J, Duez N, Cataliotti L, Klinkenbijl JH,
Westenberg HA, van der Mijle H, Snoi M,
Hurkmans C, Rutgers EJ. Sentinel node
identifi cation rate and nodal involvement in
the EORTC 10981-22023 AMAROS trial.
Ann Surg 2010;17:1854-61
Straver ME, Rutgers EJ, Rodenhuis S,
Linn SC, Loo CE, Wesseling J, Russell NS,
Oldenburg HS, Antonini N, Vrancken
Peeters MT. The relevance of breast cancer
subtypes in the outcome of neoadjuvant
chemotherapy. Ann Surg Oncol
2010;17:2411-8
Swellengrebel HA, Marijnen CA, Verwaal
VJ, Vincent A, Heuff G, Gerhards MF, van
Geloven AA, van Tets WF, Verheij M, Cats
A. Toxicity and complications of preoperative
chemoradiotherapy for locally advanced rectal
cancer. Br J Surg. 2010;98:418-26
Teertstra HJ, Loo CE, van den Bosch
MA, van Tinteren H, Rutgers EJ, Muller
SH, Gilhuijs KG. Breast tomosynthesis in
clinical practice: initial results. Eur Radiol
2010;20:16-24
Topolnjak R, Sonke JJ, Nijkamp J, Rasch
C, Minkema D, Remeijer P, van Vliet-
Vroegindeweij. Breast patient setup error
assessment: comparison of electronic portal
image devices and cone-beam computed
tomography matching results. Int J Radiat
Oncol Biol Phys 2010;78:1235-43
van Beek S, van Kranen S, Mencarelli A,
Remeijer P, Rasch C, van Herk M, Sonke
JJ. First clinical experience with a multiple
region of interest registration and correction
method in radiotherapy of head-andneck
cancer patients. Radiother Oncol
2010;94:213-7
van Blitterswijk WJ, Klarenbeek JB, van der
Luit AH, Alderliesten MC, van Lummel
M, Verheij M. Fas/CD95 down-regulation
in lymphoma cells through acquired
alkyllysophospholipid resistance: partial
role of associated sphingomyelin defi ciency.
Biochem J 2010;425:225-234
(CT 5) of RT were acquired. The mean seroma volume for CT 1 was 63cc (range: 18-
218cc). All patients were planned using the clinical SIB technique (28 x 1.81Gy to
the whole breast and additionally 28 x 0.49Gy to the boost volume planned on CT 1)
(SIB) and a SIB ART planning technique (like SIB but the fi rst 15 fractions planned on
CT 1 and the fi nal 13 on CT 3). Total dose distributions were projected and evaluated
on CT 5. Seroma reduction was measured between CT 1 and CT 3. A reduction in the
undesired volume receiving ≥95% of the prescribed dose (V excess) ≥ 50cc between the
SIB and the SIB ART-plan is clinically relevant and feasible considering the expected
seroma reduction during RT. Thereby it was set as target for a replan.
Of the 21 patients in this study, 10 patients had a V excess reduction ≥50cc. These
patients all showed an initial seroma ≥40cc and a seroma reduction ≥20cc. If
all patients with an initial seroma ≥40cc are monitored, 19% will be monitored
unnecessarily. If all monitored patients with a seroma reduction ≥20cc are
replanned, 86% will have V excess reduction ≥50cc. All patients with V excess reduction
≥50cc were monitored and replanned. In this study, the guidelines of monitoring
patients with an initial seroma ≥40cc and a replan when they show a seroma
reduction ≥20cc is safe. However, whether these thresholds are effective should be
addressed in a larger patient group in a future study.
Leaf interdigitation and smaller leafs have no significant effect on the
quality of VMAT treatment plans We investigated the effect of interdigitation
and leaf width of Elekta MLCs on volumetric modulated arc therapy (VMAT)
treatment planning in Pinnacle 3 (Phillips Medical Systems). Three types of MLCs
were modelled: 1) An MLC with 1 cm leaves without interdigitation (regular MLC),
2) an MLC with 1 cm interdigitating leaves (interdigit MLC) and 3) a 0.5 cm leaf
width MLC with interdigitating leaves (thin MLC). All MLCs have a fi eld of view of
40 by 40 cm 2 . The effect of interdigitation and leaf width is expected to be more
pronounced for irregular tumour shapes. Different tumours shapes were studied;
prostate and early-stage lung cancer as an example of more spherical PTVs, rectum
and head-and-neck tumours for more concave and irregular PTVs. Five patients
were randomly selected per tumour site. Treatment plans were generated with the
SmartArc module in Pinnacle. Healthy tissue integral dose (ID), dose conformity
(CI) and dose homogeneity (DH) in the PTV were compared, where DH is defi ned as
(D max – D min)/D mean. For the various tumour sites, the mean and maximum doses
in OARs were evaluated.
The three MLCs have statistically signifi cant different performance, although the
differences are small. The performance of the interdigit and thin MLC relative to
the regular MLC is summarized in the table; an asterix denotes degradation in
performance with respect to the regular MLC. The ID increases for both types
of MLC whereas the CI improves. The DH becomes worse for the interdigit MLC
and improves for the thin MLC. Stratifying per tumour site points out that these
differences were most prominent for rectum and prostate tumours. No statistically
signifi cant differences in mean and maximum doses in OARs are observed.
Relative difference to regular MLC
Interdigit MLC Thin MLC
Healthy tissue Integral Dose (ID) + 1.08 %* + 0.14 %*
Conformity Index V95% (CI) - 0.36 % - 2.34 %
PTV Dose Homogeneity (DH) + 2.02 %* - 10.81 %
Although statistically signifi cant differences in ID, CI and DH were observed,
the clinical relevance of the increase in ID and decrease in CI is debatable. No
tumour shape dependent improvements were observed. In the case of ID and
DH, the interdigit MLC yields poorer results than the regular MLC, in contrast to
expectations. It is not yet clear whether this is intrinsically due to the interdigit MLC
or to the design of the SmarcArc optimization module in Pinnacle.
Clinical introduction of probabilistic plan evaluation in Pinnacle An application
called UncertLite, which allows for the probabilistic evaluation of a dose distribution,
was developed and implemented in Pinnacle. UncertLite simulates the physical

and NTD (with user defi ned / ) dose distribution for a large number of potential
treatments taking into account random and systematic errors, whose distributions
are estimated from the clinical IGRT protocol. For a given dose distribution the
probability that a certain fraction of a ROI receives a certain dose is calculated within
a minute.
UncertLite was distributed in the clinic among experienced technicians, who where
asked to plan patients normally. UncertLite was used to check that CTV coverage
was suffi cient and, in particular when there was overlap between the PTV and an
OAR, it was used as an extra aid to choose the best treatment. The UncertLite report
was also given to the treating physician.
For 9 prostate patients we found that when the PTV V 95% = 99%, 99% of the CTV
received on average 95.4% (sd = 0.18%) of the prescribed dose, at 90% confi dence.
This confi rmed our currently used margins, but when introducing new techniques
or protocols, UncertLite will be used to re-evaluate them.
As an example where UncertLite helped choosing the best plan, we considered
a prostate patient, where the sigmoid colon overlapped with the PTV, making it
impossible to meet both the prescribed PTV covering (V 74.1Gy > 99%) and the colon
constraint (D max < 70 Gy). Two plans where considered, with characteristics given in
the table below. The conventional analysis would favour plan A (higher V 74.1Gy while
D max < 70Gy). However, using UncertLite, we evaluated the risk associated with
insuffi cient PTV covering and found that the expected dose in the CTV was almost
identical for both plans, while the probability that D max > 70 Gy was much higher for
plan A. Therefore plan B was the better choice for treatment.
Based on the above we concluded that analysis of the dose distribution with
UncertLite gives more insight in the true delivered dose and the risks associated
with that dose. This insight can be used to check PTV margins and to help deciding
on the best planning strategy when suffi cient PTV coverage or meeting OAR
constraints is not feasible.
Conventional analysis UncertLite analysis
V74.1Gy of PTV D max sigmoid Dose in 99% of
CTV, with 90%
confi dence
Probability that
max dose in
colon > 70Gy
Plan A 90.5 % 69.05 Gy 68.64 Gy 33.6 %
Plan B 86.8 % 68.25 Gy 68.48 Gy 19.5 %
CLINICAL APPLICATION OF IMAGE GUIDED RADIOTHERAPY
Anja Betgen, Harry Bartelink, Suzanne van Beek, José Belderbos, Nabila Bouzeya, Paula Elkhuizen,
Angelo Mencarelli, Jasper Nijkamp, Coen Rasch, Peter Remeijer, Simon Rit, Maddalena Rossi,
Christoph Schneider, Rajko Topolnjak, Marcel van Herk, Simon van Kranen, Sandra Vreeswijk,
Corine van Vliet-Vroegindeweij, Jan-Jakob Sonke
HEAD AND NECK
Plan adaptation for systematic deformations based on average patient model
A new CT is commonly acquired to adapt to progressive anatomy changes during
radiotherapy for H&N cancer patients. However, both the planning and the
repeat CT are snapshots of a variable patient pose and thus introduce systematic
deformations. The purpose of this study was to validate the application of an average
patient model.
The average patient model was obtained by the modifi cation of the planning CT with
an average deformation vector fi eld () obtained by deformable cone beam CT
to planning CT registration over multiple fractions. The following adaptive protocols
were compared: online couch shifts (as reference), single plan adaptation with one
after N scans, and weekly adaptation based on an from the previous
week. As fi gure of merit for residual systematic deformations in each protocol, we
calculated the average vector length (over all patients) of the average displacement
vector (over all fractions) of the bony (B) and soft tissue (ST) landmarks. The
effectiveness of single plan adaptation depended on the number of measurements,
with an optimum at 12, though the overall effectiveness was modest (B: 2.3�1.5
Publications (continued)
135
radiotherapy
van de Kamer JB, de Leeuw AA, Moerland
MA, Jürgenliemk-Schulz IM. Determining
DVH parameters for combined external beam
and brachytherapy treatment: 3D biological
dose adding for patients with cervical cancer.
Radiother Oncol 2010;94:248-53
van der Molen L, van Rossum MA,
Burkhead LM, Smeele LE, Rasch CR, Hilgers
FJ. A randomized preventive rehabilitation
trial in advanced head and neck cancer
patients treated with chemoradiotherapy:
feasibility, compliance and short-term effects.
Dysphagia 2010 (in press)
van Kranen S, van Beek S, Mencarelli A,
Rasch C, van Herk M, Sonke JJ. Correction
strategies to manage deformations in headand-neck
radiotherapy. Radiother Oncol
2010;94:199-205
van Leeuwen FW, Buckle T, Batteau L, Pool
B, Sinaasappel M, Jonkers J, Gilhuijs KG.
Potential value of color-coded dynamic breastspecifi
c gamma-imaging; comparing (99m)
Tc-(V)-DMSA, (99m)Tc-MIBI, and (99m)
Tc-HDP in a Mouse mammary tumor model.
Appl Radiat Isot 2010;68:2117-24
van Lummel M, van Blitterswijk WJ, Vink
SR, Veldman RJ, van der Valk MA, Schipper
D, Dicheva BM, Eggermont AMM, ten
Hagen TLM, Verheij M, Koning GA.
Enriching lipid nanovesicles with short-chain
glucosylceramide improves doxorubicin
delivery and effi cacy in solid tumors. The
FASEB Journal 2010;25:280-9
van Mourik AM, Elkhuizen PHM, Minkema
D, Duppen JC, van Vliet-Vroegindeweij.
Multiinstitutional study on target volume
delineation variation in breast radiotherapy
in the presence of guidelines. Radiother Oncol
2010;94:286-91
van Nes JGH, Putter H, van Hezewijk M,
Hille ETM, Bartelink H, Collette L, van de
Velde CJH. Tailored follow-up for early breast
cancer patients: a prognostic index that
predicts locoregional recurrence. Eur J of Surg
Oncol 2010;36:617-24
Vanneste BG, Haas RL, Bard MP, Rijna H,
Váldes Olmos RA, Belderbos JS. Involved
fi eld radiotherapy for locally advanced nonsmall
cell lung cancer: isolated mediastinal
nodal relapse. Lung Cancer 2010;70:218-20

136
radiotherapy
Publications (continued)
Verbrugge I, Maas C, Heijkoop M, Verheij
M, Borst J. Radiation and anticancer drugs
can facilitate mitochondrial bypass by CD95/
Fas via c-FLIP downregulation. Cell Death
Differ 2010;17:551-61
Verheij M, Vens C, van Triest B. Novel
therapeutics in combination with
radiotherapy to improve cancer treatment:
Rationale, mechanisms of action and clinical
perspective. Drug Resist Update2010;13:29-43
Witte MG, Heemsbergen WD, Bohoslavsky
R, Pos FJ, Al-Mamgani A, Lebesque JV, van
Herk M. Relating Dose Outside the Prostate
with Freedom from Failure in the Dutch Trial
68 Gy vs. 78 Gy. Int J Radiat Oncol Biol Phys
2010;77:131-8
Yang TI, Aukema TS, Van Tinteren H,
Burgers S, Valdes Olmos R, Verheij M.
Predicting early chemotherapy response with
technetium-99m methoxyisobutylisonitrile
SPECT/CT in advanced non-small cell lung
cancer. Mol Imaging Biol 2010;12:174-80
Yang TI, Elkhuizen PH, Minkema D,
Heemsbergen W, van Mourik AM, Cassee
J, et al. Clinical factors associated with
seroma volume reduction in breast-conserving
therapy for early-stage breast cancer: A multiinstitutional
analysis. Int J Radiat Oncol Biol
Phys 2010;76:1325-32
Yang TI, Minkema D, Elkhuizen PHM,
Heemsbergen W, van Mourik AM, van
Vliet-Vroegindeweij C. Clinical applicability
of cone-beam computed tomography in
monitoring seroma volume change during
breast irradiation. Int J Radiat Oncol Biol
Phys 2010;78:119-26
mm, ST: 3.6�2.9 mm), indicating progressive changes. Weekly plan adaptation
showed an accuracy similar to the validation series (B: 1.2 mm, ST: 2.1 mm) and
is expected to perform better on progressive changes. For patients with overall
systematic deformations ≥ 3mm, weekly adaptation reduced systematic deformations
by more than 45%. Therefore, benefi ts of the average patient model are expected to
be especially for patients with large deformations.
BREAST
Assessment of intra-fraction motion during partial breast irradiation Recently,
a pre-operative image guided accelerated partial breast irradiation (PAPBI) study has
started. The purpose of this study was to quantify intra-fraction variability of the
target area.
Prior to irradiation, a CBCT is acquired, registered to the planning CT based on
grey-values and a correction applied to align the target volume with its planned
position for eight patients. A 2nd CBCT is acquired post treatment to determine
intrafraction movement. When intrafraction motion exceeding 0.5 cm was observed
in any direction, an extra CBCT was acquired halfway within the fraction and was
also scheduled for subsequent fractions.
Intrafraction motion was largest in the anterior-posterior direction with M=-4mm,
S=3mm and s=3mm. For the other 2 directions intrafraction motion was smaller
than 2mm. Due to substantial intra-fraction motion, mid-treatment scans were
introduced to limit the impact of intrafraction motion for 50% of the patients.
LUNG
Four dimensional Cone Beam CT acquisition concurrent with VMAT delivery
Volumetric modulated arc therapy (VMAT) recently replaced IMRT in stereotactic
body radiotherapy (SBRT) for pulmonary lesions in our clinic, considerably reducing
treatment delivery times. With both CBCT and VMAT being rotational modalities,
concurrent image acquisition and dose delivery became feasible and allows effi cient
data collection for off-line decision protocols, validation of the patient geometry
during actual therapy and detection of intra-fraction motion. The purpose of this
work was to evaluate our fi rst clinical experience with simultaneous 4D-CBCT
acquisition during VMAT delivery.
Three lung cancer patients with peripheral lesions treated with stereotactic body
radiotherapy (SBRT) to 54 Gy in three fractions were included in this study. VMAT
plans using two coplanar arcs where optimized using the SmartArc module in
Pinnacle3 (Philips, Best, The Netherlands). Plans were delivered on a Synergy
treatment machine (Elekta, Crawley, UK) in about 2.5 minutes per arc. For each arc,
CBCT projection images (120kV, 20ma, 10 ms) were acquired and reconstructed
simultaneous with dose delivery. The reconstruction algorithm keeps up with the
image acquisition and produces a 4D scan directly after the delivery of each VMAT
arc. These 4D-CBCT scans were displayed over the Mid-Ventilation planning CT
scan and visually inspected for tumor misalignments. Post-treatment scans to
quantify patient stability that are part of our standard clinical protocol were omitted.
Scatter generated from the MV VMAT delivery contributed about 1-2 times as much
signal as the kV fl uence, and induced vertical streak pattern in the projection images
due to the pulsed nature of the treatment beam. Nevertheless, the 4D-CBCT images
had adequate image quality and allowed accurate 4D registration of the tumor.
Without correction for the extra scatter, the contrast of the bony anatomy relative
to soft-tissue, however, was reduced signifi cantly, which affects the automatic bony
anatomy registration algorithm. Grey value registration of the bony regions did,
however, succeed. So far, concurrently acquired 4D CBCT scans did not lead to
interventions. Observed intra-fraction variability of the time averaged tumor position
was smaller than 2mm. In conclusion, 4D-CBCT acquisition concurrent with VMAT
delivery was successfully implemented for intra-fraction monitoring of SBRT and
provides defi nitive evidence of the treatment quality.
BLADDER
Clinical experience of Image Guided Radiotherapy for bladder cancer based on
lipiodol markers Tumor delineation and localization during treatment is challenging
in radiotherapy for bladder cancer. We therefore routinely inject lipiodol markers

around the visible tumor during cystoscopy, as surrogate for the tumor position. The
purpose of this study is to evaluate our fi rst clinical experience with online adaptation
of radiotherapy based on these lipiodol markers. 14 consecutive patients were evaluated.
Lipiodol deposits were clearly visible on both planning CT and Cone Beam CT
(CBCT). For all patients, one planning CT and about 25 CBCT scans were acquired.
The CBCT scans were fi rst registered to the planning CT on bony anatomy. Then.
the lipiodol spots were registered. Both registrations were performed automatically
but the lipiodol registration was redone manually if considered unsatisfactory.
Translations of the bladder tumor after bone registration were analyzed. Tumor
translations after bone registration were 0.07 (0.14), -027 (0.63) and -0.01 (0.45) cm
in respectively LR,CC and AP direction. The maximum observed translations were
0.90, 1.95 and 2.02 cm, along the 3 axes. We conclude that online adaptation of RT
for bladder cancer by lipiodol registration is a feasible and effi cient method to reduce
the geometrical uncertainties caused by changes in bladder fi lling variations.
4DCT - high quality imaging and contrast enhancement for 3D radiotherapy
treatment planning 4D CT is widely applied to image moving tumors in the lung
or the abdomen. For treatment planning the phase closest to the time weighted
mean position of the tumor (MidV CT) is an adequate geometric and dosimetric
approximation. This procedure, however, is incapable to overcome irregular
breathing artefacts, has lower signal to noise (SNR) compared to standard 3DCT and
suboptimal contrast enhancement timing. With deformable registration, all 4DCT
data can be deformed and used to generate a time-weighted average 3DCT scan,
improving the SNR and allowing to overcome breathing artifacts (MidP CT). The
purpose of this study was to evaluate usage of a contrast enhanced expiration breath
hold (BH) CT as reference in the deformable registration procedure to deform the
BHCT to the time weighted average anatomy of the 4DCT.
Deformable registration, based on iterative multi-scale phase-based optical fl ow was
applied to 4D-CT scans of 10 patients, with a BH-scan as reference. The MidP CT
was calculated by deformation of each phase to the time weighted average anatomy
and subsequently taking the median grey-value over the phases. The MidP BHCT
was generated by directly deforming the BHCT to the time weighted average
anatomy. Anatomy representation was evaluated by comparing tumor segmentations
for each scan. Image quality was evaluated by comparing the SNR in a large vessel at
the level of the tumor between the scans.
The MidP CT and MidP BHCT visually contained less imaging artifacts compared
to the MidV CT. The average overlap between the segmented tumors in the 3 scans
was 88% (range 72-96%) with an average volume of 4.4 cc (range 0.5 - 22.2 cc). In
general distances between the segmented edges were in the order of the pixel size,
with maxima around 2-3 mm in small sub regions. The average signal in the MidV
CT, MidP CT, MidP BHCT no contrast and the MidP BHCT with contrast was 1079,
1078, 1085 and 1197 HU, respectively. The noise found in these scans was 22.8, 8.6,
11.6 and 13 HU, respectively (fi gure 7).
In conclusion, deforming all image sets derived from clinical 4D-CT-scans to a
MidP-CT is a robust technique and is about to be introduced in our clinic. Subsequently,
addition of expiration breath hold contrast enhanced imaging to create a
MidP BHCT facilitates optimal contrast timing in 4D workfl ows. The MidP BHCT
can facilitate for more accurate and reproducible delineation of involved lymph nodes.
Figure 7: Overview of the image quality in the mediastinal region when using a MidV-CT (left), a
MidP-CTRef_5 (middle) and a MidP-BH-CT (Right) at exactly the same level and window settings
137
radiotherapy

138
radiotherapy
PROSTATE
Hybrid registration technique for image-guided radiotherapy of prostate
cancer Fiducial markers are an accurate surrogate for the prostate, but they provide
little information on the position of the seminal vesicles (SVs). To reduce the SV
position uncertainty we propose a hybrid registration technique that uses fi ducial
markers to localize the prostate and subsequently performs a gray-value registration
to correct for the orientation of the SVs. Fifteen prostate cancer patients were
implanted with two or three 0.35 x 20 mm gold markers. Gray-value registration of
the delineated SVs was started from the marker registration, allowing only rotations
around the left-right (LR) axis. The center of mass of the prostate was chosen
as center of rotations. We validated the SV registration by calculating residual
registration errors for three clearly identifi able landmarks (small calcifi cations)
present in the SVs of three patients. The SVs of eight patients showed a substantial
(>5°) mean LR rotation relative to the marker registration. Overall, we found M
= -3.0°, = 6.2° and = 5.0°. These results confi rm that substantial differences
between prostate and SV orientation exist. Out of 398 scans, the SVs of 41 scans
could not be registered automatically, mostly due to motion artifacts in the CBCT
image. For most cases, there was a decrease in registration error, especially for large
differential SV rotations.
Correction, e.g. using collimator rotations, would improve target coverage of the SVs
and spare organs at risk, while preserving the planned dose to the prostate.
BREAST CANCER
Femke van der Leij, Marc van de Vijver1 , Jelle Wesseling, Kenneth Gilhuijs, Hester Oldenburg,
Claudette Loo, Adrian Begg, Wouter Vogel, Sandra Vreeswijk, Corine van Vliet-Vroegindeweij,
Harry Bartelink, Paula Elkhuizen
1 AMC, Department of Pathology, Amsterdam
DEFINING RADIOTHERAPY SENSITIVITY
A. Image guided Preoperative Accelerated partial Breast Irradiation (PAPBI)
Radiotherapy is part of breast conserving therapy (BCT) and is known to reduce LR
rates in all patients by 60-70%. So far, no patient groups can be defi ned in whom
radiotherapy would not be necessary. It is estimated that in approximately half of the
patients whole breast radiotherapy is not necessary, while in others the tumor might
be resistant to radiotherapy. If it would be possible to predict tumor response to
radiotherapy, a more tailored treatment can be advised to individual patients (higher
boost dose or primary mastectomy). To evaluate the in situ breast radiosensitivity,
the image guided accelerated partial breast irradiation (PAPBI) is the best scheme of
irradiation because of (i) low breast / ratio suggesting that breast cancer is more
sensitive to high dose per fractions; (ii) very precise breast tumor irradiation; (iii)
limited volume of irradiated area allowing larger fractions with no extra risk on late
toxicities; (iv) short treatment time course.
This trial is directed at implementing pre-operatively given image guided accelerated
partial breast irradiation without compromising local control in early breast cancer
patients. By assessing tumor response to radiotherapy, the goal of the study is to
develop a gene expression profi le that predicts the breast cancer radiosensitivity.
This gene signature of breast radiosensitivity would further design optimal
treatment strategies for individual breast cancer patients treated with BCT
To qualify for the trial, patients must be 60 years or older, and have an unifocal cT1-2
(

correlated with response to radiotherapy, defi ned as pathologic response at the time
of the lumpectomy (i.e. 6 weeks after the completion of the PAPBI). Response of
the tumor will be evaluated by MRI scan and PET (before radiotherapy and before
surgery) and classical pathology.
The trial, which started accrual yet, will accrue 120 patients in total over a period of
years. First, 60 patients will be studied as a test set to identify predictive profi les and
then, 60 patients more will be used as a validation set.
Other important aspects of this study are collections of fresh frozen tumor tissue,
blood and urine samples (i) to assess the radio-induced genetic alterations on the
surgical post-radiation specimen compared to the tumor response 6 weeks after
the end of radiotherapy; (ii) to study the early changes in gene-profi ling and (iii) to
evaluate the early functional imaging modifi cations. The Institut Gustave Roussy
(France) and the Karolinska Institutet (Sweden) will participate in this study
(Descartes Cancer Consortium).
B. Radiosensitivity breast cancer cell lines
In addition with the clinical PAPBI study, 30 human breast cancer cell lines will be
investigated for their radiosensitivity profi le. This will also be implemented in the
study described in D.
C. Case control study focusing local recurrence
Genetic profi ling will be studied in 240 breast cancer patients (79 cases with 161
matched controls). This study is in association with Institut Curie from France. The
results will also be implemented in the study described in D.
D. In the running Young Boost Trial over 1200 patients under 50 years haven
been randomized between normal boost dose vs. a higher boost dose after breastconserving
therapy. From these patients frozen material as well as paraffi n
embedded material is collected. Follow up is available for the fi rst years. The profi les
found in the studies A-B to be related with radioresponse will be evaluated on this
material whether a response profi le eventually is associated with local control. In
addition, the results from the case-control study will be evaluated on this cohort.
COMBINATION OF RADIOTHERAPY AND
CHEMOTHERAPY/BIOLOGICALS
Berthe Aleman, Harry Bartelink, Jos Beijnen, José Belderbos, Henk Boot, Annemieke Cats,
Frits van Coevorden, Ewout Courrech Staal, Otilia Dalesio, Maarten Deenen, Luc Dewit,
Johan Dikken, Ria Dubbelman, Wilma Heemsbergen, Michel van den Heuvel, Frans Hilgers,
Edwin Jansen, Rianne de Jong, Joost Knegjens, Maria Kuiper, Corrie Marijnen, Lisette van de
Molen, Coen Rasch, Maurits Swellengrebel, Renato Valdes Olmos, Johanna van Sandick,
Jan Schellens, Karijn Verschueren, Vic Verwaal, Marcel Verheij
Head and neck The CONDOR cooperative trial the University of Nijmegen targets
patients below 60 years with stage III/IV head and neck cancer and is open since
2008. Patients are fi rst to receive 3-4 courses of TPF chemotherapy followed by a
randomization between two regimens of chemoradiation. Nineteen of 70 patients
have entered the trial so far.
The fi nal report on the quality of life for patients with chemoradiation treated within
the M99RAD trial is submitted. Results remain favorable compared to historical
series with smaller tumor treated with surgery and radiotherapy. Quality of life
before treatment and after one year is predictive for (disease specifi c-) survival.
Based on our preclinical research on apoptosis-modulation, we have initiated a
clinical phase I-II trial in locally advanced head and neck cancer combining standard
cisplatin-based chemoradiotherapy with dose escalating AT-101, a small molecule
inhibitor of anti-apoptotic Bcl-2/Bcl-XL. To date, 7 patients have been included.
Gastroenterology – Esophageal cancer Over recent years a database was set
up including data on all patients treated for esophageal cancer in our institute
since 1997 (approximately 1500 patients). We evaluated the toxicity and effi cacy
of three different regimens of concurrent chemoradiation (CRT) in 94 patients
with esophageal cancer treated between 1997 and 2007. Treatment consisted of
radiotherapy to 50 Gy in 25 fractions with concurrent cisplatin and 5-fl uorouracil
(group A, n=65), radiotherapy to 50.4 Gy in 28 fractions with concurrent carboplatin
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and paclitaxel (group B, n=16) or radiotherapy to 66 Gy in 33 fractions with
low-dose cisplatin (group C, n=13). Toxicity was scored according to CTC v.3.0.
Chemoradiation was planned as neoadjuvant (n=58) or defi nitive (n=36) treatment.
Grade 3/4 hematological toxicity occurred in 18 (19%) patients and grade 3 nonhematologic
toxicity in 8 (9%) patients. During treatment, 2 patients died (1 from
duodenal ulcer bleeding and 1 from stroke). Overall, 81 (86%) patients completed
the planned treatment (86%, 94%, and 77% in groups A, B, and C, respectively).
Clinically complete or partial response was observed in 28 of 92 (30%) patients
(21%, 50%, and 54% in groups A, B, and C, respectively). After clinical and
radiologic response evaluation, treatment plan was changed in 14 (15%) patients.
A total of 45 patients underwent surgery. Pathologic complete response and
downstaging were seen in 12 (27%) and 34 (76%) operated patients, respectively.
With a median follow-up of 15 (range, 1-108) months, the 3-year survival was 41%
for all patients. We conclude that with individual treatment planning, different
regimens of chemoradiation for esophageal cancer resulted in acceptable rates of
toxicity and effi cacy.
Gastroenterology – Gastric cancer In the US Intergroup 0116 study, a signifi cant
survival benefi t in postoperative CRT was reported in gastric cancer. Based on these
results we completed three adjuvant chemoradiotherapy phase I-II trials in gastric
cancer. From these trials we identifi ed the regimen for the experimental arm in
the CRITICS study. In this international randomized phase III study patients with
operable gastric cancer receive preoperative chemotherapy, surgery and postoperative
chemotherapy, or preoperative chemotherapy, surgery and postoperative CRT. At this
moment over 300 patients have been randomized. In addition to Sweden that joined
the trial in 2008, Denmark participates as well since ultimo 2010. Furthermore,
we are running a phase I-II study together with the AMC and VUmc, in which
neoadjuvant chemoradiation with weekly paclitaxel and capecitabine is applied in
patients with inoperable gastric cancer. So far, 10 patients have been included in this
NARCIS trial.
In collaboration with Prof Van de Velde from the LUMC, we analyzed the impact of
the extent of surgery and postoperative chemoradiotherapy on recurrence patterns
in gastric cancer. We retrospectively compared survival and recurrence patterns in
our phase I/II studies evaluating more intensifi ed postoperative CRT with those
from the Dutch Gastric Cancer Group Trial (DGCT) that randomly assigned patients
between D1 and D2 lymphadenectomy. Survival and recurrence patterns of 94
patients with adenocarcinoma of the stomach who had received surgery followed by
radiotherapy combined with fl uorouracil and leucovorin (n=5), capecitabine (n=39),
or capecitabine and cisplatin (n=50) were analyzed and compared with survival and
recurrence patterns of 694 patients from the DGCT (D1, n=369; D2, n=325). With a
median follow-up of 27 months in the CRT group, local recurrence rate after 2 years
was signifi cantly higher in the surgery only (DGCT) group (17% v 5%; p=0.0015),
while no signifi cant differences were found for overall survival at 2 years (67% v
70%). Separate analysis of CRT patients who underwent a D1 dissection (n=41)
versus DGCT-D1 (n=369) showed fewer local recurrences after chemoradiotherapy
(2% v 18%; p

postoperative adjuvant capecitabine-based CRT. Blood samples were drawn on
days 22 and 43 to determine the pharmacokinetics (PK) of capecitabine, DFCR and
DFUR, and were analyzed using LC-MS/MS. 18 anal cancer patients with an intact
stomach treated with similar doses of capecitabine served as a control group. PK of
capecitabine, DFCR and DFUR showed wide inter- and low intra-patient variability.
There was no signifi cant effect of daily radiation on the exposure to capecitabine and
metabolites. Total and partial gastrectomy resulted in increased AUC and Cmax of
capecitabine and metabolites, whereas Tmax was earlier. Clinical consequences of
this variability have to be assessed.
Gastroenterology – Rectal cancer Pre-operative capecitabine-based
chemoradiotehrapy (CRT) has become standard treatment in locally advanced rectal
cancer. We evaluated acute toxicity and surgical complications in these patients
following total mesorectal excision (TME) after preoperative CRT with capecitabine.
Between 2004 and 2008, 147 consecutive patients with clinical tumour category (cT)
3-4 (with a threatened circumferential resection margin or cT3 within 5 cm of the
anal verge) or clinical node category 2 rectal cancer were treated with preoperative
CRT (50 Gy, capecitabine 825 mg/m 2 twice daily, days 1-33). TME followed 6 weeks
later. Toxicity was scored according to the CTC (version 3.0) and RTOG scoring
systems. Treatment-related surgical complications were evaluated for up to 30 days
after discharge from hospital using the modifi ed Clavien-Dindo classifi cation. The
mean cumulative dose of capecitabine was 95% and 98% of patients received at least
45 Gy. One patient died from sepsis following haematological toxicity. Grade 3-5
toxicity developed in 32 patients (22%), in particular diarrhoea (10%) and radiation
dermatitis (12%). There were no deaths within 30 days after surgery. Anastomotic
leakage and perineal wound complications developed after 13 of 47 low anterior
resections and 23 of 62 abdominoperineal resections. Surgical reintervention was
required in 30 patients. Twenty-seven patients (20%) of 138 patients who had a
laparotomy were readmitted within 30 days after initial hospital discharge. We
concluded that preoperative CRT with capecitabine is associated with acceptable
acute toxicity, signifi cant surgical morbidity but minimal postoperative mortality.
Gastroenterology – Anal cancer The feasibility and effi cacy of simultaneous
integrated boost – intensity modulated radiation therapy (SIB-IMRT) with
concomitant capecitabine and mitomycin-C was investigated in a phase I dosefi
nding study in 18 patients with locally advanced anal cancer. Patients with locally
advanced anal carcinoma were treated with SIB-IMRT in 33 daily fractions of 1.8 Gy
to the primary tumor and macroscopically involved lymph nodes and in 33 fractions
of 1.5 Gy electively to the bilateral iliac and inguinal lymph node areas. Mitomycin-C
10 mg/m 2 was administered on day 1, and capecitabine was given in a dose-escalated
fashion twice daily on irradiation days, until dose-limiting toxicity emerged in ≥ 2
of 6 patients. An additional eight patients were treated at that dose level. Patients
received a sequential radiation boost dose of 3 x 1.8 Gy if macroscopic residual tumor
was still present in week 5 of the radiation treatment. In total, 18 patients completed
the planned treatment. The MTD was capecitabine 825 mg/m 2 BID. The most
predominant acute toxicities (all grades) included dermatitis within the radiation
area (100%), fatigue (83%), pain (72%), diarrhea (67%), and leukocytopenia (61%).
Grade ≥ 3 toxicities were dermatitis (72%), fatigue (22%), and hyponatremia (11%).
Of all patients, 72% (95%-CI: 51-94%) achieved a complete response, and 28% had a
partial response. In none of the complete responders, a relapse was observed after a
median follow-up of 18 months. Longer follow-up is needed to assess the long-term
effects of this treatment.
Lung - NSCLC Combining concurrent chemotherapy with radiotherapy (CRT)
is the standard treatment of locally advanced NSCLC. Currently we are accruing
patients in the RADITUX trial (M07CCL). In this multi-center randomized phase
II trial patients with inoperable locally advanced NSCLC are randomized to our CRT
regimen (66 Gy in 24 fractions and daily dose Cisplatin 6 mg/m 2 ) with or without
the weekly addition of an EGFR monoclonal antibody (Cetuximab). The addition
of Cetuximab to our concurrent CRT was well tolerated in a phase I trial. Between
January and November 2010, 25 patients were treated within this trial in the NKI-
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Figure 8: Partial Metabolic response in
an 53 year old female with cT3N0 NSCLC
shortly after chemoratidation with
cetuximmab (M07CCL)
AVL (total inclusion: 93 patients). We expect to complete this trial in the fi rst half of
2011. In this group of patients, we studied the esophagus toxicity using FDG-PET
images to correlate radiotherapy dose to esophageal toxicity (fi gure 8).
Dose-escalation is studied by boosting radiation dose within the primary tumor
applying non-uniform radiation dose distribution using IMRT to the target based
upon biological activity on pre-treatment FDG-PET scan. In collaboration with Prof
de Ruysscher from the MAASTRO clinic the phase II PET boost trial (M09PBO)
opened in 2010. Patients are randomized to dose escalation to the planning target
volume of the primary tumor as a whole or to the 50% SUVmax of the primary
tumor (of the pre-treatment FDG-PET scan). The patients receive concurrent
chemotherapy if feasible. The primary objective is to determine the local progression
free survival at 1 year. In both treatment arms, the patients are irradiated to the
same maximum tolerated dose to the lung. Until November 2010, 8 patients were
randomized (4 patients from the NKI-AVL).
Lung - SCLC Approximately 20% of malignant tumors of the lung are due to
small cell carcinoma. In general, these patients carry a worse prognosis compared
to NSCLC patients. For limited stage small cell lung cancer (LS-SCLC), the
combination of chemotherapy and thoracic radiotherapy is the standard treatment.
Two meta-analyses have shown that thoracic radiotherapy given concurrently with
chemotherapy improves both survival and local control. Nevertheless, several
important questions including the optimal total radiation dose and radiation
fractionation are still unanswered. To establish a standard chemo-radiotherapy
regimen for LS-SCLC, an EORTC international, multicenter randomized phase III
trial started in 2009 comparing twice daily radiotherapy with high dose radiation
delivered once daily (CONVERT trial). To date, a total of 139 patients were treated
within this trial (2 patients from the NKI-AVL). For extensive stage small cell lung
cancer (ES-SCLC), chemotherapy is the cornerstone of treatment. However, over
75% of patients have persisting intra-thoracic disease after initial chemotherapy, and
about 90% manifest intra-thoracic disease progression at 1 year after completing
initial chemotherapy. In the absence of promising systemic agents that can improve
local response, the role of thoracic irradiation in patients with ES-SCLC is currently
being evaluated in a multicenter phase III randomized trial (CREST trial). The
objective of this study is to investigate whether thoracic radiotherapy can improve
1-year survival, following a response to chemotherapy. A total of 120 patients have
now been treated within this trial. At the NKI-AVL, patient accrual started in
September 2009 and to date 16 patients have been included.
BRACHYTHERAPY
Berthe Aleman, Marcel Steggerda, Baukelien van Triest, Thelma Witteveen,
Simon Horenblas, Floris Pos, Luc Moonen
Bladder preservation with brachytherapy for bladder cancer This year we have
updated the clinical outcome as part of a joint effort to analyze the results of all
bladder cancer patients treated with brachytherapy in the Netherlands. The data have
been sent to the central database, which is located at the department of Radiotherapy
in the Academic Medical Center in Amsterdam. The results of the joint analysis are
expected in 2011.
In recent years, we have updated and implemented several new techniques for
bladder cancer brachytherapy. A new initiative in close cooperation with the
department of urology is the implementation of minimal invasive surgery. In
Arnhem, they have developed a laparoscopic implantation procedure, which is
already a major improvement. The “Arnhem” procedure might be even further
improved by the implementation of robotic assisted laparoscopic implantation. This
technique is currently being developed; the fi rst patients will be treated in the fi rst
half of 2011. The impact for the mostly elderly bladder patients will be enormous. It
is expected that the hospitalization, which is usually 7 – 10 days, can be reduced to 3
days.

Focal brachytherapy for prostate cancer In the last decade, there has been a large
increase in the detection rate of prostate cancer and a signifi cant increase in the
proportion of men with very early organ confi ned disease. At present, all established
local forms of prostate cancer therapies, such as brachytherapy, aim at treating the
entire prostatic gland. Although highly effective, these treatments are associated
with signifi cant morbidity (i.e. erectile dysfunction, urinary incontinence/problems
etc.) and can seriously affect the quality of life.
Attempts to treat only one side of the prostate (based on biopsy proven unilaterality)
or exclusively the dominant cancer focus (detected by MRI scans) gain growing
interest. Preliminary results of several small studies using cryotherapy or High-
Intensity Focused Ultrasound (HIFU) applied to part of the prostate show an
important reduction of morbidity rates.
Focal or partial prostate brachytherapy seems a logical step. As no other form of
prostate cancer therapy, it is possible to focus the treatment to a part of the prostate
only. Before focal brachytherapy becomes feasible, there is need of high quality
imaging. Together with the department of radiology and in close cooperation with
Utrecht University hospital, we have implemented state of the art high quality 3
Tesla MRI sequences. We are now able to identify a dominant lesion in > 50 %
of the brachytherapy candidates. Furthermore, we have integrated MRI in the
brachytherapy process and have done focal brachytherapy treatment planning
on these patients to evaluate feasibility of the procedure. Now we are ready to
implement focal brachytherapy in the clinic. We are currently working on a phase II
study evaluating focal brachytherapy in early stage prostate cancer.
MECHANISMS, MODULATION AND PREDICTION OF
RADIATION-INDUCED CELL DEATH
Maaike Alderliesten, Wim van Blitterswijk, Jannie Borst, Albert van Hell,
Gerben Koning1 , Dayane Martins, Rogier Rooswinkel, Renato Valdes Olmos,
Baukelien van Triest, Conchita Vens, Shuraila Zerp, Marcel Verheij
1 Erasmus MC, Rotterdam the Netherlands
The translational research performed within our group focuses on (1) the
identifi cation and preclinical testing of novel targets and agents to enhance the
cytotoxic effect of radiation and on (2) the validation of new endpoints and clinical
biomarkers to quantify and predict the effi cacy and toxicity of new combination
therapies. The ultimate objective is to rapidly translate these strategies from the lab
into the clinic.
In close collaboration with several research groups within the NKI, new agents
are identifi ed on the basis of their mechanism of action and subsequently tested
for their ability to induce apoptotic cell death and to increase the cytotoxic effect
of radiation in vitro and in vivo. Current research projects focus on synthetic
alkyl-phospholipids (Edelfosine, Perifosine, Erucyl-PC), death receptor ligands
(TRAIL, CD95L/FasL/APO010), small molecule inhibitors of Bcl-2 (Gossypol/AT-
101, ABT-737) and DNA damage repair inhibitors (Olaparib, APO866). To monitor
tumor response and predict treatment outcome, we explore new functional imaging
modalities including in vivo imaging of apoptosis by annexin V scintigraphy, MIBI
SPECT-CT and ML-10 ApoSense.
In a separate project we investigate the patented concept of improved drug delivery
by short chain sphingolipid-enriched liposomes in vitro and in vivo.
Alkyl-phospholipids (APLs) APLs represent a fi rst group of compounds that has
become available for clinical application along this translational approach. These
synthetic anti-tumor agents are known to accumulate in sphingomyelin- (SM) and
cholesterol-enriched plasma membrane microdomains, also known as “lipid rafts”.
Once taken up via these membrane portals, APLs interfere with lipid metabolism,
inhibit proliferative and survival signaling, affect cell cycle distribution and
stimulate apoptosis induction in a variety of tumor cell systems. In combination
with radiation, APLs cause a synergistic apoptotic effect and reduce clonogenic cell
survival.
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The synthetic APL Edelfosine (1-O-octadecyl-2-O-methyl-rac-glycero-3phosphocholine)
induces apoptosis in S49 mouse lymphoma cells. Variant cells,
S49AR, made resistant to APL, show impaired APL uptake through lipid rafts and
are SM defi cient. These cells display cross-resistance to other apoptotic stimuli,
such as Fas/CD95 ligand (FasL) and DNA damage (etoposide, radiation), but they
can be gradually resensitized (S49ARS cells) by prolonged culturing in the absence
of APL. The resistant S49AR cells show constitutively enhanced levels of phospho-
ERK and phospho-Akt/Protein kinase B, characteristics of survival signaling.
Pharmacological inhibition of these enzymes restored apoptosis sensitivity towards
DNA damage, but not to APL or FasL. In search for a common mediator of the
cross-resistance, upstream of Akt, we found that S49AR lacked SHIP-1, an SH2
domain-containing inositol phosphatase that converts the Akt activator PtdIns(3,4,5)
P3 to PtdIns(3,4)P2. Using 32P-radiolabeling and HPLC, we confi rmed that these
phosphoinositide levels changed accordingly. Resensitized S49ARS cells regained
SHIP-1 expression and showed normalized levels of phosphoinositides and SM, like
the parental S49 cells. Down-regulation of SHIP-1 using siRNA resulted in (partial)
apoptosis cross-resistance, SM defi ciency and shift in phosphoinositide levels, much
like we found in S49AR cells. Our present and previous results suggest that SHIP-1,
in yet unresolved interplay with SM synthase-1, mediates apoptosis sensitivity of S49
lymphoma cells towards APL, FasL and DNA damage.
Perifosine (PER) is another, orally available, membrane targeting APL and Akt
inhibitor with preclinical radiosensitizing, apoptosis-enhancing and anti-angiogenic
properties. In a previous phase I study we established the MTD and optimal
scheduling of PER in combination with radiation (RT). We recently conducted
a randomized, double-blind, placebo-controlled multicenter phase II study to
assess the effi cacy of daily PER and RT in patients with locally advanced NSCLC.
Secondary objectives were to determine the toxicity of this regimen, and to evaluate
systemic exposure of the drug during RT. A total of 177 patients from 19 centers
were entered. Stages: IA-B (4.6%), IIB (9.6%), IIIA (37.3%), IIIB (48.6%). Of these
patients 95 received PER (53.7%) and 82 placebo (46.3%; PLA). 121 patients (65
PER; 56 PLA) entered the study without prior chemotherapy and 56 (30 PER; 26
PLA) had prior chemotherapy. The primary endpoint could not be evaluated as
only 15% of the patients reached the 1-year follow up. Response evaluation at day 65
showed an overall RR (CR+PR) of 40% and was not signifi cantly different between
both arms (PER: 42% and PLA: 38%). For the subgroup of patients without prior
chemotherapy TLF indicated an advantage for the PER group (mean 230 vs 165 days;
median 206 vs 126 days). Kaplan-Meier survival curves indicated an advantage for
PER patients without prior chemotherapy (p=0.088). In both groups, the majority
of patients reported treatment related AEs (PER 86.3%; PLA 76.8%). The most
frequently observed toxicity in the PER group was gastro-intestinal (grade 3-4: 14.7%
vs PLA 4.9%). Plasma concentration of PER on the fi rst and last day of RT were
comparable and in the same range as determined previously. We concluded that
the high number of patients who did not reach the 1 year follow up, mainly due to
systemic progression, compromised the power of the trial to detect an effect of the
study treatment on the local control rate. A small advantage for TLF and survival was
observed in the PER group without prior chemotherapy. Gastro-intestinal toxicity
was the most frequently observed PER-related side effect.
Other promising APL derivatives with potentially better bioavailability and
radiosensitizing properties, like the i.v. compound Erucyl-PC, have become available
and are studied in vitro and in vivo.
Other radiosensitizers in preclinical development In two separate projects
with J Borst (Division of Immunology) we study other radiosensitizers. A long term
line of research focuses on the interaction between death receptor ligands (TRAIL
and MegaFasL/APO010) and DNA damaging regimens (radiation and etoposide).
TRAIL induces apoptosis in a wide variety of tumor tissues, but lacks normal tissue
toxicity in preclinical animal models. In several leukemic and solid tumor cell lines,
we demonstrated combined (additive to synergistic) effects of TRAIL + radiation/
etoposide. The effectiveness and acceptable toxicity of the combined treatment of

TRAIL and radiation was subsequently demonstrated in vivo. These experiments
have been expanded with another death receptor ligand (MegaFasL/APO010). Our
current studies focus on the impact of TRAIL death receptor traffi cking on proapoptotic
signaling.
In collaboration with the University of Michigan and the division of Radiobiology
at the Free University of Amsterdam, we have initiated preclinical effi cacy testing
of small-molecule inhibitors of anti-apoptotic members of the Bcl-2 family (AT-
101 and ABT-737). Currently, the combination of AT-101 with cisplatin-based
chemoradiotherapy is evaluated in a clinical phase I/II study in locally advanced
head and neck cancer. So far, 7 patients have been included and no DLT has been
observed.
ABT-737 has been chemically designed to specifi cally interact with anti-apoptotic
Bcl-2 family members through binding to their BH3-interacting domains
prohibiting interactions with their pro-apoptotic relatives Bax and Bak and with
other BH3-only proteins. We are investigating under which conditions ABT-737
might synergize with radiation and etoposide using enforced expression of the 6
different anti-apoptotic proteins in Jurkat and Molt-4 acute lymphoblastic T cells.
In cases where cells became resistant to radiation, this could be counteracted by
the addition of ABT-737. For the reverse we showed that synergy only occurs upon
upregulation of specifi c Bh3-only proteins. The importance this upregulation was
confi rmed using cells that express these proteins in an inducible way. Together,
these results allow us to make an a priori prediction about which treatments to
combine with ABT-737 based on the bcl-2 protein status of a tumor.
In collaboration with C Vens (Division of Experimental Therapy) we explore the
interaction between radiation and inhibitors of DNA repair. The NAD+-depleting
agent APO866 was found to enhance radiation-induced cell death and act as a
radiosensitizer in various models, including 2 prostate cancer cell lines. Restoration
of cellular NAD levels abolished the cytotoxic effect. In vivo MTD of APO866 was
defi ned as the highest non-lethal dose with a reduction in body weight of less than
10%, and was determined at 10 mg/kg, administered every 12 hours for 4 times.
APO866 induced NAD-depletion is thought to inhibit PARP, that is involved in
DNA repair. PARP inhibitors are also good candidates for combined use with DNA
damaging agents. The main mechanism by which both radiation and cisplatin
kill tumor cells is by an accumulation of un- or misrepaired DNA damage. PARP
inhibitors increase radiation and chemotherapy (Cisplatin) response in preclinical
studies. PARP inhibitors have been shown to specifi cally kill homologous
recombination defi cient tumor cells as single agent. ATM mutations are expected
to affect DSB repair and homologous recombination status therefore amplifying
damage induced by the combined PARP inhibitor radiation (Cisplatin) treatment.
Thus tumor targeted treatment and radio-chemosensitization could be achieved in
the presence of frequently observed ATM gene mutations. We have designed 3 phase
I-II studies evaluating the safety and tolerability of Olaparib in combination with
(cisplatin-based chemo-) radiotherapy in locally advanced breast cancer, NSCLC and
head and neck cancer. Olaparib exhibits low systemic toxicity profi les when given as
monotherapy. When combined with chemotherapeutic agents more toxicity is seen.
Prediction of tumor response Radiation, like most anti-cancer treatments,
achieves its therapeutic effect by inducing different types of cell death in tumors.
To monitor treatment effi cacy a variety of routine anatomical imaging modalities
is available. However, changes in tumor function (e.g., metabolism, proliferation,
hypoxia) often precede these volumetric alterations and may refl ect tumor responses
to treatment more accurately. Therefore, reliable biomarkers and imaging modalities
that could assess treatment responsiveness in an early phase would be very useful to
identify responders and/or avoid ineffective, toxic therapies. We previously evaluated
two non-invasive cell death imaging techniques in collaboration with the department
of nuclear medicine (Valdés Olmos, Division of Diagnostic Oncology): 99mTc-
Annexin V scintigraphy (TAVS) and 99mTc-MIBI single photon emission computed
tomography (MIBI SPECT-CT). Our results indicated that both TAVS and MIBI
SPECT-CT might be useful as non-invasive predictive tests for treatment outcome,
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ultimately allowing an early adaptation of the initiated therapy and help to design
novel (combined modality) strategies. Currently, we focus on another potential
imaging biomarker for radiation-induced cell death: 18F-ML-10. We designed a
clinical protocol in rectal cancer to determine the optimal timing of ML-10 imaging
for predicting treatment outcome after preoperative 5x5 Gy or capecitabine-based
chemoradiotherapy (25x2 Gy).
Improved in vitro and in vivo anti-tumor efficacy of glucosylceramideenriched
liposomal doxorubicin Anti-cancer therapy is often suboptimal due to
inability to deliver suffi cient levels of cytostatics into tumor cells. We identifi ed a
class of short-chain sphingolipids that, when inserted in the tumor cell membrane,
facilitates the translocation of certain drugs of well-defi ned amphiphilicity over
the plasma membrane. One of these sphingolipids, N-octanoyl-glucosylceramide
(GC), co-formulated with liposomal doxorubicin, enhanced the accumulation of
doxorubicin into cultured tumor cells and into xenograft tumor tissue in vivo.
This translated in enhanced anti-tumor effi cacy of the GC-enriched doxorubicin
liposomes in an A431 xenograft tumor model. Next, we examined this novel concept
of GC-directed membrane permeabilization for doxorubicin in a spontaneous
mouse tumor model that mimics human Invasive Lobular Carcinoma (ILC) breast
cancer. We found that GC-enriched liposomal doxorubicin enhanced the uptake of
doxorubicin in ILC cell cultures at least fi ve-fold, and inhibited tumor outgrowth
in vivo much more effectively than either of the two clinically applied formulations
of doxorubicin, (free, non-liposomal) Adriamycin® or (standard/conventional
liposomal) Caelyx®. Mice treated with GC-liposomal doxorubicin showed an
extended overall survival (up to 2-fold) compared to conventional liposomes (fi gure
9). No increase in normal tissue toxicity was observed upon the addition of GC. The
presented data hold promise for widening the therapeutic window of amphiphilic
anti-cancer agents, including doxorubicin, using this plasma membrane modulation
strategy. We are currently continuing the GC-enriched liposomal doxorubicin
towards clinical investigation. Moreover, we investigate the cellular and molecular
mechanisms of GC-directed membrane permeabilization. The facilitation of
doxorubicin infl ux by short-chain sphingolipids appeared dependent on the
(cellular) lipid membrane environment. Insertion of the short-chain sphingolipids
into plasma membranes does not result in enhanced doxorubicin accumulation in
any cell type. Cardiac myocytes, for example, do not respond to GC exposure, in
contrast to all malignant cells that have been tested. In order to pinpoint the specifi c
membrane requirements that are involved, we have set-up a membrane model
system that allows studying the translocation of doxorubicin over lipid membranes
of various compositions. In addition, we are examining the specifi city of the GC for
particular cell types, focusing on the in vivo accumulation of doxorubicin in heart
and (spontaneous) tumor tissue, cardiac function upon repeated administration, and
the overall toxicity profi le of this new GC-enriched doxorubicin formulation.
Figure 9: GC co-formulation improves the overall survival in mice with progressing ILC. Mice received
no treatment (control, purple line), once weekly free doxorubicin (bleu line), conventional liposomal
doxorubicin (yellow line) or the GC-modifi ed liposomal doxorubicin (green line). A body weight
decrease to 80% of the initial value at start of treatment or a tumor volume exceeding 1500 mm3 served
as predefi ned parameters for mouse study exit.

DIVISION OF SURGICAL ONCOLOGY
IMAGE GUIDED SURGERY
Theo Ruers, Marc van Beurden, Henk van der Poel, Michel Wouters, Germaine Relyveld, Jelle
Wesseling, Sven Rottenberg, Danny Evers, Jarich Spliethoff, Ronni Wessels, Diederik Grootendorst,
Breast Surgery group, Lung Surgery Group, Urology group, Head and Neck Oncology group
This research line, which has started recently, is aiming at optimizing surgical
procedures by better surgical guidance during operative procedures. To this end
new imaging technologies are developed and tested to improve tumor mapping
and staging pre and intra-operative. These imaging and surgical guidance
procedures should lead to more radical resections while sparing normal tissue
and organ function. The research line is a strong collaboration between the NKI-
AVL, Technical University Twente and industrial partners. For the moment three
projects are running. In the fi rst project we are developing a tool for optical biopsies
by means of spectroscopy and fl uorescence techniques. An optical needle was
developed together with industry. Ex vivo tissue specimens from more than hundred
patients have been analyzed with an accuracy of over 90%. In vivo testing is planned
early in 2011. In a second project, in cooperation with the group Biomedical Physics
and Biomedical Photonics of AMC, optical coherence tomography is tested for
improved tissue diagnosis in vulvar neoplasia, penis cancer and skin malignancies.
A third project concentrates on the use of photoacoustic imaging for diagnosis of
lymph node metastases and is running in close collaboration with the Biomedical
Photonic Imaging group of the University of Twente. We recently showed that
photoacoustic imaging is able to detect microscopic tumor foci within lymph
nodes of melanoma patients. The research performed in these projects should lead
to strong synerchy with the new innovative minimal invasive operating theatre
complex planned to be build in 2013 and should result in a technology platform that
can be used for further clinical studies.
GYNAECOLOGY
Gemma Kenter, Marc van Beurden, Willemien van Driel, Petra Biewenga,
Jan Lange, Lotti Lubsen-Brandsma, Monique Brood, Pia Kvistborg
The department focuses on innovative treatment for ovarian cancer, on interventions
to improve quality of life for premature (iatrogenic) menopausal symptoms and for
cervical carcinoma and immunotherapy for HPV related (premalignant) genital
neoplasms. Research takes place in close cooperation with the other centers from the
Center for Gynaecologic Oncology Amsterdam (CGOA).
Ovary In a randomized multicenter phase III clinical trial for stage III ovarian
carcinoma coordinated by the NKI-AVL the effect of secondary debulking surgery
with or without hyperthermic intraperitoneal cisplatinum is being studied.
Inclusion started march 2007 and currently runs in 5 centers in The Netherlands. It
is expected that 4 additional centers in Belgium and France will participate. During
interval debulking patients are randomized between interval debulking alone or
in combination with infusion of cisplatinum under intra-operative hyperthermic
condition. Primary endpoint of this study is progression free survival (KWF
CKTO2006-16).
In a multicenter trial the effect of different hormonal replacement regimen on
bone density, breast density and quality of life after prophylactic bilateral salpingo
oophorectomy are examined in a randomized control trial (M05HIR Hirise).
The effect of hormonal replacement therapy on menopausal complaints related
to biochemical changes in surgically and naturally postmenopausal women is
investigated in a prospective observational comparative study (M06HRT Novaria).
A multicenter randomized trial to investigate the effect of cognitive behavioral
therapy (CBT) and physical exercise for climacteric symptoms in breast cancer
Division head Theo Ruers
147
surgical oncology
Board
Theo Ruers MD PhD Head
Marc Van Beurden MD PhD Academic staff
Michiel Van den Brekel MD PhD Academic staff
General Surgical Oncology
Emiel Rutgers MD PhD Head
Arend Aalbers MD Academic staff
Daphne Hompes MD Academic staff
Frits van Coevorden MD PhD Academic staff
Hester Oldenburg MD PhD Academic staff
Houke Klomp MD PhD Academic staff
Johanna Van Sandick MD PhD Academic staff
Jos Van Der Hage MD PhD Academic staff
Koen Peeters MD Academic staff
Marianne Piek-den Hartog MD Academic staff
Marie-Jeanne Baas-Vrancken Peeters MD PhD
Academic staff
Michel Wouters MD Academic staff
Omgo Nieweg MD PhD Academic staff
Theo Ruers MD PhD Academic staff
Vic Verwaal MD PhD Academic staff
Hidde Veenstra MD Research physician
Martijn Stuiver Physiotherapist
Ronnie Wessels Research assistant
Ewout Courrech Staal MD Research physician
Lotte Nieuwenhuis PhD student
Mila Donker MD Research assistant
Rachel Numan PhD student
Ronald de Vreeze MD Research physician
Sjoerd Bruin MD Research physician
Danny Evers MD Research physician
Karen Bloemendal PhD student
Martine Bloemer PhD student
Stella Mook MD Research physician
Tjeerd Aukema MD Research physician
Erik von Meyenfeldt MD Research physician
Eva Schaake Research physician
Jarich Spliethoff PhD student
Yvonne Klaver Reseach physician

148
surgical oncology
Head and Neck Oncology and Surgery
Michiel Van den Brekel MD PhD Head
Annemiek Ackerstaff PhD Psychologist
Corina van As-Brooks PhD Academic staff
Alfons Balm MD PhD FRCS FACS Academic
staff
Maarten Borgemeester MD Research physician
Cindy van den Boer MD Research physician
Renee Clapham MSc PhD student
Renske Fles Msc Researcher
Tom Geurts MD PhD Research physician
Frans Hilgers MD PhD Academic staff
Carlijn Holland MD Research physician
Luuk Janssen MD PhD Research physician
Irene Jacobi PhD Phoniatrician
Baris Karakullukcu MD Academic staff
Martin Klop MD PhD Academic staff
Menno Krap DDS Academic staff
Annemarijn Kreeft MD Research physician
Nicoline Leunis MD Research physician
Michiel Lieshout DDS Academic staff
Wouter Lodder MD Research physician
Peter Lohuis MD PhD Academic staff
Lisette Van der Molen MSc PhD student
Peter van Maanen MD Research physician
Marlies Maatje MD Research physician
Hester van Monsjou MD Research physician
Saar Muller PhD Academic staff
Jimmy Pramana MD Research physician
Renske Scheenstra MD Research physician
Noortje Schwandt MD PhD Research physician
Ludi Smeele MD DDS PhD Academic staff
Rob Van Son PhD Post-doc
Bing Tan MD PhD Academic staff
Noortje Theunissen Academic staff
Adriaan Timmers DDS Academic staff
Caroline Verhagen MD PhD student
Dalith van der Vlies MD Research physician
Maarten Wildeman MD Research physician
Marloes Wondergem MD Research physician
Volkert Wreesmann MD Research physician
Charlotte Zuur MD PhD Academic staff
Urologic Oncology
Simon Horenblas MD PhD FEBU Head
Axel Bex MD PhD Academic staff
Wim Meinhardt MD PhD Academic staff
Henk Van de Poel MD PhD Academic staff
Richard Meijer MD Fellow
Chantal Nunnink MD Resident
Niels Graafl and Research physician
Gynaecology
Gemma Kenter MD PhD Head
Jan Lange MD Academic staff
Lottie Lubsen-Brandsma MD Academic staff
Marc van Beurden MD PhD Academic staff
Monique Brood MD Academic staff
Petra Biewenga MD Academic staff
Pia Kvistborg MD Post-doc
Willemien van Driel MD PhD Academic staff
patients experiencing treatment-induced menopause is fi nalized, the results of
which are analyzed (KWF 2006-3470).
The benefi cial effect of a laparoscopy in order to predict the operability in high stage
ovarian carcinoma will be studied in a multicenter randomized trial coordinated by
the CGOA (ZON MW doelmatigheid 80-82310-97-11056).
Vulva Participation in the GROINSS-V II study, an international multicenter observational
study on patients with vulvar cancer. Patients with positive lymph nodes,
diagnosed by sentinal node technic, undergo radiation without full lymphadenectomy.
Endpoints of this study are recurrence free survival and quality of life.
For patients with locally advanced carcinoma of the vulva an effi cacy study is
ongoing during which patients are treated with induction chemoradiation and if
necessary followed by surgery in order to reduce postoperative morbidity (AMC
locally advanced vulvar cancer effi cacy study).
The benefi t of wearing therapeutic elastic stockings in order to prevent lymphedema
for patients treated with inguinal lymph node dissection is conducted in a nonblinded,
randomized controlled trial (M06PRO Kousen study). Inclusion was
fi nalized and results will be available in 2010.
Optical coherence tomography (OCT) is an emerging biomedical optical imaging
technique that performs high resolution, cross sectional tomographic imaging
generating pictures that resemble histopathological examination. We will investigate
the potential role of OCT as “optical biopsy” device in patients with (pre)malignant
vulvar disease.
We reported on the clinical effi cacy in 58% of imiquimod for treatment of usual type
vulvar intraepithelial neoplasia (uVIN). We have assessed numbers of
immunocompetent cells, expression of p16INK4a in relation to HPV clearance in
relation to clinical response. Data indicate that imiquimod-induced clearance of
HPV, is confi rmed by normalization of p16INK4a expression and in normalization
of immunocompetent cells in the dermis which is strongly correlated to histologic
regression.
Patients with VIN III take part in a multicenter randomized trial to study the
effect of vaccination with synthetic long HPV 16 E6/E7 peptides with or without
imiquimod on the vaccination site.
Cervix A multicenter trial has recently been approved by the CCMO to study the
effi cacy of combined chemo-immunotherapy in high stage or recurrent carcinoma
of the cervix.
A multicenter trial has been prepared to study the effi cacy of combined chemoimmunotherapy.
Furthermore, preparations for a phase 1 trial with DNA HPV 16 E7
vaccination in patients with HPV related disorders of the genital region or head and
neck region are being made.
BREAST CANCER
Emiel Rutgers, Hester Oldenburg, Marie-Jeanne Vrancken Peeters,
Jos van der Hage, Mila Donker, Stella Mook, Tjeerd Aukema.
Adjuvant! Online (www.adjuvantonline.com) calculates a 10-year survival probability
based on the patient’s age, tumor size, grade and estrogen receptor. To investigate
whether the predictions by Adjuvant are applicable to the Dutch breast cancer
population, we conducted a retrospective validation study in 5380 patients. Our
results show that Adjuvant! predicts both prognosis and treatment benefi t accurate
in the whole group and in most subgroups, with an overestimation in patients under
40 years of age with an ER+ tumor.
Mammographic screening has led to a proportional shift towards earlier stage
breast cancers at presentation. The aim of our study was to assess whether method
of detection provides prognostic information beyond standard prognostic factors.
In our study cohort of 2592 invasive breast cancer patients screen-detection was
an independent prognostic factor for reduced all-cause- and breast cancer-specifi c
mortality, compared to non-screening-related carcinomas. Therefore, method of
detection should be taken into account when estimating individual prognosis.

We assessed the infl uence of neoadjuvant chemotherapy for invasive breast cancer
on short-term complications after skin sparing mastectomy and immediate
prosthetic reconstruction. Therefore the surgical outcome in patients treated
with neoadjuvant chemotherapy was compared to patients without preoperative
chemotherapy. Since the overall complication rate was similar, we concluded that
neoadjuvant chemotherapy does not increase the rate of immediate postoperative
complications after skin sparing mastectomy and immediate reconstruction.
We developed a new surgical technique to evaluate the axillary nodal response by
Marking of the Axillary lymph node with Radioactive Iodine (MARI) seeds prior
to neoadjuvant chemotherapy and selectively remove them after the neoadjuvant
chemotherapy. Since the identifi cation rate of the MARI node was high (95%) and
the MARI node gave an accurate indication for the response in the additional nodes
in 95%, we concluded that this procedure is feasible and promising for selecting
patients for a more conservative axillary treatment after neoadjuvant chemotherapy.
In cooperation with the PSOE we performed a RCT (EVA) evaluating the
effectiveness of cognitive behavioural therapy, physical exercises and the
combination of both interventions on premature menopause in breast cancer
patients. First analysis of the fi nal results show signifi cant effect sizes for e.g.
menopausal symptoms and hot fl ushes.
We are preparing an RCT (POWER) to study the axillary recurrence rate in primary
operable breast cancer patients that have a positive sentinel node and do not undergo
completion locoregional axillary treatment.
GASTRO-INTESTINAL CANCER
Vic Verwaal, Theo Ruers, Johanna van Sandick, Frits van Coevorden, Marie-Jeanne Vrancken
Peeters, Arend Aalbers, Ewout Courrech Staal, Martine Bloemer, Karen Bloemendal, Maurits
Swellengrebel, Sjoerd Bruin, Danny Evers, Loes Velthuizen, Jarich Spliethoff
Oesophagus Several studies were performed to investigate whether biological
markers can improve individual treatment planning in patients with non-metastatic
oesophageal cancer. The tumour-stroma ratio was examined on archival biopsy
material and proved to be an independent prognostic factor for survival. In an
other study, the incidence of mutations in EGFR, KRAS and BRAF was studied in
oesophageal cancer biopsies from patients who were treated with chemoradiotherapy
and surgery. In none of the 30 patients, mutations were found. Therefore, prudence
is warranted when introducing targeted therapies on the basis of EGFR, KRAS and
BRAF mutations in the treatment of oesophageal cancer.
We reviewed the literature on quality aspects of oesophageal cancer surgery.
There was strong evidence that both hospital and surgeon volume are important
determinants of postoperative mortality. The most commonly reported process
measures were determinants of patient selection for surgery. The level of evidence
for pathological outcome measures was high. This review indicated that there is a
need for uniformity in the evaluation of quality of care. A study was undertaken to
investigate the quality of care for 821 oesophageal cancer patients who were seen
at the NKI-AVL between 2003 and 2008. By evaluating different dimensions of
healthcare quality, we have identifi ed which steps in the multidisciplinary care path
need more attention in order to raise the whole level of care.
Hipec The most eye catching study performed by the HIPEC group is a study in
which a new classifi cation of peritoneal metastases is made based on cell atypia,
mitosis index and percentage of mucous on one side and survival on the other side.
PM could be categorized into four groups: low-grade, well-differentiated mucinous
tumor (DPAM); intermediated-grade mucinous carcinoma (PMCA-i); high-grade
mucinous carcinoma (PMCA); and high-grade nonmucinous carcinoma (PCA).
Multivariate analysis showed that histological classifi cation, gender, number of
segments affected, completeness of cytoreduction, and HIPEC as primary treatment
were signifi cant related to OS and DFS. The 5-year OS was 64% in the DPAM group,
36% in the PMCA group, and 24% in the PCA group. Of PM originating from an
appendix tumor, 29% were of non-DPAM type. Of primary colorectal tumors, 37%
resulted in mucinous PM, and another 26% of PM of colorectal origin had partly
149
surgical oncology
Plastic and Reconstructive Surgery
J Joris Hage MD PhD Head
Arjen van Turnhout MD Academic staff
Carolien Wever MD Academic staff
Leonie Woerdeman MD PhD Academic staff
Marieke van der Berg MD Academic staff
Martine van Huizum MD Academic staff
Brigitte Drost MD
Anesthesiology
Peter Schutte MD Head
Dirk Buitelaar MD Academic staff
Katina Efthymiou MD Academic staff
Christoph Hahn MD PhD Academic staff
Sandra Huissoon MD Academic staff
Lenie Hulshoff MD Academic staff
May Ronday MD Academic staff
Michael Sˇrámek MD PhD Academic staff
Julia ten Cate MD Academic staff
Ingeborg Vergouwe MD Academic staff
Dermatology
Wietze Van der Veen MD PhD Head
Inka Nieuweboer-Krobotova MD Academic staff
Biljana Zupan-Kajcovski MD Academic staff
Germaine Relyveld MD PhD Academic staff

150
surgical oncology
Publications
Adam R, Bhangui P, Poston G, Mirza D,
Nuzzo G, Barroso E, Ijzermans J, Hubert C,
Ruers T, Capussotti L, Ouellet JF, Laurent
C, Cugat E, Colombo PE, Milicevic M. Is
perioperative chemotherapy useful for solitary,
metachronous, colorectal liver metastases?
Ann Surg 2010;252:774-787
Ahmed AKJ, Hahn D, Hage JJ, Bleiker E,
Woerdeman LAE. Temporary banking of
the nipple-areola complex in 97 skin sparing
mastectomies. Plast Reconstr Surg 2010 (in
press)
Alderliesten T, Loo C, Paape A, Muller
S, Rutgers E, Peeters MJ, Gilhuijs K. On
the feasibility of MRI-guided navigation to
demarcate breast cancer for breast-conserving
surgery. Med Phys. 2010;37:2617-26
Alkhateeb SS, Van Rhijn BW, Finelli A, van
der Kwast T, Evans A, Hanna S, Vajpeyi
R, Fleshner NE, Jewett MA, Zlotta AR.
Nonprimary pT1 nonmuscle invasive bladder
cancer treated with bacillus Calmette-Guerin
is associated with higher risk of progression
compared to primary T1 tumors. J Urol.
2010;184:81-6
Aukema TS, Kappers I, Olmos RA,
Codrington HE, van Tinteren H, van Pel
R, Klomp HM, for the NEL Study Group.
Is 18F-FDG PET/CT useful for the early
prediction of histopathologic response to
neoadjuvant erlotinib in patients with
non-small cell lung cancer? J Nucl Med.
2010;51:1344-8
Aukema TS, Olmos RA, Korse CM, Kroon
BB, Wouters MW, Vogel WV, Bonfrer JM,
Nieweg OE. Utility of FDG PET/CT and
brain MRI in melanoma patients with
increased serum S-100B level during followup.
Ann Surg Oncol. 2010;17:1657-61
Aukema TS, Rutgers EJ, Vogel WV, Teertstra
HJ, Oldenburg HS, Vrancken Peeters MT,
Wesseling J, Russell NS, Valdés Olmos
RA. The role of FDG PET/CT in patients
with locoregional breast cancer recurrence:
a comparison to conventional imaging
techniques. Eur J Surg Oncol. 2010;36:387-92
Aukema TS, Straver ME, Peeters MJ, Russell
NS, Gilhuijs KG, Vogel WV, Rutgers EJ,
Olmos RA. Detection of extra-axillary lymph
node involvement with FDG PET/CT in
patients with stage II-III breast cancer.
Eur J Cancer. 2010
mucinous histology. The conclusion of this study was that histology is a signifi cant
predictive factor of OS and DFS in PM treated with surgical cytoreduction and
HIPEC. Due to collaboration between the HIPEC centres in the Netherlands a
large epidemiologic study (3356 patients) is performed on the change in survival of
metastasized colon cancer over the last decades. This study showed that the median
survival of patients with liver metastasis changed from 34 (1995 – 2000) to 51 weeks
(2001 – 2006) where as the survival of patient with PM stayed the same (35 in 1995
– 2000 and 30 weeks in 2001 – 2006). A second fi nding was that the most patients
with peritoneal carcinomatosis were still treated with systemic chemotherapy only,
in stead of with HIPEC.
Colorectal liver metastases Research is focused on imaging and local tumour
destruction. The results of an international randomized study on the use of RFA for
unresectable colorectal liver metastases were presented as an oral at ASCO 2010.
Further research is directed at immunological cell death and immunostimulation
after local tumour destruction (e.a. HIFU) within a CTMM consortium. Together
with the Technical University Twente gold nanoshells, in combination with
NIR light, are used for tumour detection and ablation of resection margins. In
collaboration with industrial partners the use spectroscopy for tumor diagnosis and
guidance of minimal invasive procedures is investigated. The research line is funded
by KWF, the UTwente, EORTC and industrial partners.
MELANOMA
Jos van der Hage, Bin Kroon, Omgo Nieweg, Martijn Stuiver,
Hidde Veenstra, Ronnie Wessels, Michel Wouters
The research activities of the melanomologists concern the anatomy and physiology
of the lymphatic system, implications for dissemination, implementation of new
forms of imaging techniques and other new forms of diagnostic and staging
methods, sentinel node biopsy, aspects of inguinal node dissection, in transit
metastases and aspects of regional isolation perfusion.
Eight years ago, we found a 23% incidence of in transit metastases in patients with
an involved sentinel node. This high percentage was reason for concern. Now, in our
much larger patient population, we found no predisposition for in-transit metastases
and we consider this issue resolved defi nitively.
Melanoma surgeons are struggling with the question whether every patient with
a tumour-positive sentinel node needs a completion node dissection. Of interest,
patients with a subcapsular metastasis smaller than 0.1 mm have a 91% fi ve-year
overall survival rate and a 9% incidence of involvement of other lymph nodes.
One may consider sparing these patients a completion node dissection. Lymph
node dissections, especially in the groin are associated with substantial morbidity.
Identifying the patients at risk for major wound complications (i.e. dehiscence)
pre-operatively could infl uence operative techniques and peri-operative care. In a
retrospective study of 236 groin dissections these risk factors were identifi ed and
peri-operative measures to decrease morbidity in these patients, like minimal
invasive operative techniques, are now under consideration.
There is one prospective randomized trial looking at whether lymphatic mapping
improves survival. A recent interim analysis of this RCT (led by Dr. D.L. Morton)
confi rmed that the mean number involved nodes in patients with an involved
sentinel node is 1.4, yet this number increases to 3.2 by the time lymph nodes
become palpable (p=0.0001). Melanoma specifi c survival is signifi cantly better in
the patients with a tumor-positive sentinel node: 61% versus 42% in the patients
who were initially observed and who came back with palpable lymph nodes later on
during follow up (p-value 0.01).
Several locoregional treatment options for advanced melanoma patients exist.
Different treatment modalities range from local excision to isolated limb perfusion.
Although isolated limb perfusion is a very effective therapy, it is a highly complex
procedure. Another modality is CO2 laser therapy. CO2 laser therapy is a simple
and effective therapy applicable to patients with multiple in transit metastases
without signifi cant morbidity. In 2010, patients who underwent this type of therapy

in the past 4 years in the Netherlands Cancer Institute were analyzed. CO2 laser
was demonstrated to be a save and effective treatment modality associated with an
almost 100% limb salvage rate.
LIPOSARCOMA STUDY GROUP
Frits van Coevorden, Daphne de Jong, Petra Nederlof, Rick Haas,
Harm van Tinteren, Ronald de Vreeze
We analyzed our liposarcoma database of 325 patients(1977-2007) and subjected the
material to additional tests for improved classifi cation and grading. Adding clinical
information of these cases created a set of data for further study and analysis. The
occurrence of combined patterns of well-differentiated liposarcoma(WDLS) and
myxoid/roundcell liposarcoma(MRLS) designated as mixed-type liposarcoma pose
a conceptual problem as this feature may have consequences for treatment choice
and prognosis. We have dissected the molecular relation of tumor components
in cases of mixed-type liposarcoma. Based on heterogeneous preoperative MRI
features, 8 cases were selected. Preoperative biopsies and resection specimens
were analyzed including molecular and immunohistochemical analysis on all
components. As controls, cases with homogeneous MRI features and uniform
aspects of 10 MRLS and 5 WDLS were studied. All patients with heterogeneous MRI
features showed morphological components of MRLS and WDLS. RTPCR showed
FUS-DDIT3 fusion in both components in 5/8 cases in the absence (0/5) of MDM2
and CDK4 amplifi cation. In 3/8 patients, MDM2 and/or CDK4 were overexpressed,
and amplifi cation was shown by MLPA in the absence of MRLS translocations. All
control patients showed a molecular pattern consistent with their morphological
features. Therefore, mixed-type liposarcomas should not be regarded as collision
tumors, but as an extreme variant of the morphological spectrum within a single
biological entity, explaining the biological contradiction of mixed-type liposarcoma.
For treatment stratifi cation, detailed classifi cation including molecular support
should be performed in tumors with heterogeneous MRI features.
The classifi cation of multifocal myxoid/round cell liposarcoma (MRLS), which is
defi ned as tumor presentation in at least two separate sites before manifestation in
the lungs, as either metastasis or as a second primary tumor, has essential clinical
consequences. MRLS is characterized by t(12;16)(q13;p11) or t(12;22)(q13;q12), and
various exon fusion transcripts are described with varying incidences, which permits
their use as markers for clonality. Moreover, in solid tumors, analysis of loss of
heterozygozity(LOH) is valuable for clonality analysis. Therefore, fi fteen multifocal
MRLS-patients with 2-5 metachronous (n=12) or synchronous (n=3) localizations
were investigated. Using RT-PCR, the detailed molecular characteristics of the FUS-
CHOP and EWS-CHOP breakpoints were determined. LOH-analysis at twelve loci
was then used to further analyze clonal relationships. In all patients, tumor sites
showed identical FUS-CHOP fusion products. In six patients, identical rare fusion
transcripts were found, supporting a clonal relationship. Nine patients had the
common exon5-FUS/exon2-CHOP fusion transcript, and two of these were identifi ed
as clonally related by LOH-analysis. In all other patients, LOH-analysis was highly
suggestive of a clonal relationship, and no evidence for interpretation of a second
primary tumor was found. This study supports the metastatic nature of apparent
multifocal myxoid/round cell liposarcoma.
THORACIC CANCER SURGERY
Houke Klomp, Michel Wouters, Johanna van Sandick, Erik von Meyenfeldt,
Eva Schaake, Tjeerd Aukema
Molecular imaging may play a critical role in the evaluation and treatment
planning for NSCLC. The M06NEL study investigated the role of 18F-FDG PET/
CT for the early identifi cation of response to therapy in patients who were treated
with a molecular-targeted agent (EGFR-TKI erlotinib). 18F-FDG PET/CT was
used to monitor the disease before and at one week after administration of EGFR-
TKI. Changes in tumor 18F-FDG uptake during treatment were measured by
Publications (continued)
151
surgical oncology
Aukema TS, Valdés Olmos RA, Korse CM,
Kroon BBR, Wouters MWJM, Vogel WV,
Bonfrer JMG, Nieweg OE. Utility of FDG
PET/CT and brain MRI in melanoma
patients with increased serum S-100B
level during follow-up. Ann Surg Oncol
2010;17:1657-1661
Aukema TS, Valdés Olmos RA, Wouters
MW, Klop WM, Kroon BB, Vogel WV,
Nieweg OE.Utility of preoperative 18F-FDG
PET/CT and brain MRI in melanoma
patients with palpable lymph node
metastases. Ann Surg Oncol. 2010;17:2773-8
Axwijk PH, Kluijt I, de Jong D, Gille H,
Teertstra J, Horenblas S. Hereditary
causes of kidney tumours. Eur J Clin Invest.
2010;40:433-9
Bex A, Jonasch E, Kirkali Z, Mejean A,
Mulders P, Oudard S, Patard JJ, Powles
T, van Poppel H, Wood CG. Integrating
Surgery with Targeted Therapies for Renal
Cell Carcinoma: Current Evidence and
Ongoing Trials. Eur Urol. 2010
Bex A, Sonke GS, Pos FJ, Brandsma D,
Kerst JM, Horenblas S. Symptomatic brain
metastases from small-cell carcinoma of the
urinary bladder: The Netherlands Cancer
Institute experience and literature review.
Ann Oncol. 2010;21:2240-5
Bex A, Van der Veldt AA, Blank C, Meijerink
MR, Boven E, Haanen JB. Progression of
a caval vein thrombus in two patients with
primary renal cell carcinoma on pretreatment
with sunitinib. Acta Oncol. 2010;49:520-3
Bex A, Vermeeren L, de Windt G, Prevoo
W, Horenblas S, Olmos RA. Feasibility
of sentinel node detection in renal cell
carcinoma: a pilot study. Eur J Nucl Med Mol
Imaging. 2010;37:1117-23
Bex A, Vermeeren L, Meinhardt W,
Prevoo W, Horenblas S, Valdés Olmos RA.
Intraoperative sentinel node identifi cation
and sampling in clinically node-negative
renal cell carcinoma: initial experience in 20
patients. World J Urol. 2010
Biewenga P, Mutsaerts MA, Stalpers LJ,
Buist MR, Schilthuis MS, van der Velden J.
Can we predict vesicovaginal or rectovaginal
fi stula formation in patients with stage
IVA cervical cancer. Int J Gynecol Cancer.
2010;20:471-5
120

152
surgical oncology
Publications (continued)
Biewenga P, van der Velden J, Mol BW,
Stalpers LJ, Schilthuis MS, van der Steeg JW,
Burger MP, Buist MR. Prognostic model for
survival in patients with early stage cervical
cancer. Cancer. 2010
Boström PJ, Alkhateeb S, van Rhijn BW,
Kuk C, Zlotta AR. Optimal timing of radical
cystectomy in T1 high-grade bladder cancer.
Expert Rev Anticancer Ther. 2010;10:1891-
902
Bruin SC, Verwaal VJ, Vincent A,
van ‘t Veer LJ, van Velthuysen ML.
A clinicopathologic analysis of peritoneal
metastases of colorectal and appendiceal
origin. Ann Surg Oncol 2010;17:2330-2340
Cevizci R, et al. Laser excision of a typical
carcinoid tumor of the larynx: a case report.
Kulak Burun Bogaz Ihtis Derg,
2010;20:305-8
Chai X, van Herk M, van de Kamer JB,
Remeijer P, Bex A, Betgen A, De Reijke
TM, Hulshof MC, Pos FJ, Bel A. Behavior
of lipiodol markers during image guided
radiotherapy of bladder cancer. Int J Radiat
Oncol Biol Phys. 2010;77:309-14
Chakera AH, Hesse B, Burak Z, Ballinger J,
Britten A, Caracó R, Cochran AJ,
Cook MG, Drzewiecki KT, Essner R,
Even-Sapir E, Eggermont AMM, Gmeinar
Stopar T, Ingvar C, Mihm MC, McCarthy
SW,Mozzillo N, Nieweg OE, Scolyer R,
Starz H, Thompson JF, Trifi ró G, Viale G,
Vidal-Sicart S, Uren RF, Waddington W,
Chiti A, Spatz A, Testori A. EANM-EORTC
general recommendations for sentinel node
diagnostics in melanoma. Ann Surg Oncol
(in press)
Courrech Staal EFW, Aleman BMP,
Boot H, van Velthuysen MF, van Tinteren
H, van Sandick JW. Systematic review
of the benefi ts and risks of neoadjuvant
chemoradiation for oesophageal cancer.
Br J Surg 2010;97:1482-96
Courrech Staal EFW, Aleman BMP,
van Velthuysen MLF, Cats A, Boot H,
Jansen EPM, van Coevorden F, van Sandick
JW. Chemoradi ation for esophageal cancer:
institutional experience with three different
regimens. Am J Clin Oncol 2010
standardized uptake values. The results of the study suggest that early during the
course of therapy, 18F-FDG PET/CT can predict response to treatment in patients
with NSCLC. In locally advanced stage IIIA-N2 NSCLC, evaluation of induction
chemotherapy followed by either surgical resection or radiotherapy, showed that
outcome was favourable after lobectomy, but not after pneumonectomy (5-yr survival
43% versus 16%, 5-yr survival after radiotherapy 16%). Newer induction protocols
combining (daily low-dose) chemotherapy with high-dose radiation (66 Gy) are
ongoing as the M07CCL (Raditux) study using cetuximab in combination with
concurrent chemoradiotherapy. A collaborative study will investigate the role of
surgery following such intensifi ed chemoradiation protocols.
Our department participated in the EORTC 08031 phase II trial investigating the
feasibility of trimodality therapy that consisted of induction chemotherapy followed
by extrapleural pneumonectomy and postoperative radiotherapy in patients with
malignant pleural mesothelioma. Although feasible, trimodality therapy in patients
with mesothelioma was not completed within the strictly defi ned timelines of this
protocol and adjustments are necessary.
Local treatment of pulmonary metastases is considered in patients with (oligo)
metastatic disease, in whom primary tumour control has been achieved. The role of
local treatment by either thoracotomy, video-assisted thoracoscopic procedures and
local ablation techniques for long-term disease control in oligometastatic disease has
been evaluated (for several primary tumor types).
Variation in quality of care provided by different institutions is the subject of an
intensive debate in the Netherlands. A study of the Quality of Cancer Care taskforce
of the Dutch Cancer Society showed that outcome of bladder, colorectal, breast
and lung cancer patients in the Netherlands varies by region and the hospital their
cancer was diagnosed in. However, hospital characteristics like a high diagnostic
volume, teaching status or availability of radiotherapy facilities proved no guarantee
for optimal treatment rates.
HEAD AND NECK SURGERY AND ONCOLOGY
Alfons Balm, Michiel van den Brekel, Frans Hilgers, Martin Klop, Peter Lohuis,
Ludi Smeele, Bing Tan, Baris Karakullukcu, Charlotte Zuur
The department of head and neck surgery and oncology is a national referral centre
and one of the larger clinical departments in this fi eld treating about 600 new
patients annually. The department is active in clinical and translational research.
Currently, 3 full professors are part of the department and staff has part-time
positions in the Academic Medical Centre (AMC) of the University of Amsterdam.
Being a multidisciplinary fi eld, there are many clinical and research connections
within the institute and also in the AMC.
Translational Research In 2010 the head and neck department was involved in
several translational projects. Together with Adrian Begg and Conchita Vens we
studied the Fanconi pathway in oral cancer samples as well as cell lines. In about
15% of cell lines there is a phenotype of Fanconi defi ciency. Currently some 100
patient samples are sequenced. This fi nding opens the way to start looking for
alternative treatment strategies such as s PARP inhibitors. In a project together with
Marcel Verheij on the BCL2 inhibitor gossypol, a clinical phase two study has been
started in patients treated with cisplatin based chemoirradiation. Together with
Jacques Neefjes and the department of pulmonology we have set up a protocol for
short term cultures. These can be used to test chemosensitivity pretreatment and
also be used to test the chemical library of Huib Ovaa to look for new targets. In
cooperation with the NWHHT (Dutch Cooperative Head and Neck Group) genetic
markers predicting progression from dysplasia to cancer are studied. This year an
expression profi le predicting lymph node metastases as developed by Roepman et al,
has been validated in a large Dutch patient cohort. Early detection and monitoring
treatment outcome of nasopharyngeal cancer using (anti-)Epstein-Barr Virus (EBV)
based tumor markers is studied in the Netherlands, UI-Jakarta, UGM-Yogyakarta
together with the VUmc. With the same group a project on lytical induction therapy
in recurrent nasopharyngeal cancer has been approved by the KWF.

Photodynamic Therapy The department is active in research on photodynamic
therapy and several grants have been obtained (VENI, ZonMW, NWO, KWF).
Currently the focus of this research is on interstitial PDT. In several projects
treatment planning and dosimetry for interstitial PDT is studied by means of
developing pretreatment planning tools and intraoperative imaging guidance. In a
national study with participation of all academic medical centers in the Netherlands,
the cost effectiveness of Foscan PDT for incurable recurrent head and neck cancers
is being studied. Monitoring of tissue oxygen saturation and Foscan concentration
during PDT to optimize treatment parameters is another project.
Rehabilitation Rehabilitation research focuses on several topics. Prevention
of trismus, swallowing and speech problems in patients treated with chemoradiation
for advanced head and neck cancer is one fi eld of research. An evidence
based rehabilitation program has been set up in 2010. In a project on pulmonary
rehabilitation after laryngectomy, in cooperation with ATOS, new heat-and-moistexchangers
are being developed and tested using an artifi cial airway system, but
also in patients. Mucociliairy transport in the trachea after laryngectomy is another
fi eld of interest. Together with the university of Amsterdam centre for language
and communication (ACLC), an automatic speech analyzer has been developed for
pathologic voices, such as after laryngectomy or chemoradiation. A project to use
these systems in rehabilitation was started.
Clinical Research Clinical research is quite diverse. Fields of interest are sentinel
node detection, imaging, prediction of inoperability clinical trials using novel
chemotherapy regimens and clinical and biological aspects of young patients with
head and neck cancer. The use of SPECT-CT and intraoperative imaging using both
the Sentinella and fl uorescence is assessed and has been found useful in oral cancer
and melanoma. Studies on modelling tumor resection and functional outcome
prediction are done in cooperation with the Technical University Twente and VUmc.
UROLOGIC ONCOLOGY
Simon Horenblas, Axel Bex, Wim Meinhardt, Henk van de Poel,
Richard Meijer, Chantal Nunnink, Niels Graafl and
Research in urologic oncology has been centred around the following themes:
Improved staging of urologic tumours, organ and function sparing, fundamental
research in prostate, kidney and penis cancer.
Improved staging of urologic tumours Research has been focussed on the role
of sentinel node biopsy in kidney, prostate, testicular and penile cancer. In all
above mentioned tumours, apart from kidney cancer, sentinel node biopsy has
proven to be extremely useful in minimizing the morbidity of surgery of the lymph
nodes and at the same time maximizing the detection rate of occult lymph node
metastases. The collaboration with the department of nuclear physics has been very
fruitful. Especially the use of SPECT /CT scans and intra-operative imaging with a
mobile gamma camera (Sentinella®) has proven to be a clinical useful adjunct. In
prostate cancer no false negative fi ndings were observed. The sentinel node strategy
together with the Sentinella was effective in localizing sentinel nodes in a variety of
anatomical locations, otherwise not removed during lymph node dissection.
The scope of research has been widened by collaboration with the department of
clinical physics. A comparative trial was started, comparing patent blue, ICG and
ICG with technetium. Using a fl uorescence camera ICG is seen after i.v. injection.
In testicular cancer also no false negative fi ndings have been observed. Accrual in
this clinical study is very slow unfortunately, mainly because of the fact that stage I
germ cell tumours are mostly been treated outside of NKI-AVL.
In penile cancer the false negative rate dropped from 20% to an acceptable 5.1%.
For renal cancer we continue with the sentinel node study, approved in 2008,
with the aim of elucidating the lymphatic pathways of renal cancer and the
immunological effect of renal cancer in the sentinel nodes.
Publications (continued)
153
surgical oncology
Courrech Staal EFW, Smit VT, van
Velthuysen MLF, Spitzer-Naaykens JMJ,
Wouters MWJM, Mesker WE, Tollenaar
RAEM, van Sandick JW. Reproducibility
and validation of tumour stroma ratio scoring
on oesophageal adenocarcinoma biopsies. Eur
J Cancer 2010
Courrech Staal EFW, van Sandick JW,
van Tinteren H, Cats A, Aaronson NK.
Health-related quality of life in long-term
esophageal cancer survivors after potentially
curative treatment. J Thorac Cardiovasc Surg
2010;140:777-83
Courrech Staal EFW, Wouters MWJM,
Boot H, Tollenaar RAEM, van Sandick JW.
Quality of care in the surgical treatment of
esophageal cancer: a review. Eur J Sur g Oncol
2010;36:1035-43
Courrech Staal EFW, Wouters MWJM,
van Sandick JW, Takkenberg MM,
Smit VTHBM, Junggeburt JMC, Spitzer-
Naaykens JMJ; Karsten T; Hartgrink HH,
Mesker WE, Tollenaar RAEM. The stromal
part of adenocarcinomas of the oesophagus:
does it conceal targets for therapy?
Eur J Cancer, 2010;46:720-728
De Jong MC, et al. CD44 expression predicts
local recurrence after radiotherapy in larynx
cancer. Clin Cancer Res, 2010;16):5329-38
De Jong MC, et al. HPV and high-risk
gene expression profi les predict response to
chemoradiotherapy in head and neck cancer,
independent of clinical factors. Radiother
Oncol, 2010;95:365-70
De Ronde JJ, Hannemann J, Halfwerk H,
Mulder L, Straver ME, Vrancken Peeters
MJ, Wesseling J, van de Vijver M, Wessels
LF, Rodenhuis S. Concordance of clinical
and molecular breast cancer subtyping in
the context of preoperative chemotherapy
response. Breast Cancer Res Treat.
2010;119:119-26
De Vos van Steenwijk PJ, Heusinkveld M,
Ramwadhdoebe TH, Löwik MJ,
van der Hulst JM, Goedemans R,
Piersma SJ, Kenter GG, van der Burg SH.
An unexpectedly large polyclonal repertoire
of HPV-specifi c T cells is poised for action in
patients with cervical cancer Cancer Res.
2010 Apr Cancer Res. 2010;70:2707-17

154
surgical oncology
Publications (continued)
De Vreeze RS, de Jong D, Koops W,
Nederlof PM, Ariaens A, Haas RL,
van Coevorden F. Oncogenesis and
classifi cation of mixed-type liposarcoma:
A radiological, histopathological and
molecular biological analysis. Int J Cancer.
2010
De Vreeze RS, de Jong D, Koops W,
Nederlof PM, Ariaens A, Haas RL,
van Coevorden F J Multifocal myxoid
liposarcoma-metastasis or second primary
tumor?: a molecular biological analysis.
Mol Diagn. 2010;12:238-43
De Vries RR, Nieuwenhuijzen JA, Vincent A,
van Tinteren H, Horenblas S. Survival
after cystectomy for invasive bladder cancer.
Eur J Surg Oncol. 2010;36:292-7
De Vries RR, Visser O, Nieuwenhuijzen
JA, Horenblas S; Members of the
Urological Oncology Working Group of the
Comprehensive Cancer Centre Amsterdam.
Outcome of treatment of bladder cancer:
a comparison between low-volume hospitals
and an oncology centre. World J Urol.
2010;28:431-7
Del Turco MR, Ponti A, Bick U,
Biganzoli L, Cserni G, Cutuli B, Decker
T, Dietel M, Gentilini O, Kuehn T, Mano
MP, Mantellini P, Marotti L, Poortmans
P, Rank F, Roe H, Scaffi di E, van der Hage
JA, Viale G, Wells C, Welnicka-Jaskiewicz
M, Wengstvam Y, Cataliotti L. Quality
indicators in breast cancer care. Eur J Cancer.
2010;46:2344-56
Dirven R, Kooijman PG, Maal TJ, Hilgers FJ,
Bergé SJ, Marres HA. An external neck
brace to support the peristomal fi xation
of an automatic stoma valve (ASV):
3D sterophotogrammetrical assessment.
Acta Otolaryngol 2010;130:851-858
Dovern E, de Hingh IH, Verwaal VJ,
van Driel WJ, Nienhuijs SW. Hyperthermic
intraperitoneal chemotherapy added to
the treatment of ovarian cancer. A review
of achieved results and complications.
Eur J Gynaecol Oncol 2010;31:256-261
Duijts SF, Faber MM, Oldenburg HS, van
Beurden M, Aaronson NK Effectiveness of
behavioral techniques and physical exercise on
psychosocial functioning and health-related
quality of life in breast cancer patients and
survivors-a meta-analysis. Psychooncology.
2010
Organ and function sparing The role of cytoreductive surgery in metastatic renal
cancer is further being investigated in a randomized EORTC study comparing
immediate versus deferred nephrectomy for patients with synchronous metastatic
renal cell carcinoma and the primary tumour in situ. This study (E 30073)
coordinated by Axel Bex, followed the experiences of the phase II study with neoadjuvant
Sutent (tyrosine kinase inhibitor).
In bladder cancer we continue to expand our experiences with the so called
Sexuality Preserving Cystectomy and Neobladder (SPCN). In a recent publication
excellent continence and potency rates were seen without any danger of increased
recurrences. Despite international scepsis we continue to advocate this procedure in
well selected patients. In line with the wish to decrease the morbidity of the surgery
robotic assisted cystectomy was started in 2010.
Improving early urine continence after robot assisted laparoscopic prostatectomy
(RALP) led to a randomized study on the role of ventral suspension using the vas
deference. A previous randomized study was completed showing no benefi t for
reconstruction of the median fi brous raphe in men after RALP.
We are involved in the active surveillance study for prostate cancer (Prias, initiated
by Rotterdam) and have entered more patients than any one else.
We are partners together with Rotterdam and Groningen in the PCMM consortium
regarding prostate cancer and prostatectomy
TRANSLATIONAL RESEARCH IN PROSTATE, KIDNEY AND
PENILE CANCER
Prostate cancer The role of Pim-1 expression in prostate cancer progression is
investigated. Pim-1 was earlier shown to enhance the growth response of prostate
cancer cells in vitro to low levels of DHT. A possible explanation for this may be
the downregulation by Pim-1 of the 17-beta-hydroxysteroid-dehydrogenase type 2
gene product (HSD17B2). HSD17B2 is involved in the enzymatic down regulation
of DHT and testosterone in breast and prostate cancers. Its down regulation by
Pim-1 overexpression may aid prostate cancer cells to survive in low testosterone
environments, such as in castrate resistant prostate cancer (CRPC).
The role of mTOR inhibition in chemotherapy is being assessed in a phase I study in
collaboration with Andre Bergman, medical oncologist. In this study the combined
use of everolimus (mTOR inhibitor) and cyclophosphamide in CPRC will be tested.
In collaboration with Prof. Ton Schumacher the role of T-cell receptors in prostate
cancer model vaccination studies was addressed.
Penis cancer The role of HPV in penis cancer was further investigated in close
collaboration with the department of pathology of the Free University medical
centre. Using molecular and serological analyses for a wide range of HPV types and
comparing serological fi ndings with age-matched male controls. Primarily HPV 16
infection is directly involved in penile carcinogenesis. Based on earlier micro-array
results a renewed analysis was started comparing HPV+ and HPV- tumors.
Kidney cancer In collaboration with the department of immunology, the
department of angiogenesis of Arjan Griffi oen at the Free University medical centre
and Eric Jonasch, MD Anderson Cancer Center, Houston, kidney cancer tissue is
analysed, specifi cally looking into neo-vascularization and immune modulation.
Also together with the department of immunology analysis of rapid expansion of
TIL cells was started.
Improved quality control of treatment results Using prospective data collection,
almost all uro-surgical treatments at the NKI-AVL can be analysed now. Extensive
use of these databases was made for prostate, bladder and penile cancer. Quality
of life analysis was done of in patients with localized prostate cancer managed by
brachytherapy or robot assisted laparoscopic prostatectomy (RALP).
Prospective data are analysed for risk estimation of treatment or active surveillance
for prostate cancer within the framework of the IMPACT-study, funded by ZonMW.
Together with the Comprehensive Cancer Center Amsterdam a comparative study
was published looking at recurrences in bladder cancer as a quality control tool.
Treatment related parameters were analysed in low volume and high volume

hospitals and compared with the results in the NKI-AVL. Although the recurrence
rate did not differ between the NKI-AVL (high volume hospital) and the other
hospitals, the early post –operative death was less than in other hospitals (0.6%
versus 4,3%).
ANESTHESIOLOGY AND INTENSIVE CARE MEDICINE
Peter Schutte, Dirk Buitelaar, Katina Efthymiou, Christoph Hahn, Sandra Huissoon,
Lenie Hulshoff, Michael Sˇrámek, Julia ten Cate, Ingeborg Vergouwe
The principal aim of the Department of Anesthesiology and Intensive Care Medicine
is to deliver the highest standard of anesthesiological and intensive care.
Presently our research focuses mainly on the role of peripheral nerve and central
neuraxial blockades as an adjuvant to general anesthesia. The purpose of these
blockades is threefold: a reduction in perioperative opioid consumption, the
prevention of chronic pain and shortening of length of hospital stay.
In collaboration with the Departments of Surgical Oncology, Head and Neck Oncology
and Urological Oncology, innovations are/have been implemented. E.g. superfi cial
cervical plexus blockade in patients undergoing head and neck surgery, fi eld blockade
and paravertebral blockade in breast surgery and transversus abdominis plane
blockade (TAP-block) in, for instance, robot assisted laparoscopic prostatectomies.
The results of the N07TAP–trial are evaluated and submitted for publication.
Furthermore two more study protocols are currently being developed. One study
focuses on perioperative pain treatment in patients undergoing laryngectomies
and commando resections. In this study the benefi cial effects of intravenous S(+)-
Ketanest and lidocaïne will be studied. The other study focuses on the intraoperative
position of patients undergoing thoracotomies in relation to the prevention of
postoperative ipsilateral postthoracotomy shoulder pain.
Future focus of interest is the role of regional and general anesthesia in the
prognosis of surgically treated oncologic patients.
PLASTIC AND RECONSTRUCTIVE SURGERY
Marieke van den Berg, Brigitte Drost, J Joris Hage, Martine van Huizum,
Arjen van Turnhout, Carolien Wever, Leonie Woerdeman
The research of the Department of Plastic and Reconstructive Surgery is focused
predominantly on innovative reconstructive techniques after ablative surgery by
other specialists. Additionally, ongoing vascular and functional research was done
in collaboration with the Department of Plastic Surgery at the University Medical
Centre Utrecht (prof. dr. M. Kon).
Vascular anatomy and clinical application of internal mammary artery
perforator flaps Internal mammary artery perforator (IMAP) pedicled or free fl aps
allow for superior skin cover of regional oncological defects and primary closure of
the parasternal donor site associated with limited associated donor site morbidity.
In collaboration with the UMCU, the dominant and non-dominant perforators
and their vascular territories were objectifi ed. Mobilization of the vascular pedicle
resulted in an increase of its length of 4.7 cm and, consequently, to double that
increase of the arc of rotation of the pedicled IMAP fl ap. The clinical reliability of the
IMAP free fl ap was proven.
Immediate prosthetic breast reconstruction after neoadjuvant chemotherapy
Neoadjuvant chemotherapy is gaining acceptance as an option for breast cancer
treatment but it is not known whether preoperative chemotherapy hampers
wound healing after skin sparing mastectomy and immediate prosthetic breast
reconstruction. The outcome of such combined surgery in women treated with
neoadjuvant chemotherapy from 2006 through 2009 was compared to that in
women without preoperative chemotherapy. The overall rate of postoperative
complications was less among neoadjuvantly treated women than in the control
Publications (continued)
155
surgical oncology
Elferink MA, Krijnen P, Wouters MW,
Lemmens VE, Jansen-Landheer ML,
van de Velde CJ, Langendijk JA, Marijnen
CA, Siesling S, Tollenaar RA. Variation in
treatment and outcome of patients with rectal
cancer by region, hospital type and volume in
the Netherlands. Eur J Surg Oncol. 2010;36
Suppl 1:S74-82
Elferink MA, Wouters MW, Krijnen P,
Lemmens VE, Jansen-Landheer ML,
van de Velde CJ, Siesling S, Tollenaar RA.
Disparities in quality of care for colon cancer
between hospitals in the Netherlands.
Eur J Surg Oncol. 2010;36 Suppl 1:S64-73
Elshof LE, Rutgers EJ, Deurloo EE, Loo
CE, Wesseling J, Pengel KE, Gilhuijs KG.
A practical approach to manage additional
lesions at preoperative breast MRI in patients
eligible for breast conserving therapy: results.
Breast Cancer ResTreat. 2010;124:707-15
Evers DJ, Verwaal VJ. Indication for
oophorectomy during cytoreduction for
intraperitoneal metastatic spread of colorectal
or appendiceal origin. Br J Surg 2010
Faries MB, Thompson JF, Cochran A,
Elashoff R, Glass EC, Mozzillo N, Nieweg
OE, Roses DF, Hoekstra HJ, Karakousis
CP, Reintgen DS, Coventry BJ, Wang HJ,
Morton DL; for the MSLT Cooperative
Group.The Impact on Morbidity and Length
of Stay of Early Versus Delayed Complete
Lymphadenectomy in Melanoma: Results of
the Multicenter Selective Lymphadenectomy
Trial (I). Ann Surg Oncol. 2010
Fles R, et al. Knowledge of general
practitioners about nasopharyngeal cancer
at the Puskesmas in Yogyakarta, Indonesia.
BMC Med Educ, 2010;10:81
Freling NJ, et al. Imaging fi ndings in
craniofacial childhood rhabdomyosarcoma.
Pediatr Radiol, 2010;40:1723-38;quiz 1855
Geurts TW, et al. Resection of secondary
pulmonary malignancies in head and
neck cancer patients. J Laryngol Otol,
2010;124:1278-83
Gooiker GA, van Gijn W, Post PN, van de
Velde CJ, Tollenaar RA, Wouters MW. A
systematic review and meta-analysis of the
volume-outcome relationship in the surgical
treatment of breast cancer. Are breast
cancer patients better of with a high volume
provider? Eur J Surg Oncol. 2010;36 Suppl
1:S27-35

156
surgical oncology
Publications (continued)
Gooiker GA, Veerbeek L, van der Geest LG,
Stijnen T, Dekker JW, Nortier JW, Marinelli
AW, Struikmans H, Wouters MW, Tollenaar
RA. The quality indicator ‘tumour positive
surgical margin following breast-conserving
surgery’ does not provide transparent
insight into care. Ned Tijdschr Geneeskd.
2010;154:A1142
Goossens-Laan CA, Visser O, Wouters MW,
Jansen-Landheer ML, Coebergh JW, van de
Velde CJ, Hulshof MC, Kil PJ. Variations in
treatment policies and outcome for bladder
cancer in the Netherlands. Eur J Surg Oncol.
2010;36 Suppl 1:S100-7
Graafl and NM, Lam W, Leijte JA, Yap T,
Gallee MP, Corbishley C, van Werkhoven E,
Watkin N, Horenblas S. Prognostic Factors
for Occult Inguinal Lymph Node Involvement
in Penile Carcinoma and Assessment of the
High-Risk EAU Subgroup: A Two-Institution
Analysis of 342 Clinically Node-Negative
Patients. Eur Urol. 2010
Graafl and NM, Leijte JAP, Valdés Olmos
RA, van Boven HH, Nieweg OE, Horenblas
S. Repeat dynamic sentinel node biopsy in
locally recurrent penile carcinoma. Br J Urol
Int 2010
Graafl and NM, Valdés Olmos RA,
Meinhardt W, Bex A, Van dr Poel HG,
Van Boven HH, Nieweg OE, Horenblas
S. Nodal staging in penile carcinoma by
dynamic sentinel node biopsy after previous
therapeutic primary tumor resection. Eur
Urol 2010;58:748-751
Graafl and NM, Valdés Olmos RA, Teertstra
HJ, Kerst JM, Bergman AM, Horenblas S.
18F-FDG PET/CT for monitoring induction
chemotherapy in patients with primary
inoperable penile carcinoma: fi rst clinical
results. Eur J Nucl Med Mol Imaging.
2010;37:1474-80
Graafl and NM, van Boven HH, van
Werkhoven E, Moonen LM, Horenblas
S. Prognostic signifi cance of extranodal
extension in patients with pathological
node positive penile carcinoma. J Urol.
2010;184:1347-53
Graafl and NM, Verhoeven RH, Coebergh
JW, Horenblas S. Incidence trends and
survival of penile squamous cell carcinoma
in the Netherlands. Int J Cancer.
2011;128:426-32
group (0.15 vs. 0.29; p = 0.042) and resulted in less loss of prostheses (0.08 vs. 0.11;
p = 0.566). Immediate prosthetic reconstruction is an attractive treatment option for
this particular group of patients also.
Reconstruction of oncological perineo-vulvar defects: An algorith of sequence
Because both VIN and vulvar carcinoma have a tendency towards local recurrence,
future reconstructive options should be reckoned with during treatment of the
primary and all subsequent perineo-vulvar lesions. A proposal of sequence of
reconstructive options for these defects was called for.
Local skin fl aps are preferably designed so as not to sever the branches of the
internal pudendal vascular system. Initial use of the pudendal thigh fl ap is preferred
over the infragluteal fl ap to preserve the infragluteal fl ap for future use. Only when
these two fl aps no longer are suffi cient should the gluteal thigh fl ap be applied.
Myocutaneous fl aps are rarely indicated.
DERMATOLOGY
Esther Tjin, Debbie Konijnenberg, Gabrielle Krebbers, Henk Mallo, Jan Wouter Drijfhout,
Kees Franken, Chantal van der Horst, Jan Bos, Omgo Nieweg, Bin Kroon, John Haanen,
Kees Melief, Florry Vyth-Dreese, Rosalie Luiten
Integrated analyses of melanoma – T cell interactions: relevance for immunotherapy
Clinical studies have shown that immunotherapy of melanoma by vaccination
or adoptive transfer of effector T-cells can mediate tumor regression. However, a
signifi cant number of melanoma patients either respond poorly or show relapse.
This might be either caused by tumor resistance to T-cell-mediated lysis or impaired
effector T-cell function. We analyzed the presence of melanoma antigen-specifi c T
cells in the melanoma tissue, as compared to T cells in the adjacent skin tissue and
in the peripheral blood. More specifi c T cells were found in the tumor than in the
adjacent skin or blood. Interestingly, functional T cell analysis revealed 2 groups of
patients: 1) patients with functional CD8+ T cell responses and 2) patients with nonfunctional
CD8+ T cell responses. More importantly, although no correlation was
found between the T cell effector function and patients’ survival, patients with nonfunctional
T cell responses showed loss of HLA expression in the melanoma more
frequently, whereas the other escape mechanisms did not differ between the groups.
These fi ndings have major implications for selecting therapeutic strategies to treat
melanoma patients, in particular, which patients may benefi t from immunotherapy.
Hansje-Eva Teulings, Esther Tjin, Ludmilla Nieuweboer-Krobotova, Jos van der Hage,
Michel Wouters, Wietze van der Veen, Jan Bos, Kees Melief, John Haanen and Rosalie Luiten
Local immunotherapy by the synergism of monobenzone and imiquimod cream
(MI) for cutaneous metastases in stage III-IV melanoma patients Melanoma
is a good candidate for treatment with immunotherapy, during which vitiligo
development is a favourable prognostic sign. At the department of Dermatology
of the AMC, we have developed a new therapy for melanoma, based on the potent
vitiligo-inducing effect of monobenzone combined with the immunostimulatory
adjuvant imiquimod (MI therapy). MI therapy induces strong melanoma-reactive
immunity, which effectively eradicated established melanoma in mice (van den
Boorn al, PLoS one 2010).
Based on these data, we will start a phase II clinical trial of monobenzoneimiquimod
(MI) therapy in melanoma patients at the NKI-AVL in 2011. The MI
regimen is a low-cost, simple therapy, which is applicable in broad range of patients
regardless of HLA-haplotype. It does not require elaborate patient-specifi c in vitro
cultures nor non-myeloablative lymphodepletion, reducing patient treatment burden.
The MI compounds have each already been used in humans, making it readily
available and easily applicable in the clinic. In this trial, we will investigate the local
antitumor effect of MI therapy in melanoma patients as well as the induction of
melanocyte/melanoma-specifi c immunity.

Publications (continued)
Hage JJ, van Beurden M. Reconstruction of
aquired perineo-vulvar defects: A proposal of
sequence. Semin Plast Surg 2011 (in press)
Hage JJ. Use of vascularized jejunum fl ap for
vaginal reconstruction: A word of caution.
Microsurg 2010;30:672-673
Heller DS, van Seters M, Marchitelli C, Moyal-
Barracco M, Preti M, van Beurden M. Update on
intraepithelial neoplasia of the vulva: proceedings
of a Workshop at the 2009 World Congress of the
International Society for the Study of Vulvovaginal
Diseases, Edinburgh, Scotland, September 2009. J
Low Genit Tract Dis 2010;14:363-73
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Proc Natl Acad Sci U S A. 2010;107:11895-9
Wiering B, Adang EM, van der Sijp JR, Roumen
RM, de Jong KP, Comans EF, Pruim J, Dekker
HM, Ruers TJ, Krabbe PF, Oyen WJ. Added value
of positron emission tomography imaging in the
surgical treatment of colorectal liver metastases.
Nucl Med Commun 2010;31:938-944
Wind B, Meesters A, Kroon M, Beek J, Van
der Veen J, Nieuweboer-Krobotova L, Bos J,
Wolkerstorfer A. Punchgraft testing in vitiligo;
effects of UVA, NB-UVB and 632.8 nm Helium-
Neon Laser on the outcome. J Eur Acad Dermatol
Venereol 2010
Wind BS, Kroon WM, Beek JF, Van der Veen
JPW, Nieuweboer-Krobotová L, Meesters
AA, Bos JD, Wolkerstorfer A. Home versus
outpatient narrowband UVB therapy for
the treatment of non-segmental vitiligo: a
retrospective questionnaire study. Brit J Dermatol
2010;162:1142-4
Wind BS, Kroon MW, Beek JF, Vander Veen JP,
Nieuweboer-krobotova L, Meesters AA, Bos JD,
Wolkerstorfer A. Non-ablative 1,550 nm fractional
laser therapy versus triple topical therapy for the
treatment of melasma: a randomized controlled
split-face study. Lasers Surg Med 2010;42:607-12
Wouters MW, Jansen-Landheer ML, van de Velde
CJ. The Quality of Cancer Care initiative in the
Netherlands. Eur J Surg Oncol. 2010;36 Suppl
1:S3-S13
Wouters MW, Siesling S, Jansen-Landheer
ML, Elferink MA, Belderbos J, Coebergh JW,
Schramel FM. Variation in treatment and
outcome in patients with non-small cell lung
cancer by region, hospital type and volume in the
Netherlands. Eur J Surg Oncol. 2010;36 Suppl
1:S83-92
Zlotta AR, Cohen SM, Dinney C, Droller M, van
der Kwast TH, van Rhijn BW, Bochner B, Ameil
G, Jewett MA. BCAN Think Tank session 1:
Overview of risks for and causes of bladder cancer.
Urol Oncol. 2010;28:329-33

Peer-reviewed papers in press/on line
Baijens L, Speijer R, Roodenburg N, Hilgers F.
Prosthetic tracheoesophageal voice production
and swallowing function in a patient following
circumferential pharyngolaryngectomy and
neopharyngeal reconstruction with a jejunal free
fl ap. Dysphagia online April 3, 2010
Scheenstra RJ, Muller SH, Vincent A, Hilgers FJ.
Heat and moisture exchange capacity of the upper
respiratory tract and the effect of tracheotomy
breathing on endotracheal climate. Head Neck,
online August 4, 2010
Scheenstra RJ, Muller SH, Hilgers FJ.
Endotracheal temperature and humidity in
laryngectomized patients in a warm and dry
environment; are heat and moisture exchangers
still effective? Head Neck online Oct 27, 2010
Scheenstra RJ, Muller SH, Vincent A, Ackerstaff
A, Jacobi I, Hilgers FJ. The effect of a newly
developed heat and moisture exchanger for
pulmonary rehabilitation of laryngectomized
patients on the endotracheal temperature and
humidity. Respiratory Care
Van der Molen L, Van Rossum MA, Burkhead
LM, Smeele LE, Rasch CR, Hilgers FJ. A
randomized preventive rehabilitation trial in
advanced head and neck cancer patients treated
with chemo-radiotherapy: feasibility, compliance
and short-term effects. Dysphagia, online July 11,
2010
Van Kalkeren TA, Van der Houwen EB, Duits
ME, Hilgers FJ, Hebe A, Mostafa BE, Lawson
G, Martinez Z, Woisard V, Marioni G, Ruske D,
Schultz P, Post WJ, Verkerke GJ, Van der Laan
BF. A worldwide, multi center study of peristomal
geometry and morphology in laryngectomees and
its clinical effects. Head Neck 2010
Book chapters
Hilgers FJ, Balm AJ, Van den Brekel MW,
Tan IB. Chapter 13. Surgery for larynx and
hypopharyngeal cancer; f. Voice restoration. In:
Surgery of Larynx and Trachea. Editors: Remacle
and Eckel. SpringerVerlag, Stuttgart;2010; pp
245-256
Hilgers FJ, Van den Brekel MW. Chapter 113:
Vocal and Speech Rehabilitation Following
Laryngectomy. In: Cummings Otolaryngology:
Head and Neck Surgery. 5th Edition. Editors:
Flint, Haughey, Richardson, Robbins, Thomas,
Niparko, and Lund. Elsevier, Philadelphia; pp
1594-1610
161
surgical oncology

162
biometrics department
Head of department Otilia Dalesio
Otilia Dalesio MSc Head
Harm Van Tinteren PhD Academic staff
Andrew Vincent PhD Academic staff
Erik Van Werkhoven MSc Academic staff
Tinie Benraadt MD Academic staff
Jolanda Appelman Technical staff
Robin Arens Technical staff
Danny Baars Technical staff
Nathalie Barbier Technical staff
Frauwkje Bessels Technical staff
Henk Botma Technical staff
Audi Boucher Technical staff
Monique Carreno Technical staff
Jolien Claassen Technical staff
Saskia Cooke Technical staff
Gerda de Jong Technical staff
Sebastiaan de Vries Technical staff
Marjolijn De Waal MSc Technical staff
Brigitte Dufournij Technical staff
Marlou Eilers Technical staff
Ernesto Fernandez Salcedo MSc Technical
staff
Christiane Hagenaars Technical staff
Song-Hieng Hau MSc Technical staff
Roos Hennink Technical staff
Annelies Hiemstra Technical staff
Paula Hoekstra MSc Technical staff
Eduard Ivanov Technical staff
Aarti Jibodh Technical staff
Irene Jonkers Technical staff
Joop Jonkman MSc Technical staff
Josien Kant Technical staff
Lies Kolmschate Technical staff
Lisette Lorist Technical staff
Marianne Mahn MSc Technical staff
Ingrid Mandjes MSc Technical staff
Bojana Milojkovic MDTechnical staff
Carla Modder Technical staff
Pietje Muller Technical staff
Elvira Nuijten Technical staff
Cecile Paulus van Pauwvliet Technical staff
Loes Pronk MSc Technical staff
Harriet Rehorst Technical staff
Vera Reijnders Technical staff
Anneke Reinders Technical staff
Daniel Roberts Technical staff
Marielle Roskam Technical staff
Martin Ryan Business advisor
Dea Storm Technical staff
Beata Sznajder PhD Technical staff
Ria Tilgenkamp Technical staff
Alex Torres Acosta Technical staff
CLINICAL STUDIES AND OTHER
COLLABORATIONS
We have developed collaborations with several cooperative groups; academic groups,
industry and clinical research organizations, and we function as partner for clinical
studies and clinical trials, being involved from the generation of the idea, protocol
setting, the planning, and providing project management, randomization services,
development of (electronic) case record forms, data handling, monitoring and quality
assurance, and statistical expertise. In addition, the tools that we have developed
within our ICT projects are attracting new associations. With the help of the
training kits that we have developed, personnel of clinical trial coordinating centers
can be incorporated along with our own form designers in the production of the
applications for their own clinical trials.
Diagnostic studies In collaboration with the Department of Nuclear Medicine
of the Free University in Amsterdam we have been examining reproducibility
of positron emission tomography (PET) data. The objective being to compare
reproducibility of different PET techniques and lay the foundation for guidelines
equivalent to the RECIST rules for CT scans.
From an analysis point of view, we wish to determine the 95%-coeffi cient-ofrepeatability
(CR95) of the ratio of the two test-retest observations. The dataset
consists of individual-level data from fi ve separate studies. The major complicating
issues being the strong association between the repeatability of the ratio of two
test-retest observations and their mean, and second, the differences between studyspecifi
c CR95s can be quite pronounced. We address these issues by modeling the
square of the normalized ratio using a general-linear-mixed model with a Gamma
density function. The square of the normalized (Geary–Hinkley transformed)
ratio is chi-squared, and thus Gamma distributed. The use of a log-link and a
log-transformed mean implies that, in place of the usual linear regression, the
determined variance-mean association is an allometric relation. The differences
between studies are accounted for with the use of a mixed effects structure. The
overall variance-mean association is back transformed onto the original scale and
used to calculate the association between the percentage change in two observations
and their mean.
Figure 1: (A) Test-retest observations color coded by study. (B) Ratio (normalized) vs. mean
(log-transformed) of test-retest data. Dotted lines are the observed standard deviations of the four
quartiles of the data pooled. The solid line is the estimated standard deviation of the pooled data using
a GLM with a Gamma density distribution. (C) Study-specifi c estimates of standard deviations (dotted
lines) and overall estimate of standard deviation including study as a random effect. (D) Percentage
change of the testretest
observations
plotted against their
(geometric) mean.
The solid line being the
CR95.

NVALT cancer studies The Department functions as Data Centre for the cancer
studies of the Dutch Chest Physician Association (NVALT) and we collaborate
in planning, designing and running of their clinical trials in lung cancer and
mesothelioma. More than 75 hospitals in the Netherlands participate in the clinical
trials. In 2010 the Dutch Cancer Society has granted support for the management
of data of 2 new studies of the group; the NVALT12 and the NVALT14. The
NVALT12 is a randomized study of docetaxel, cisplatin, bevacizumb with or without
nitroglycerine patches in patients with stage IV non squamous non small cell lung
cancer. The NVALT14 is a randomized trial comparing longstanding indwelling
pleural catheters with pleurodesis as a frontline treatment for malignant pleural
effusion.
In total 8 studies of the 14 trials that have or are being run by the group have been at
least partially supported by the Dutch Cancer Society.
In 2010 patients accrual to the NVALT 5, a randomized phase III trial of the
antiangiogenic agent Thalidomide in patients with malignant pleural mesothelioma
after fi rst line chemotherapy, was completed. Final analysis and publication will be
completed in 2011.
Also this year, specimens and pathology slides were collected and analyzed and
the fi nal statistical analysis of the NVALT4 trial has been performed. A total of 561
patients were randomized into this placebo-controlled study of docetaxel/carboplatin
with celecoxib or placebo in patients with locally advanced or metastatic non-small
cell lung cancer. The publication is expected next year.
Mid 2010 NVALT8A was closed to patient entry due to low accrual. Indeed,
the accrual to the 2 studies in resectable NSCLC low and high risk patients
as determined by preoperative PET SUVmax (NVALT8 A and B), has been
disappointing. The NVALT8B study has been modifi ed allowing entry also of
low risk patients.
The accrual to NVALT 10 continued at a consistent pace and 138 had already been
randomized in this study comparing erlotinib vs erlotinib and chemotherapy
combination in pretreated patients with advanced NSCLC.
In the NVALT11 study the value of prophylactic cranial irradiation (PCI) versus
observation is studied in radically treated patients with stage III non-small cell lung
cancer. It is done as a cooperation of the NVALT, the Dutch Lung Cancer Research
Group (DLCRG) and the National Platform for Radiotherapy in Lung Tumors
(LPRL). A total of 75 have been included in this study.
The SIOP-RTSG The Renal Tumor Study Group of the International Society
of Pediatric Oncology (SIOP) has its origin in the late sixties when European
pediatricians joined forces in the treatment of children with nephroblastoma
(Wilms’ tumors). In 1993, the sixth randomized controlled trial was launched and
the statistical secretariat moved to Amsterdam. The SIOP 9301 trial was the fi rst
to study a reduction of therapy in Wilms’ tumors. Over the years the number of
participating centers and countries has progressively increased and in 2001 the
UK and Brazil joined for the seventh randomized trial. The statistical group of the
Department is involved in the continuous checking, completing and analyzing
the 1993 and the 2001 database. The latter is a compilation of 6 different regional
databases with patients from over 28 countries in Europe and around the world.
The randomized part of the current SIOP 2001 study again evaluates a reduction of
therapy and was closed by the end of 2009 with 584 patients enrolled. Early 2011 the
data will be analyzed and presented.
The SIOP 2001 study introduced also several new treatment strategies for patients
not eligible for randomization compared to previous practice. The data on these
patients are currently studied for the development of new prognostic factors
or described in detail. The coming two years also extra effort will be put in the
collection of tumor specimen for the studies on genotyping.
163
biometrics department
Ludy Valkenet MD Technical staff
Marjolijn van den Haak MSc Technical staff
Emile Van der Donk Technical staff
Tony Van de Velde Technical staff
Gabry Van Netten Technical staff
Wil Van Waardenberg Technical staff
Steven Vanhoutvin Technical staff
Anneke Wals Technical staff
Lidwina Wever Technical staff
Yvonne Wijnands Technical staff
Els Willemse Technical staff
Jeroen Zandbergen MSc Technical staff

164
biometrics department
Publications
Annema JT, Bohoslavsky R, Burgers S,
Smits M, Taal B, Venmans B, Nabers H,
van de Borne B, van Balkom R, Haitjema
T, Welling A, Staaks G, Dekkers OM, van
Tinteren H, Rabe KF. Implementation
of endoscopic ultrasound for lung cancer
staging. Gastrointest Endosc 2010;71:64-70
Aukema TS, Kappers I, Olmos RA,
Codrington HE, van Tinteren H, van Pel
R, Klomp HM. Is 18F-FDG PET/CT useful
for the early prediction of histopathologic
response to neoadjuvant erlotinib in patients
with non-small cell lung cancer? J Nucl Med
2010;51:1344-8
Bueno de Mesquita JM, Nuyten DS,
Wesseling J, van Tinteren H, Linn SC, van
de Vijver MJ. The impact of inter-observer
variation in pathological assessment of
node-negative breast cancer on clinical risk
assessment and patient selection for adjuvant
systemic treatment. Ann Oncol 2010;21:40-7
Correa C, McGale P, Taylor C, Wang Y,
Clarke M, Davies C, Peto R, Bijker N, Solin
L, Darby S. Overview of the randomized
trials of radiotherapy in ductal carcinoma in
situ of the breast. J Natl Cancer Inst Monogr
2010;2010:162-77
Courrech Staal EF, van Sandick JW, van
Tinteren H, Cats A, Aaronson NK. Healthrelated
quality of life in long-term esophageal
cancer survivors after potentially curative
treatment. J Thorac Cardiovasc Surg
2010;140:777-83
Courrech Staal EF, Aleman BM, Boot H,
van Velthuysen ML, van Tinteren H, van
Sandick JW. Systematic review of the benefi ts
and risks of neoadjuvant chemoradiation
for oesophageal cancer. Br J Surg
2010;97:1482-96
de Vries RR, Nieuwenhuijzen JA, Vincent A,
van Tinteren H, Horenblas S. Survival after
cystectomy for invasive bladder cancer. Eur J
Surg Oncol 2010;36:292-7
Dingemans AM, de Langen AJ, van den
Boogaart V, Marcus JT, Backes WH,
Scholtens HT, van Tinteren H, Hoekstra
OS, Pruim J, Brans B, Thunnissen FB,
Smit EF, Groen HJ. First-line erlotinib
and bevacizumab in patients with locally
advanced and/or metastatic non-small-cell
lung cancer: a phase II study including
molecular imaging. Ann Oncol 2010
The SIOP 9301 and 2001 register now includes data on almost 7000 nephroblastomas
and other renal malignancies, such as renal cell carcinomas, clear cell carcinomas
and rhabdoid tumors. In localized disease, which is the case in about 80% of
patients, 5-years event-free survival is now 87% and overall survival almost 95%.
Other studies Collaboration of the statistical team with the Dutch Colorectal
Cancer Group (DCCG) had resulted in several large phase III randomized studies
in patients with advanced colorectal cancer previously untreated. In 2010 a
confi dential interim analysis of the CAIRO 3 study was prepared to be discussed by
the Independent Data Monitoring Committee. The study compares maintenance
treatment with capecitabine and bevacizumab versus observation after induction
treatment with capecitabine, oxaliplatin, and bevacizumab as fi rst-line treatment.
For this study, we have developed an e-CRFs application. End 2010 453 patients have
been randomized by 61 centers.
We collaborate with medical departments in our Institute in carrying out, handling
data and SAEs and providing statistical support for the multi center clinical
trials they coordinate. We coordinate with the radiotherapy department a large
randomized study in young women with early breast cancer. In excess of 2100
patients have already been entered by participating centers in the Netherlands,
France and Germany. Tumor material is being collected for translational research
analyses. Cosmetics results are being evaluated with pictures taken in series.
With the surgeons and medical oncologists we collaborate in several studies, like
the OVHIPEC, which is a study of intraperitoneal chemotherapy and hyperthermia
in ovarian cancer, the Matador and the TRAIN study in breast cancer, the Tyvtax
in head and neck cancer and the Raditux study in lung cancer. We also collaborate
with the pharmacology department as statistical center for 2 randomized studies.
One is a double blind randomized study on cardio-protection of breast cancer
patients treated by Herceptin for which our statistical team has produced this year
the fi rst confi dential interim analysis report to be discussed by the independent data
monitoring committee. The second is a randomized double blind study of treatment
of hot fl ashes in breast cancer patients in menopause following chemotherapy
for their breast cancer. Accrual has been beginning 2010, the fi nal analysis was
performed during this year and a publication has been submitted.
The statisticians have collaborated with other departments of the institute and other,
academic and not academic institutes in the region in a variety of studies many of
which resulted in co-authorships as can be seen in the publication list.
ICT PROJECTS
We have been involved in the development of tools for clinical trials during the
last 20 years and we have developed the most modern tools to support clinical
trial organization. The most important being ALEA, which is a system to register,
randomize and automatically distribute notifi cations of the randomization; and
an electronic case record forms (eCRF) system, that allows the development of
applications for studies, by which entry of data from the participating centers
directly into the Data Center databases can be done. The eCRF system allows
detection of inconsistencies at the source resulting in less data queries at later times
and speeding up the statistical analysis and reporting.
The system includes an audit trail in compliance with the requirements of the
International Committee for Harmonization and produces standard based export
facilities (CDISC, ODM and SDTM).
TENALEA is a service that provides these online tools to support the data
management process for clinical trials. The TENALEA Initial Deployment
project is an EU funded project which aims to provide academic clinical trial
coordination centers with industry grade online tools to support the clinical trial
data management process. The project is carried out with a consortium of 10
academic clinical trial coordination centers in Germany, Poland, France, UK and
the Netherlands, a software company and the UK Cochrane Center. We are the
initiators and coordinators of the project. The focus of the project is deployment

of a randomization service and an electronic Case Report Forms (eCRF) service.
The randomization service allows clinical trial coordination centers to setup a
randomization procedure for a clinical trial, and allow investigators and their staff to
perform randomization of patients over the internet using a standard browser. The
eCRF service enables the replacement of the traditional paper case report forms by
an electronic dossier.
TENALEA Initial Deployment is nearing its completion, which is scheduled for June
2011. After a consolidation of the TENALEA use of smaller trial coordination centers
to larger centers, we are currently running a total of 192 open studies serving 31
Clinical Trial Coordination centers with the TENALEA Randomisation and the
TENALEA eCRF service.
Figure 2. TENALEA Operational Environment – DTAP
During 2010, the operational environment of TENALEA has been further improved.
TENALEA now runs the development and the test at the NKIAVL, while the
acceptance and production are operated in a commercial data center at InterConnect
BV in Den Bosch. During 2010, TENALEA has completed its migration from
VMWare virtualization to Microsoft Hyper-V based virtualization. The operational
environment as well as the TENALEA Software Development Life Cycle has passed
its periodic external audit.
With TENALEA Randomisation and eCRF, a study defi nition user works on
the Development environment to create the forms, user accounts and any other
elements required for online randomization and / or eCRF. Once completed, the
study designer publishes the study to the test environment, where internal staff
of the Trial Coordination center, such as data managers and statisticians, can test
whether the randomization program or eCRF work as required. If the internal
staff approves the system, the study defi nition is published to Acceptance, where
the staff of participating study sites such as study nurses, investigators and local
pharmacy staff can test whether the system works properly from their respective
IT environment. Once acceptance has been obtained from study sites, the study
defi nition is published to Production, where the system will be running live for the
study. The publishing mechanism, in combination with the strict separation of these
four environments, makes the system working in compliance with legislation as well
as with methodologies used in similar development paradigms, such as controlled
document management and software development.
The primary long term goal of the project is that the Services can continue to be
available to Academics after the completion of the TENALEA Initial Deployment
project at a moderate marginal cost. Early 2011 different modalities will be discussed
within the partners and larger users of the Services.
165
biometrics department
Publications (continued)
Geurts TW, Klomp HM, Burgers SA,
van Tinteren H, Roukema BY, Balm
AJ. Resection of secondary pulmonary
malignancies in head and neck cancer
patients. J Laryngol Otol 2010;1-6
Jongmans P, Wempe TG, van Tinteren H,
Hilgers FJ, Pols LC, As-Brooks CJ. Acoustic
analysis of the voiced-voiceless distinction in
Dutch tracheoesophageal speech. J Speech
Lang Hear Res 2010;53:284-97
Quarles van Ufford HM, van Tinteren H,
Stroobants SG, Riphagen II, Hoekstra OS.
Added value of baseline 18F-FDG uptake
in serial 18F-FDG PET for evaluation of
response of solid extracerebral tumors to
systemic cytotoxic neoadjuvant treatment: a
meta-analysis. J Nucl Med 2010;51:1507-16
Rizvi SN, Comans EF, Boellaard R, van
Tinteren H, Hoekstra OS. Two decades
at the cross-roads of biology, physics and
epidemiology: lessons learned in [18F-]FDG
positron emission tomography in oncology.
Eur J Cancer 2010;46:2150-8
Straver ME, Glas AM, Hannemann J,
Wesseling J, van de Vijver MJ, Rutgers
EJ, Vrancken Peeters MJ, van Tinteren
H, Van ‘t Veer LJ, Rodenhuis S. The 70gene
signature as a response predictor for
neoadjuvant chemotherapy in breast cancer.
Breast Cancer Res Treat 2010;119:551-8
Teertstra HJ, Loo CE, van den Bosch
MA, van Tinteren H, Rutgers EJ, Muller
SH, Gilhuijs KG. Breast tomosynthesis in
clinical practice: initial results. Eur Radiol
2010;20:16-24
Tulner LR, Van Campen JP, Kuper IM,
Gijsen GJ, Koks CH, Mac Gillavry MR,
van Tinteren H, Beijnen JH, Brandjes
DP. Reasons for undertreatment with oral
anticoagulants in frail geriatric outpatients
with atrial fi brillation: a prospective,
descriptive study. Drugs Aging 2010;27:39-50
van der Eerden P, Simmons M, Zuur K,
van Tinteren H, Vuyk H. Full-thickness skin
grafts and perichondrial cutaneous grafts
following surgical removal of cutaneous
neoplasms of the head and neck. Eur Arch
Otorhinolaryngol 2010;267:1277-83

166
biometrics department
Publications (continued)
Verschuur AC, Vujanic GM, van Tinteren H,
Jones KP, de Kraker J, Sandstedt B. Stromal
and epithelial predominant Wilms tumours
have an excellent outcome: the SIOP
93 01 experience. Pediatr Blood Cancer
2010;55:233-8
Yang TJ, Aukema TS, van Tinteren H,
Burgers S, Valdes Olmos R, Verheij M.
Predicting early chemotherapy response with
technetium-99m methoxyisobutylisonitrile
SPECT/CT in advanced non-small cell lung
cancer. Mol Imaging Biol 2010;12:174-80
Graafl and NM, Lam W, Leijte JA, Yap T,
Gallee MP, Corbishley C, van Werkhoven
E, Watkin N, Horenblas S. Prognostic
Factors for Occult Inguinal Lymph Node
Involvement in Penile Carcinoma and
Assessment of the High-Risk EAU Subgroup:
A Two-Institution Analysis of 342 Clinically
Node-Negative Patients. Eur Urol 2010
Graafl and NM, van Boven HH, van
Werkhoven E, Moonen LM, Horenblas
S. Prognostic signifi cance of extranodal
extension in patients with pathological
node positive penile carcinoma. J Urol
2010;184:1347-53
Buikhuisen WA, Burgers JA, Vincent
AD, Schellens JH, Beijnen JH, Smit EF,
Joerger M. Pemetrexed pathway-associated
germline polymorphisms: a useful tool for
treatment individualization? J Clin Oncol.
2010;28:e482-3
Palma DA, Senan S, Haasbeek CJ,
Verbakel WF, Vincent A, Lagerwaard F.
Radiological and Clinical Pneumonitis after
Stereotactic Lung Radiotherapy: A Matched
Analysis of Three-dimensional Conformal
and Volumetric-modulated Arc Therapy
Techniques. Int J Radiat Oncol Biol Phys.
2010
Helgason HH, Engwegen JY, Zapatka
M, Vincent A, Cats A, Boot H, Beijnen
JH, Schellens JH. Identifi cation of serum
proteins as prognostic and predictive markers
of colorectal cancer using surface enhanced
laser desorption ionization-time of fl ight
mass spectrometry.Oncol Rep. 2010;24:57-64
Bruin SC, Verwaal VJ, Vincent A, van ‘t Veer
LJ, van Velthuysen ML. A clinicopathologic
analysis of peritoneal metastases of colorectal
and appendiceal origin.Ann Surg Oncol.
2010;17:2330-40
Scheenstra RJ, Muller SH, Vincent A, Ackerstaff
AH, Jacobi I, Hilgers FJ. Short-term endotracheal
climate changes and clinical effects of a heat
and moisture exchanger with an integrated
electrostatic virus and bacterial fi lter developed for
laryngectomized individuals.Acta Otolaryngol.
2010;130:739-46
Scheenstra RJ, Muller SH, Vincent A,
Sinaasappel M, Hilgers FJ. Infl uence of breathing
resistance of heat and moisture exchangers
on tracheal climate and breathing pattern
in laryngectomized individuals.Head Neck.
2010;32:1069-78
de Vries RR, Nieuwenhuijzen JA, Vincent A,
van Tinteren H, Horenblas S. Survival after
cystectomy for invasive bladder cancer.Eur J Surg
Oncol. 2010;36:292-7
Zuur JK, Muller SH, Vincent A, Sinaasappel M,
de Jongh FH, Hilgers FJ. The infl uence of a heat
and moisture exchanger on tracheal climate in a
cold environment.Med Eng Phys. 2009;31:852-7
Straver ME, Rutgers EJ, Russell NS, Oldenburg
HS, Rodenhuis S, Wesseling J, Vincent A,
Peeters MT. Towards rational axillary treatment
in relation to neoadjuvant therapy in breast cancer.
Eur J Cancer. 2009
Snoeren N, Voest EE, Bergman AM, Dalesio
O, Verheul HM, Tollenaar RA, van der Sijp JR,
Schouten SB, Rinkes IH, van Hillegersberg
R. A randomized two arm phase III study in
patients post radical resection of liver metastases
of colorectal cancer to investigate bevacizumab in
combination with capecitabine plus oxaliplatin
(CAPOX) vs CAPOX alone as adjuvant treatment.
BMC Cancer. 2010;10:545
Accepted publications
Vollebergh MA, Lips EH, Nederlof PM, Wessels
LF, Schmidt MK, van Beers EH, Cornelissen S,
Holtkamp M, Froklage FE, de Vries EG, Schrama
JG, Wesseling J, van de Vijver MJ, van Tinteren
H, de Bruin M, Hauptmann M, Rodenhuis S,
Linn SC. An aCGH classifi er derived from BRCA1mutated
breast cancer and benefi t of high-dose
platinum-based chemotherapy in HER2-negative
breast cancer patients. Ann Oncol 2010
Gadiot J, Hooijkaas AI, Kaiser AD, van Tinteren
H, van Boven H, Blank C. Overall survival and
PD-L1 expression in metastasized malignant
melanoma. Cancer 2010
Richard W, Timmer F, van Tinteren H, de Vries
N. Complications of hyoid suspension in the
treatment of obstructive sleep apnea syndrome. Eur
Arch Otorhinolaryngol 2010
Knijn N, Tol J, Koopman M, Werter MJ, Imholz
AL, Valster FA, Mol L, Vincent AD, Teerenstra
S, Punt CJ. The effect of prophylactic calcium
and magnesium infusions on the incidence of
neurotoxicity and clinical outcome of oxaliplatinbased
systemic treatment in advanced colorectal
cancer patients. Eur J Cancer. 2010
Swellengrebel HA, Marijnen CA, Verwaal VJ,
Vincent A, Heuff G, Gerhards MF, van Geloven
AA, van Tets WF, Verheij M, Cats A. Toxicity and
complications of preoperative chemoradiotherapy
for locally advanced rectal cancer. Br J Surg. 2010
Palma DA, van Sörnsen de Koste J, Verbakel
WF, Vincent A, Senan S. Lung Density Changes
After Stereotactic Radiotherapy: A Quantitative
Analysis in 50 Patients. Int J Radiat Oncol Biol
Phys. 2010
Scheenstra RJ, Muller SH, Vincent A, Hilgers FJ.
Heat and moisture exchange capacity of the upper
respiratory tract and the effect of tracheotomy
breathing on endotracheal climate. Head Neck.
2010

CLINICAL TRIALS IN THE
NETHERLANDS CANCER INSTITUTE
Type of Study
cancer study coordinator Activated
(nick name) Title in NKI-AVL Phase (closed)
ALL SITES
M05RTB A phase III international randomized trial of single versus M Verheij III 10-1-2006
multiple fractions for re-irradiation of painful bone metastases (22-12-2010)
M07KUC A phase I, open-label, study to assess the safety and tolerability
of KU-0059436 in combination with Carboplatin, KU-0059436 in
combination with Paclitaxel/Carboplatin doublet and KU-0059436
in combination with Paclitaxel in the treatment of patients with
advanced solid tumors
JHM Schellens I 25-6-2007
M07MKC A phase I dose escalating study evaluating MK-1775 in both
monotherapy and in combination with Gemcitabine, Cisplatin
or Carboplatin in adult patients with advanced solid tumors
JHM Schellens I 21-2-2008
M07OTS A phase I study of oral topotecan in subjects with cancer and
impaired renal function
JHM Schellens I 18-2-2008
M07PFU Pharmacogenomic and pharmacokinetic safety and cost-saving
analysis in patients treated with fl uoropyrimidines
JHM Schellens other 16-5-2007
M08CIP Chemotherapy induced peripheral neuropathy outcome W Boogerd other 11-3-2009
(CI-PERINOMS) measures standardisation study
M08HYT A phase I open-label study of the safety, tolerability and
pharmacokinetics of two schedules of oral topotecan in
combination with pazopanib in subjects with advanced solid tumors
JHM Schellens I 15-9-2008
M08MEK Open-label, multicenter, dose-escalation phase I study with
extension to evaluate safety, pharmacokinetics and activitiy of
RO4987655, a MEK inhibitor, administered orally as monotherapy
in patients with advanced tumors
JHM Schellens I 4-2-2009
M09BGJ A phase I open-label, multicenter, dose escalation study of oral
BGJ398, a pan FGF-R kinase inhibitor in adult patients with
advanced solid malignancies
JHM Schellens I 10-12-2009
M09DAZ A phase I open label multicenter study to assess the safety and
tolerability, pharmacokinetics, preliminary anti-tumor activity of
ascending doses of AZD4547 in patients with advanced solid
malignancies
JHM Schellens I 14-10-2009
M09GDC A phase Ib open label, dose-escalation study of the safety and
pharmacology of GDC-0941 in combination with erlotinib with
advanced solid tumors
JHM Schellens I 12-8-2009
M09GSK Phase I open label, dose-escalation study of the phosphoinositide
3-kinase inhibitor (GSK2126458) in subject with solid tumor or
lymphoma
JHM Schellens I 31-3-2010
M09LAP An open label phase Ib continuation study of lapatinib monotherapy JHM Schellens I 26-11-2009
(E111767) or lapatinib in combination with other anti-cancer treatment in
patients with solid tumors
167
clinical trials

168
clinical trials
Type of Study
cancer study coordinator Activated
(nick name) Title in NKI-AVL Phase (closed)
M09NIB The NIB-Cohort study, therapeutic drug monitoring of tyrosine
kinase inhibitors
JHM Schellens other 9-6-2009
M09OCP A phase I clinical study of oral CP-4126 in patients with JHM Schellens I 2-4-2009
advanced solid tumor (29-4-2010)
M09RGD A phase II, open label, non-randomized, multicenter, pilot, effi cacy
study of [F-18]RGD-K5 Positron Emission Tomography (PET) as a
tool to monitor response to an anti-angiogenic drug
TJM Ruers II 20-5-2010
M09RIF An open-label phase I study to assess the effect of rifampicin on the JHM Schellens I 1-12-2009
(Bold 111) pharmacokinetics of eribulin mesylate (E7389) in subjects with
advanced solid tumors
(22-6-2010)
M10AZD A phase I open-label, multicenter study to assess the safety,
tolerability, pharmacokinetics and preliminary anti-tumor activity
of ascending doses of AZD5363 under adaptable dosing schedules
in patients with advanced solid malignancies
JHM Schellens I 2-12-2010
N06PFB Pleural fl uid bank MM van den Heuvel other 27-9-2006
N07DOW Weekly administration of oral Docetaxel in combinaton with Ritonavir JHM Schellens I 14-11-2007
N07NEX Phase I study of gemcitabine and carboplatin plus sorafenib in
patients with advanced solid tumors
JHM Schellens I 29-1-2008
N08BPM 24 hour ambulatory blood pressure monitoring in patients with JHM Schellens other 2-9-2008
malignancies enrolled in phase I oncological studies (4-5-2010)
N08CER An open-label, non-randomized, single-center study to determine JHM Schellens I 2-2-2009
(BOLD 103) the metabolism and elimination of Carbon-14 labeled eribulin
acetate (14-C-eribulin) in patients with advanced solid tumors
N08CTC Validation of circulating tumor cells (CTC) detection techniques
in patients with advanced solid tumors
JHM Schellens other 31-7-2008
N08EDO Phase I interaction study of docetaxel with supplementation of
St. John's wort or Echinacea
JHM Schellens I 3-2-2009
N08KER An open-label phase I study to evaluate the pharmacokinetics JHM Schellens I 2-2-2009
(BOLD 109) and tolerance of co-administration of oral multiple dose of
ketoconazole and an IV (bolus) infusion of Eribulin in patients
with advanced solid tumors
(14-5-2010)
N08NPM NVALT Palliative care Protocol on malignant pleural effusion MM van den Heuvel other 13-3-2008
N10BOM Weekly administration of (bi-) daily Oral Docetaxel in combination
with Ritonavir
JHM Schellens I 17-5-2010
N10EFP Feasibility of ejection fraction measurement with gallium-68
citrate PET/CT (EF-PET)
WV Vogel other 11-6-2010

Type of Study
cancer study coordinator Activated
(nick name) Title in NKI-AVL Phase (closed)
BRAIN / CNS
M10BEL Randomized phase II study of bevacizumab versus bevacizumab D Brandsma II 27-9-2010
(BELOB) plus lomustine versus lomustine in patients with recurrent
glioblastoma, the BELOB trial
N05THB Totale hersenbestraling met een eenmalige boost bij patiënten
met solitaire hersenmetastase van een epitheliale tumor
- een registratiestudie
LGH Dewit other 7-4-2005
BREAST
E10031 A phase III trial evaluating the role of ovarian function suppression GS Sonke III 20-7-2005
(SOFT) and the role of exemestane as adjuvant therapies for
premenopausal women with endocrine responsive breast cancer;
tamoxifen versus ovarian function suppression + tamoxifen versus
ovarian function suppression + exemestane
(13-7-2010)
E10041 Micro-array in node-negative disease may Avoid Chemotherapy: EJTh Rutgers III 2-1-2007
(MINDACT) a prospective randomized study comparing the 70-gene signature
with the common clinical-pathological criteria in selecting patients
for adjuvant chemotherapy in node-negative breast cancer
E10981 After mapping of the Axilla: radiotherapy or surgery? EJTh Rutgers III 21-12-2000
(AMAROS) (29-4-2010)
E22051 SUPREMO, an MRC phase III randomised trial to assess the role NS Russell III 27-2-2007
(SUPREMO) of adjuvant chest wall irradiation in 'intermediate risk' operable
breast cancer following mastectomy
M03RBC Radiation dose intensity study in breast cancer in young women: GMM Bartelink III 29-3-2004
(YOUNG a randomized phase III trial of additional dose to the tumor bed
BOOST) ("young boost")
M04MAT Microarray Analysis in breast cancer to Tailor Adjuvant Drugs SC Linn III 26-4-2004
(MATADOR) Or Regimens (MATADOR)
M05BRI Long term risk of breast cancer following treatment of NS Russell other 5-1-2006
(BRIGHT) Hodgkin's disease
M05HIR Hormonal substitution after prophylactic adnectomy in women M van Beurden other 31-5-2005
(HIRISE) with an increased risk for breast- and ovarian cancer due to a
genetic predisposition: HIRISE (High-Risk women and hormonal
Substitution Exposure)
M06HER Prospective randomized pharmacological intervention study; JHM Schellens III 18-6-2007
(CANDY) evaluating the effect of angiotensin II-receptor (AT1) blocker
candesartan versus placebo in prevention of trastuzumabassociated
cardiotoxicity in patients with primary breast cancer
treated with trastuzumab
M06TM2 A. Multicentre, prospective phase II trial investigating the effi cacy SC Linn III 26-9-2006
(TEAM II) of neoadjuvant hormonal therapy with exemestane for six months
B. Randomised, multicentre, prospective, phase III trial
investigating the effi cacy and safety of the addition of ibandronate
to adjuvant hormonal therapy in postmenopausal women with
hormone receptor positive early breast cancer.
169
clinical trials

170
clinical trials
Type of Study
cancer study coordinator Activated
(nick name) Title in NKI-AVL Phase (closed)
M07ATX Phase II randomized trial of combination therapy of paclitaxel and SC Linn II 12-12-2007
(ATX) bevacizumab versus paclitaxel, capecitabine and bevacizumab as
fi rst-line treatment for locally recurrent or metastatic breast cancer
patients with HER2/neu negative tumor
(6-12-2010)
M07CBE Late effects of chemotherapy on brain functioning in the elderly SSB Schagen other 1-7-2008
M07FEP A randomized double-blind phase 2 trial of Fulvestrant plus GS Sonke II 12-9-2007
Enzastaurin versus Fulvestrant plus placebo in aromatase
inhibitor-resistant metastatic breast cancer
(26-4-2010)
M07LET IDEAL: Investigation on the duration of extended adjuvant EJTh Rutgers III 16-11-2007
(IDEAL) letrozole treatment. An open lable, randomized phase III trial
comparing 2,5 year duration of letrozole (Femara) treatment with
5 year duration in patients previously treated for endocrine
sensitive early breast cancer
M08BCP Prospective and Retrospective register study of the German SC Linn other 27-3-2008
(BOOG Adjuvant Cancer Study Group (GABG) for diagnosis and treatment
2003- 04) of breast cancer in pregnancy
M08CPE A two-arm randomized open label phase 2 study of CP-751,871
in combination with exemestane versus exemestane alone as fi rst
line treatment for postmenopausal patients with hormone receptor
positive advanced breast cancer
SC Linn II 27-1-2009
M08HAT A randomized phase II study of concomitant trastuzumab,
bevacizumab with paclitaxel versus trastuzumab and bevacizumab
followed by the combination of trastuzumab, bevacizumab and
paclitaxel at progression as fi rst-line treatment of patients with
metastatic breast cancer with Her2-neu overexpression
SC Linn II 20-4-2009
M08MAM An exploratory clinical study for initial validation of a novel small RA Valdes Olmos other 29-4-2009
(MARI) ring device for positron emission mammotomography
M08MUL Multicenter feasibility study of the sentinel node procedure in HSA Oldenburg other 16-4-2009
(MULTISENT) patients with multiple breast tumors (MULTISENT)
M08PBI Image guided Preoperative Accelerated partial Breast Irradiation PH Elkhuizen other 1-10-2009
(PAPBI) (PAPBI): defi ning radiotherapy sensitivity
M08TRA Trastuzumab in a neoadjuvant regimen for Her2+ breast cancer – GS Sonke II 18-9-2008
(TRAIN) the TRAIN study
M09MLA An open label study to examine the effects of low-fat and high-fat JHM Schellens I 26-11-2009
(EGF111582) meals on the pharmacokinetics of orally administered lapatinib in
metastatic Erb2 positive breast cancer patients
M09SRB Sentinal Node and recurrent breastcancer; regional staging and EJTh Rutgers other 27-10-2009
(SNARB) registration (SNARB)
M09TNM Randomized phase II/III study of individualized neo-adjuvant S Rodenhuis II/II 7-1-2010
(Neo-TN) chemotherapy in triple negative breast tumors
M10DCI A randomized phase III study of radiation doses and
fractionation schedules for ductal carcinoma (DCIS) of the Breast
NS Russell III 9-6-2010

Type of Study
cancer study coordinator Activated
(nick name) Title in NKI-AVL Phase (closed)
N04POM Tailored preoperative chemotherapy in stage II or III breast cancer
with either a primary tumor over 3 cm in size or a clinically
tumor-positive axilla.
S Rodenhuis II 21-3-2005
N04RTB Effecten van bestraling op bloedvaten NS Russell other 5-10-2004
N05MIB 99mTc-methoxyisobutylisonitrile (MIBI) for imaging of apoptosis
and prediction of tumor respons to chemotherapy and/or
radiotherapy in cancer patients
RA Valdes Olmos other 7-6-2005
N06GLB Phase I study of gemcitabine plus lapatinib (GW572016) in women
with advanced breast cancer
JHM Schellens I 16-5-2007
N06IAA Randomized phase II/III study of intensifi ed alkylating agent S Rodenhuis II/II 8-1-2007
chemotherapy with peripheral blood progenitor cell support in the
preoperative chemotherapy of breast tumors that are defi cient for
homologous recombination
(11-2-2010)
N06TRZ The effect of Trastuzumab treatment on B-cell kinase activities JHM Schellens other 11-12-2006
assessed by microarray derived phosphorylation profi les (28-4-2010)
N06VNI Meting van de functie van de arm en de bijdrage daaraan van de
grote borstspieren voor en na de tweezijdige implantatie van een
endoprothese ten behoeve van een borstreconstructie die direct
aansluitend aan de huidsparende borstoperatie wordt uitgevoerd
JJ Hage other 26-9-2006
N07BOS Genetic determinants of survival and second breast cancer EJTh Rutgers other 12-12-2007
(BOSOM) development in premenopausal breast cancer patients
N07MAN Randomized phase II/III study of second-line endrocrine S Rodenhuis II/II 3-4-2008
(Mandjes treatment followed by capecitabine versus capecitabine followed
studie) by endrocrine treatment in patients with metastatic ER positive
breast cancer
N08AFT A randomized prospective trial of 2-6 weeks pre-operative SC Linn II 4-8-2008
(AFTER) hormonal treatment for hormone receptor positive breast cancer:
Anastrozole +/- fulvestrant or tamoxifen exposure - response in
molecular profi le (AFTER-study)
N08RMB Tumorresponse monitoring in patients with breast cancer treated
with primary systemic therapy: towards predicting response in both
the primary tumor and in axillary lymph nodes
MTFD Vrancken Peeters other 23-9-2008
N09PRF Analgesia and nerve function following pulsed radiofrequency for A Lukas 2-6-2010
(PRF4PMPS) postmastectomy pain
N09TOL Long-term safety of trastuzumab in patients with HER2-positive JHM Schellens 22-10-2009
breast cancer (27-4-2010)
N10BES
(BEST-Rx)
Biomarker evaluation and selection of targeted therapy JHM Schellens other 15-11-2010
N10RFS Observational study into specifi c in vivo human discrimination TJM Ruers other 25-10-2010
(OpSpect) between benign and malignant tissue using a combination of
diffuse refl ectance and fl uorescence spectroscopy
171
clinical trials

172
clinical trials
Type of Study
cancer study coordinator Activated
(nick name) Title in NKI-AVL Phase (closed)
GASTRO INTESTINAL
M05RAX Pre-operative chemoradiotherapy regimen with capecitabine and A Cats II 20-3-2006
(RAX) bevacuzimab in locally advanced rectal cancer. A feasability
study (RAX)
M06CRI A multicenter randomized phase III trial of neo-adjuvant M Verheij III 11-1-2007
(CRITICS) chemotherapy followed by surgery and chemotherapy or by
surgery and chemoradiotherapy in resectable gastric cancer
(CRITICS-study: ChemoRadiotherapy after Induction chemo
Therapy In Cancer of the Stomach)
M06RCS A phase II study evaluating short course radiotherapy, VJ Verwaal II 22-2-2007
(M1 study) neoadjuvant bevacizumab,capecitabine and oxaliplatin and
radical resection of primary tumor and metastases in primary
stage IV rectal cancer
(8-1-2010)
M06SCR A multicenter phase III randomised trial comparing total A Cats III 9-5-2006
(SCRIPT) mesorectal excision with pre-operative radiotherapy with or
without post-operative oral capecitabine in the treatment of
operable primary rectal cancer
M07CBO Maintenance treatment with capecitabine and bevacuzimab A Cats III 4-9-2007
(CAIRO3) versus observation after induction treatment with capecitabine,
oxaliplatin and bevacuzimab as fi rst-line treatment in patients with
advanced colorectal carcinoma, a randomised phase III study
(CAIRO3)
M07DDO Development of a docetaxel, oxaliplatin, capecitabine combination A Cats I 23-7-2007
(D-DOCS) schedule in patients with advanced cancer of the stomach or the
gastro-esophageal junction, a phase I study
(8-10-2010)
M07HBT Feasibility study of external beam radiation therapy followed by B van Triest I/II 25-7-2007
(HerBerT) high-dose rate endorectal brachytherapy (HDBRT) in inoperable
rectal cancer patients
M07HEP A randomized two arm phase III study in patients post radical A Cats III 28-4-2008
(HEPATICA) resection of liver metastasis of colorectal cancer to investigate
Bevacizumab (q3w) in combination with capecitabine plus Eloxatin
(XELOX) (q3w) as adjuvant chemotherapy versus XELOX alone
(q3w) as adjuvant treatment (the HEPATICA study: HEPatic
resection with Adjuvant Therapy In colorectal Carcinoma metastasis)
(21-10-2010)
M07NAR Neo-Aduvant Radiotherapy-Chemotherapy In Stomach Cancer. E Jansen I/II 28-1-2008
(NARCIS) Induction therapy with carboplatin, paclitaxel and radiotherapy in
patients with locally advanced gastric cancer. The "NARCIS" study
M07RBV Clinical pilot study: radiolabeled bevacizumab as a tracer of VEGF
expression in patients with colorectal liver metastases. Selecting
patients who may benefi t from anti-VEGF therapy and visualizing
the response
TJM Ruers I 12-2-2008
M07RCM Simultaneous integrated boost radiation therapy with concomitant A Cats I 31-1-2008
capecitabine and mitomycin-C chemotherapy for locally advanced
anal carcinoma
(9-9-2010)
M08ACL Accelerated growth of synchronous colorectal liver metastases: TJM Ruers II 21-2-2008
(SILENT) effects of neo-adjuvant therapy

Type of Study
cancer study coordinator Activated
(nick name) Title in NKI-AVL Phase (closed)
M08CEL Capsule endoscopy in Lynch Syndrome for small intestinal tumor A Cats other 13-7-2010
(CELSIUS) screening: the CELSIUS study
M08DCS Tumor destruction and DC activation in situ: towards in vivo
loaded DC vaccines
TJM Ruers pilot 18-9-2008
M08GPO
(PROFOC)
Genetic and protein profi ling in patients with oesophageal cancer JW van Sandick other 1-9-2008
M08LYN
(CHROMOLYN)
Chromoendoscopy in Lynch syndrome patient A Cats other 13-7-2010
M09OCB A pilot evaluating response to induction chemotherapy with
oxaliplatin, capecitabine and bevacizumab in patients with
extensive peritoneal carcinomatosis of colorectar origin
VJ Verwaal pilot 25-3-2010
M10STE A comparison of two expandable metal stents for the palliation of H Boot other 10-8-2010
(STEMA) malignant esophageal disease
N02ECC A single institution phase II study of ECC (Epirubicin, Cisplatin, H Boot II 29-8-2002
Capecitabin) in locally advanced or metastatic gastric cancer and
adenocarcinoma of the oesophago-gastric junction and distal
oesophagus
(21-10-2010)
N05MCT A phase II study of treatment with the combination of unlabelled H Boot II 5-12-2005
("cold") and radioactive ("hot") meta-iodobenzylguanidin (MIBG)
in metastatic carcinoid tumors
(17-12-2010)
N05STP Serum and tissue protein profi ling and tumor genetic analysis
in patients with potential premalignant conditions or colorectal
cancer
ME Smits other 19-1-2006
N08ICG Pilot study on the use of fl uorecence imaging of lymph nodes
during colorectal lymphadenectomy using indocyanine green
TJM Ruers pilot 30-12-2008
N08RCT Deformation of target volume during radiotherapy for rectal cancer,
a repeat CT study
M Verheij other 14-1-2009
N10RFS Observational study into specifi c in vivo human discrimination TJM Ruers other 25-10-2010
(OpSpect) between benign and malignant tissue using a combination of
diffuse refl ectance and fl uorescence spectroscopy
GYNAECOLOGICAL
M05HIR Hormonal substitution after prophylactic adnectomy in women M van Beurden other 31-5-2005
(HIRISE) with an increased risk for breast- and ovarian cancer due to a
genetic predisposition: HIRISE (High-Risk women and hormonal
Substitution Exposure)
M05PPO Proteomic patterns in blood and tissue of ovarian cancer patients WJ van Driel other 12-1-2006
M05SNV GROningen International study on sentinel nodes in vulvar cancer WJ van Driel other 26-3-2007
(GROINSS-V II) (GROINSS-V) II
M06HRT The effect of hormonal replacement therapy on menopausal CM Korse other 25-9-2006
(NOVARIA) complaints related to biochemical changes in surgically and
naturally postmenopausal women. A prospective observational
comparative study
173
clinical trials

174
clinical trials
Type of Study
cancer study coordinator Activated
(nick name) Title in NKI-AVL Phase (closed)
M06OVH Phase III randomised clinical trial for stage III ovarian carcinoma WJ van Driel III 4-1-2006
(OVHIPEC) randomising between secondary debulking surgery with or without
hyperthermic intraperitoneal chemotherapy (OVHIPEC-1)
M06RTE Randomised phase III trial comparing concurrent chemoradiation B van Triest III 29-3-2007
(PORTEC-3) and adjuvant chemotherapy with pelvic radiation alone in high risk
and advanced stage endometrial carcinoma: PORTEC-3
M07RCV Phase II study of defi nitive radiochemotherapy for locally
advanced squamous cell cancer of the vulva: an effi cacy study
WJ van Driel II 26-6-2007
M09OLA A phase II open label, randomized, comparative multicentre study GS Sonke II 22-4-2010
to compare the effi cacy and tolerability of olaparib in combination
with paclitaxel and carboplatin versus paclitaxel and carboplatin
alone in patients with platinum sensitive advanced serous ovarian
cancer
(30-6-2010)
M09PSO Phase II randomised, double blind, multicentre study to assess the GS Sonke II 13-8-2009
effi cacy of AZD2281 in the teatment of patients with platinum
sensitive serous ovarian cancer following two or more platinum
containing regimens
(22-2-2010)
M10MKO Phase II and pharmacological study with WEE-1 inhibitor MK-1775
combined with carboplatin in patients with p53 mutated epithelial
ovarian cancer
JHM Schellens II 8-7-2010
M10MKT A two Part, phase I-IIa study evaluating MK1775 in combination
with Topotecan/Cisplatin in adult patients with cervical cancer
JHM Schellens I/II 26-5-2010
N05CGO Randomized clinical pharmacological dose-escalation study to
explore both safety and preliminary effi cacy and pharmaco-kinetics,
-dynamics and -genomics of fi xed dose rate gemcitabine and
30-minute standard gemcitabine infusion both administered in
combination with carboplatin as second-line treatment in patients
with adv ovarian ca
JHM Schellens I 8-9-2005
N10OCT Optical vulvar biopsy by optical coherence tomography (OCT) M van Beurden other 24-8-2010
HEAD AND NECK
M07CMD Treatment of myogenic cranio mandibular dysfunction (CMD):
a prospective randomised clinical trial, comparing a mechanical
stretching device (Therabite) with standard physiotherapy
FJM Hilgers III 29-8-2007
M08CON A randomized study of docetaxel/cisplatin/5-fl uorouracil (TPF) JP de Boer II 2-2-2009
(CONDOR) as neoadjuvant chemotherapy followed by concomitant
chemoradiotherapy (CRT) with conventional radiotherapy (RT)
versus concomitant CRT with accelerated RT in patients with
locally advanced head and neck squamous cell cancer (HNSCC)
in good condition. (CONDOR)
M08DDL Docetaxel versus Docetaxel and Lapatinib in recurrent or JP de Boer II 12-2-2009
(TYVTAX) metastatic squamous cell carcinoma of the head and neck
(SCCHN); an open label multicenter randomized phase II study
M08EBV Standardized early detection of primary and recurrent
nasopharyngeal carcinome (NPC) using (anti-) EBV based
tumor markers in The Netherlands
IB Tan other 29-4-2009

Type of Study
cancer study coordinator Activated
(nick name) Title in NKI-AVL Phase (closed)
M08MSH Measuring shoulder disability after neck dissection; a comparison
of 4 self report scales
M Stuiver other 25-3-2009
M08SNU Ultrasound guided fi ne needle aspiration cytology and sentinel M van de Brekel other 26-6-2008
(SNUS) node biopsy in the detection of occult lymph node metastases of
early oral and oropharyngeal cancer (SNUS)
M09HZT Effi cacy of adding hyperbaric oxygen therapy to the treatment of LM Smeele III 23-11-2009
(HBOT) late radiation damage of the lower jaw (osteonecrosis)
M09NST Clinical feasability of a new surgical tool for primary or secondary
tracheoesophageal puncture and voice prosthesis insertion for
prosthetic voice rehabilitation after total laryngectomy
FJM Hilgers other 3-12-2009
M09OSA Prospective cohort study on the prevalence of obstructive sleep
apnea in patients with head and neck cancer treated wither either
radiotherapy, chemoradiation or surgery
M van de Brekel other 2-12-2009
M09PDT A multi centre cost-effectiveness evaluation of a novel treatment
option in the netherlands: photo dynamic therapy with temoporfi n
for the treatment of advanced incurable head and neck cancers,
for whom prior conventional treatments have failed
IB Tan other 6-8-2009
N04RTB Effecten van bestraling op bloedvaten NS Russell other 5-10-2004
N05HME De kortetermijninvloed van een Heat and Moisture Exchanger op
de endotracheale temperatuur en luchtvochtigheid bij
gelaryngectomeerden
JK Zuur other 1-9-2005
N07CRH Phase I/II study of combined treatment with AT-101, cisplatin and FJP Hoebers I/II 16-2-2010
(compa) radiotherapy in patients with locally advanced head and neck cancer
N07MCP Microcirculatory changes during photodynamic therapy (PDT) in
squamous cell carcinoma of the oral cavity and oropharynx
MP Copper other 21-5-2007
N07VIN Transoral resection of stage I-II carcinomas of the oropharynx by
robot-assisted surgery: a feasability study
M van de Brekel other 12-3-2007
N08HHF Validation of hypoxia imaging of cancer in the head and neck
area with FAZA-PET
WV Vogel II 22-10-2008
N08PTR Phase I study of radiotherapy dose escalation on the FDG-PET O Hamming-Vrieze I 28-1-2010
(PET01RAD) avid region of the primary tumor in locally advanced oropharynx
and oral cavity tumors eligible for concurrent chemoradiation
N09BIO Prospective study of changes in the oral biofi lm and the
composition of salivary proteins in patients, being irradiated for
a tumor in the head-and-neck area
AJM Balm other 25-3-2010
N10BAS Clinical feasibility of a new adhesive base plate (placeholder for
stoma appliances) for rehabilitation after total laryngectomy
FJM Hilgers other 17-5-2010
N10OPT Optimising photodynamic therapy for the treatment of head and
neck cancer
B Karakullukcu other 1-11-2010
N10VMO Evaluation of tumor variability with MRI during radiotherapy
treatment in patients with an oropharyngeal or oral cavity carcinoma
O Hamming-Vrieze other 8-11-2010
175
clinical trials

176
clinical trials
Type of Study
cancer study coordinator Activated
(nick name) Title in NKI-AVL Phase (closed)
LEUKAEMIA / MDS
M05H68 A randomized phase III study in previously untreated patients with M Kerst III 21-3-2006
(HOVON 68) biological high-risk CLL: fl udarabine + cyclophophamide (FC)
versus FC + low dose alemtuzumab
(12-11-2010)
M10OMB Ofatumumab maintenance treatment versus no further treatment M Kerst III 10-11-2010
(HOVON 101) in relapsed CLL responding to induction therapy
LUNG
E08061 Phase II study of sunitinib (SU11248) in patients with small cell P Baas II 22-2-2010
lung cancer who are either chemo-naïve (extensive disease) or
have a "sensitive" relapse
(30-6-2010)
E08072 Concurrent Once-daily Versus twice-daily RadioTherapy: a 2-arm JL Knegjens III 11-11-2009
(CONVERT) randomised controlled trial of concurrent chemo-radiotherapy
comparing twice-daily and once-daily radiotherapy schedules in
patients with limited stage small cell lung cancer (SCLC) and good
performance status (CONVERT)
M03THA Phase III trial of the antiangiogenic agent Thalidomide in patients P Baas III 18-2-2004
(NVALT5) with malignant pleural mesothelioma after fi rst line chemotherapy
(NVALT5)
(14-1-2010)
M05ATD Accurate target defi nition of non-small cell lung tumors using J Stroom other 5-1-2006
pathology-validated PET and CT imaging for the optimization of
radiation treatment
(2-9-2010)
M05GCP Pharmacogenomic and pharmacokinetic study in patients with JHM Schellens other 13-9-2005
advanced non small-cell lung cancer (NSCLC) treated with fi rst-line
gemcitabine / platinum combination chemotherapy
(5-2-2010)
M06NEL Effi cacy of neoadjuvant erlotinib in patients with clinical stage I/II HM Klomp I/II 8-11-2006
(NEL) non-small cell lung cancer (NSCLC)
M07CCL Open-label, randomised multi-center study investigating Cetuximab, MM van den Heuvel I/II 13-3-2008
(Raditux) in combination with concurrent chemo-radiotherapy in locally
advanced non-small cell lung carcinoma (RADITUX)
M07OSM A phase III randomized, double blind, placebo controlled trial of P Baas II/II 9-10-2007
(OSM) oral suberoylanilide hydroxamic acid (L-001079038) in patients
with advanced malignant pleural mesothelioma previously treated
with systemic chemotherapy
M07STN A multi-center phase III randomized, double-blind placebo- MM van den Heuvel III 13-12-2007
(START) controlled study of the cancer vaccine Stimuvax (L-BLP25 or
BLP25 liposome vaccine) in non-small cell lung cancer (NSCLC)
subjects with unresectable stage III disease
M08EMD An open-label, phase Ib, dose-escalation trial on the safety, MM van den Heuvel I 9-4-2009
(Selectikine) tolerability, pharmacokinetics, immunogenicity, biological effects
and antitumor activity of EMD 521873 in combination with local
irradiation (20Gy) of primary tumors or metastases in subjects with
NSCLC stage IIIb with malignant pleural effusion or stage IV with
disease control (PR or SD) after application of 4 cycles of
platinum-based, fi rst-line chemotherapy
(7-9-2010)

Type of Study
cancer study coordinator Activated
(nick name) Title in NKI-AVL Phase (closed)
M09AVT A randomized open label multinational phase III comparing JA Burgers III 19-8-2009
(ACT-1) amrubicin versus topotecan in patients with extensive or limited
and sensitive or refractory small cell lung cancer after failure of fi rst
line chemotherapy
(18-2-2010)
M09BPL Open label study of bevacizumab maintenance therapy with or JA Burgers III 14-12-2009
(AVAPERL) without pemetrexed after a fi rst line treatment chemotherapy with
bevacizumab-cisplatin pemetrexed in patients with advanced,
metastatic or recurrent non-squamous non-small cell lungcancer
(28-6-2010)
M09CRE Randomized trial on chest irradiation in extensive disease small JL Knegjens III 19-3-2009
(CREST) cell lung cancer
M09LST Evaluation of molecular sputum test diagnostics for lung cancer JA Burgers other 18-5-2009
M09N10 A randomized phase II study of erlotinib compared to single agent JA Burgers II 11-9-2009
(NVALT10) chemotherapy-erlotinib combination in pretreated patients with
advanced NSCLC (NVALT10 study)
M09PBO Dose escalation by boosting radiation dose within the primary JSA Belderbos II 26-11-2009
(PET-BOOST) tumor on the basis of a pre-treatment FDG-PET-CT scan in stage
II and III NSCLC: a randomised phase II trial
M09PCI Prophylactic Cranial Irradiation(PCI) versus observation in radically JSA Belderbos III 20-10-2009
(NVALT11) treated patients with stage III non-small cell lung cancer: a phase III
randomized study (NVALT 11)
M09ROS A randomized clinical trial of radiosurgery ( stereotactic JSA Belderbos III 28-4-2009
(ROSEL) radiotherapy) or surgery in patients with IA NSCLC who are fi t
to undergo primary resection
M10GEF Observaties bij langdurige respons op gefi tinib (Iressa) in het JA Burgers 7-6-2010
Expanded Access Programma (30-8-2010)
M10LUC Registration phase III study of LucanixTM( Belagenpumatucel-L)
in advanced non-small cell lung cancer: an international multicenter,
randomized, double-blind, placebo-controlled study of LucanixTM
maintenance therapy for stages III/IV NSCLC subjects who have
responded to or have stable disease following one regimen of
front-line platinum-based combination chemotherapy
MM van den Heuvel III 30-6-2010
M10SON A phase II study of sorafenib in patients with locally advanced
and/or metastatic (stage IIIb or IV) non small cell lung cancer
(NSCLC) with a K-RAS mutation
JA Burgers II 8-7-2010
N00ALF Endoscopic detection op pre-neoplastic lesions and carcinoma in P Baas pilot 30-6-2000
the bronchial tree with delta-aminolevunic acid fl uorescence (10-3-2010)
N04LSN Feasibility of lymphoscintigraphy and sentinel node biopsy in
patients with non-small lung cancer
HM Klomp pilot 8-9-2004
N05MIB 99mTc-methoxyisobutylisonitrile (MIBI) for imaging of apoptosis
and prediction of tumor respons to chemotherapy and/or
radiotherapy in cancer patients
RA Valdes Olmos other 7-6-2005
N07NRA A phase I/II study with NAMI-A, a Novel Ruthenium Anticancer
Agent, and gemcitabine combination second-line therapy in
NSCLC patients
JHM Schellens I/II 31-7-2008
177
clinical trials

178
clinical trials
Type of Study
cancer study coordinator Activated
(nick name) Title in NKI-AVL Phase (closed)
N08CPA A randomized phase I/II study of standard chemotherapy (cisplatin
and pemetrexed) with or without Axitinib in patients with malignant
mesothelioma: interim biopsy analysis to determine effi cacy
P Baas I/II 22-5-2009
N09MLN Quantifi cation of mediastinal lymph node position variability by
implanted fi ducial markers using cone beam computer tomogram
in non-small cell lung cancer patients treated with radical irradiation
JSA Belderbos other 8-4-2010
N10ILM designing and testing new intervention therapies for lung cancer
and mesothelioma
P Baas other 30-8-2010
N10RFS Observational study into specifi c in vivo human discrimination TJM Ruers other 25-10-2010
(OpSpect) between benign and malignant tissue using a combination of
diffuse refl ectance and fl uorescence spectroscopy
LYMPHOMA – HODGKIN’S DISEASE
E20012 BEACOPP (4 cycle's escalated + 4 cycle's baseline) vs ABVD JW Baars III 29-3-2004
(8 cycle's) in Stage III & IV Hodgkin's Lymphoma (8-1-2010)
E20051 The H10 EORTC/GELA randomized Intergroup trial on early JW Baars II 23-11-2006
(H10) PET-scan guided treatment adaptation versus standard combined
modality treatment in patients with supradiaphragmatic stage I/II
Hodgkin's lymphoma
LYMPHOMA – NON-HODGKIN’S
E20971 A phase III study on low-dose totasl body irradiation and involved RLM Haas III 2-2-2004
fi eld radiotherapy in patients with localized, stages I and II, low
grade non-hodgkin's lymphoma
(30-9-2010)
M05H55 Effi cacy of maintenance therapy with rituximab after induction JP de Boer III 20-7-2005
therapy (R-CHOP versus R-FC) for elderly patients with mantle
cell lymphoma not suitable for autologous stem cell transplantation
(22-10-2010)
M07H80 Phase II study on the feasability and effi cacy of R-DHAP-MTX JW Baars II 10-9-2007
(HOVON80) combined with i.t. rituximab and autologous SCT in patients with
a recurrent aggressive B-cell NHL with CNS localisation
M07H84 Randomized phase III study on the effect of early intensifi cation JW Baars III 22-1-2008
(HOVON84) of rituximab in combination with 2-weekly CHOP chemotherapy
followed by rituximab maintenance in elderly patients with DLBCL
M09ORC Ofatumumab versus Rituximab salvage chemoimmunotherapy JW Baars III 13-7-2010
(ORCHARRD) followed by ASCT in relapsed or refractory DLBCL
M09TAM Treatment induced alterations in microenvironment in follicular D de Jong other 27-5-2009
(TAMIL) lymphoma. A multicenter descriptive study
M10H105 Rituximab in primary central nervous system lymphoma. JW Baars III 23-11-2010
(HOVON105) A randomized HOVON/ALLG intergroup study.
N03OFP A phase II study of eradication therapy with additional oral JP de Boer II 6-10-2003
fl udarabine in t(11;18) positive gastric MALT lymphoma (28-1-2010)
N05MIB 99mTc-methoxyisobutylisonitrile (MIBI) for imaging of apoptosis
and prediction of tumor respons to chemotherapy and/or
radiotherapy in cancer patients
RA Valdes Olmos other 7-6-2005

Type of Study
cancer study coordinator Activated
(nick name) Title in NKI-AVL Phase (closed)
MELANOMA / SKIN
E18071 Adjuvant immunotherapy with anti-CTLA-4 monoclonal antibody
(ipilimumab) versus placebo after complete resection of high-risk
III melanoma: a randomized, double-blind phase 3 trial of the
EORTC Melanoma Group
JBAG Haanen III 27-4-2010
M05MSL A phase III multicenter randomized trial of sentinel OE Nieweg III 11-9-2006
(MSLT-II) lymphadenectomy and complete lymph node dissection versus
sentinel lymphadenectomy alone in cutaneous melanoma patients
with molecular or histopathological evidence of metastases in the
sentinel node
M07DNA Intradermal naked DNA vaccination for mounting tumor-specifi c
immunity in stage IV melanoma patients: a phase I clinical study
JBAG Haanen I 9-1-2009
M09ADM A phase II, double-blind randomised study to assess the effi cacy JBAG Haanen II 6-10-2009
(AZD6244) of AZD6244 (Hyd-Sulfate) in combination with Dacarbazine
compared with Dacarbazine alone in fi rst line patient with BRAF
mutation positive advanced cutaneous or unknown primary
melanoma
(26-2-2010)
M09BRI A randomized, open-label, controlled, multicenter, phase III study JBAG Haanen III 16-3-2010
(BRIM 3) in previously untreated patients with unresectable stage IIIc or IV
melanoma
(17-12-2010)
N03LAM Longitudinal analysis of melanoma-specifi c immunity in stage III
and IV melanoma patients
JBAG Haanen II 22-8-2003
N06TIS Integrated analyses of melanoma-T cell interactions; relevance
for immunotherapy
JBAG Haanen other 29-8-2006
N10BIO Novel targets for medication in melanoma patients who are
progressive after treatment with BRAF inhibitor RO5185426 or
dacarbazine
JBAG Haanen other 12-7-2010
N10TCR Donor leukapheresis for T-cell receptor gene therapy for
treatment of metastatic melanoma (skin cancer)
JBAG Haanen pilot 5-8-2010
MISCELLANEOUS
M09BOU The role of microparticles bearing active tissue factor in cancer M Tesselaar other 11-2-2010
(BOUILLAUD) and thrombosis: "the Bouillaud-study"
M09XLT An international, randomized, double-blinded, phase III effi cacy
study of XL184 versus placebo in subjects with unresectable,
locally advanced or metastatic medullary thyroid cancer
JP de Boer III 8-7-2009
M10RTW To enhance return-to-work in cancer patients - a randomised trial
(return to work)
WJ van Driel other 14-9-2010
N01RIT Identifi cations of molecular mechanisms involved in radiation- NS Russell other 7-5-2002
induced telangiectasia (17-12-2010)
N03THY Therapeutic management of thymoma and thymic carcinoma:
- a prospective registration study based on preoperative risk
assessment of local failure.
LGH Dewit pilot 25-11-2003
179
clinical trials

180
clinical trials
Type of Study
cancer study coordinator Activated
(nick name) Title in NKI-AVL Phase (closed)
N05CHO The effi cacy of cordotomies in patients with chest pain due to A Lukas II 17-1-2006
primariy localized malignancies of the chest (22-12-2010)
N09DRF Intraoperative real time imaging with a dual radioactive/
fl uorescence modality for sentinel node localization. A feasibility
study including reproducibility of multimodality lymphatic mapping
with a cocktail tracer containing 99mTc nanocolloid/ICG
RA Valdes Olmos other 8-6-2009
SOFT TISSUE / OSTEOSARCOMA
E62012 Randomised trial of single agent doxorubicin versus doxorubicin S Rodenhuis III 9-7-2003
plus ifosfamide in the fi rst line treatment of advanced or metastatic
soft tissue sarcoma
(11-5-2010)
E62063 A phase III randomized study evaluating surgery of residual F van Coevorden III 18-2-2009
(SURGIST) disease in patients with metastatic gastro-intestinal stromal tumor
responding to Imatinib mesylate
E62072 A randomized double blind phase III trial of Pazopanib versus M Kerst III 18-11-2008
(PALETTE) placebo in patients with soft tissue sarcoma whose disease has
progressed during or following prior therapy
(15-1-2010)
M01ROS Phase II study of rosiglitazone in advanced liposarcoma S Rodenhuis II 24-8-2001
(30-9-2010)
M09RTP Phase I clinical study of a combined modality treatment of RLM Haas I 24-6-2010
(PASART-1) sarcomas of the extremities with radiotherapy (RT) and doseescalation
of Pazopanib (PASART-1)
N10DMY Dose reduction of preoperative radiotherapy in Myxoid RLM Haas II 15-12-2010
(DOREMY) liposarcomas
N10PPS Perfusion PET sarcoma; feasability of tumor perfusion
quantifi cation with Gallium-68-citrate PET/CT in sarcoma
and radiotherapy.
WV Vogel other 3-12-2010
URO-GENITAL
E30072 A phase III randomised double blind study comparing sorafenib
with placebo in patients with resected primary renal cell carcinoma
at high or intermediate risk of relapse
M Kerst III 21-7-2009
E30073 Randomized phase III trial comparing immediate versus deferred
nephrectomy in patients with synchronous metastatic renal cell
carcinoma
A Bex III 17-3-2010
M00LMT Identifi cation of occult lymph node metastases in testicular cancer
to select patients for adjuvant treatment. Feasability of a
laparoscopic selective retroperitoneal lymphadenectomy.
S Horenblas pilot 14-11-2000
M06HFP Hypofractionated irradiation for prostate cancer: a randomized F Pos III 8-3-2007
(HYPRO) multicenterphase III study (HYPRO) (6-12-2010)
M06LAN Late Adverse effects in Dutch testicular cancer survivors: M Kerst other 12-2-2007
(LANCE) a Nationwide case-control study on Cardiovascular Events
M06SIL Phase I dose-escalation trial for the combination of sorafenib with
interleukin-2 treatment in patients with clear cell renal carcinoma
JBAG Haanen I 20-11-2006

Type of Study
cancer study coordinator Activated
(nick name) Title in NKI-AVL Phase (closed)
M07PGC Phase II trial of paclitaxel, gemcitabine and cisplatin in patients
with relapsing germ cell cancer after fi rst line chemotherapy
M Kerst II 8-1-2008
M08PZD A phase III, placebo-controlled, double-blind study to assess the HG van der Poel III 9-3-2009
(M15) effi cacy and safety of once-daily orally administered ZD4054 10 mg
in non-metastatic hormone-resistant prostate cancer patients
M09PRG Evaluation of PROTEX absorbable injectable hydrogel when used B van Triest other 20-7-2009
(protex) to maintain space between rectum and prostate in men undergoing
radiation therapy for stage T1-T2 prostate cancer:
a non-randomized single-arm clinical study
M09PSR Study VEG108844 a study of pazopanib versus sunitinib in the JBAG Haanen III 27-5-2009
(COMPARZ) treatment of subjects with locally advanced and/or metastatic renal
cell cancer
(16-4-2010)
M09RRC An open label multi center expanded access study of RAD001 in C Blank IV 28-4-2009
(REACT) patients with metastatic carcinoma of the kidney who are intolerant
of or have progressed despite any available vascular endothelial
growth factor receptor tyrosine kinase inhibitor therapy
(1-4-2010)
M10AZP A phase I, open-label, multicenter study to assess the safety, JHM Schellens I 23-8-2010
(SARAFTip) tolerability, pharmacokinetics and preliminary anti-tumor activity of
ascending doses of AZD3514 in patients with castration-resistant
prostate cancer
M10IPI A randomized, double-blind, phase III trial comparing ipilimumab A Bergman III 14-12-2010
(CA 184-043) vs placebo following radiotherapy in subjects with castration
resistant prostate cancer that have received prior treatment with
docetaxel
M94SAL Salvage regimen incorporating repeated ablative chemotherapy
with autologous PSCT, a phase II study
S Rodenhuis II 4-7-1994
N06MPM Samarium-153-EDTMP (QUADRIMET) versus docetaxel for HG van der Poel II 14-3-2007
(QUADRIMET) multiple painful oseous metastases in prostate cancer
N06SUN Sunitinib prior to nephrectomy in patients with metastatic renal A Bex II 16-5-2007
cell carcinoma and the primary in situ (2-11-2010)
N08API Analysis of prostate-specifi c immunity in stage III and IV prostate
cancer patients
JBAG Haanen other 22-1-2009
N08SNR Site and distribution of sentinel lymph nodes in renal cell
carcinoma, a phase II study
A Bex II 19-3-2009
N09IGF Pilot study on the use fl uorescence imaging of lymph nodes
during laparoscopic pelvic sentinel node dissection for prostate
cancer using indocyanine green
HG van der Poel pilot 14-7-2009
N09QPB Quantative FDG-PET/CT of primary bladder cancer WV Vogel pilot 8-6-2009
N09VDU Urethral suspension using vas deferens for prevention of urin HG van der Poel II 11-6-2009
(RALP 2009) incontinence after robot assisted laparascopic prostatectomy
(RALP)
(3-6-2010)
181
clinical trials

182
invited speakers
INVITED SPEAKERS 2010
Genevieve Almouzni, Paris, France
Non-coding RNA and pericentric heterochromatin in mouse cells
Allan Balmain, San Francisco, USA
Systems genetics analysis of infl ammation and cancer
susceptibility in mouse models
Alberto Bardelli, Candiolo, Italy
Cancer mutations and targeted therapies in cells, mice and
patients
Yves Barral, Zurich, Switzerland
The NoCut checkpoint and its role in the coordination of late
mitotic events
Anton Berns, Amsterdam, The Netherlands
The mouse as an oncogenomic tool
David Boettiger, Philadelphia, USA
Mechanical and chemical control of cell adhesion
Matthew Bogyo, Stanford, CA, USA
Imaging protease activity in cancer and infl ammation using
small molecule activity based probes
Anne Brunet, Stanford CA, USA
Mechanisms of aging and longevity
George Calin, Houston, USA
Non-coding RNAs in clinical practice - from bench to bedside
Paul Coffer, Utrecht, The Netherlands
Divide, differentiate or die? A novel PKB-FOXO node
coordinating regulation of metabolism and autophagy
Job Dekker, Worcester, MA, USA
Three-dimensional architecture of the genome
Caroline Dive, Manchester, UK
Circulating biomarkers for early clinical trials in oncology
Douglas Easton, Cambridge, UK
Genome-wide association studies in cancer - what have we learnt
and where next?
Luis Ferreira Moita, Lisbon, Portugal
shRNA-based dissection of innate immune responses
Eileen Furlong, Heidelberg, Germany
Gene regulatory networks during development: predicting cisregulatory
activity
Nick Gilbert, Edinburgh, United Kingdom
The effect of DNA supercoiling on chromatin structures
Thomas Gingeras, Woodbury, NY, USA
Eukaryotic transcriptomes: complex, multifunctional, and
compartmentalized
Joost Gribnau, Rotterdam, The Netherlands
Activation of X inactivation
Gerald de Haan, Groningen, The Netherlands
Gene(tic) networks in hematopoietic stem cells
Matthias Hentze, Heidelberg, Germany
Translational control by miRNAs
Sander van den Heuvel, Utrecht, The Netherlands
Proliferation, differentiation and asymmetric cell division
Frank Holstege, Utrecht, The Netherlands
Understanding regulatory circuitry through expression-profi le
phenotypes
Lukas Huber, Innsbruck, Austria
Regulation of cell migration and focal adhesions by endosomal
MAPK scaffold complexes
Toshihisa Ishikawa, Yokohama, Japan
SmartAmp2, the world’s fastest SNP detection method: Its
molecular mechanism and clinical applications
Lee Josephson, Charlestown, MA, USA
Multimodal imaging agents: Problems and opportunities
René Ketting, Utrecht, The Netherlands
RNAi-like pathways in germ cells and early development
David Komander, Cambridge, UK
Atypical ubiquitin chains and their hydrolysis by deubiquitinases
Antonis Koromilas, Quebec, Canada
Translational control by the eIF2alpha phosphorylation pathway
in response to stress and its implications in cancer
Tony Kouzarides, Cambridge, UK
Chromatin modify enzymes: their function and role in cancer
Lee Kraus, Ithaca NY, USA
Nuclear signaling, chromatin structure, and gene regulation by
estrogens and NAD+
Wouter de Laat, Utrecht, The Netherlands
Uncovering genome structure and function
Titia de Lange, New York, USA
Protection and maintenance of mammalian telomeres
Edward Marcotte, Austin TX, USA
Insights from proteomics into cellular evolution and surprising
disease models
John Martens, Rotterdam, The Netherlands
Exploring and integrating the breast cancer transcriptome

Christine Mayr, New York, USA
Control of 3’UTR length by alternative cleavage and
polyadenylation
Danesh Moazed, Boston, USA
RNAi-based mechanisms for assembly and propagation of
heterochromatin
Andrew Morris, Lexington, USA
Role of Autotaxin (Lysophospholipase D) in cardiovascular and
metabolic regulation
James Nelson, Stanford, CA, USA
Functional evolution of the Cadherin-Catenin complex and
regulation of the actin cytoskeleton
Huib Ovaa, Amsterdam, the Netherlands
Chemistry of ubiquitin-mediated proteolysis and antigen
presentation
Yves Pommier, Bethesda, MD, USA
DNA topoisomerases and genomic stability
Sridhar Ramaswamy, Cambridge MA, USA
Functional genomic approaches to cancer metastasis, dormancy,
and drug resistance
John Radford, Manchester, UK
Approaches to reducing the impact of late effects of treatment
for Hodgkin lymphoma
Emma Rawlins, Cambridge, UK
Lung epithelial progenitor cells in development and repair
Hans Schreiber, Chicago IL, USA
Tumor stroma and the immune response
Ali Shilatifard, Kansas City, Missouri, USA
Lessons learned from yeast about human leukemia
Colin Stewart, Singapore
Architecture of the cell’s nucleus in development aging and
disease
Henk Stunnenberg, Nijmegen, The Netherlands
Genome wide (epi)genetic profi ling provides molecular insight
into differentiation and transformation pathways
Francoise Stutz, Geneva, Switzerland
Antisense RNAs and transcriptional gene silencing in yeast
Crislyn D’Souza-Schorey, Notre Dame, Indiana, USA
Cellular models for epithelial morphogenesis and cell invasion
Jussi Taipale, Helsinki, Finland
Systems biology of cancer
Christian Ungermann, Osnabrück, Germany
Function of tethering complexes in the endolysosomal system
Roel Verhaak, Boston, MA, USA
Cancer Genome Sequencing
183
invited speakers
Jose Villadangos, Melbourne, Australia
Regulation of antigen presentation in the dendritic cell network
Marian Walhout, Worcester, MA, USA
Gene-centered regulatory networks
Kevin White, Chicago, IL, USA
Nuclear receptors, transcriptional networks and cancer
Niels de Wind, Leiden, The Netherlands
Mutatis mutandis: Roles of mutagenic replication of damaged
DNA in fi tness and disease
Tony Wynshaw-Boris, San Francisco, CA, USA
Dishevelled: in vivo analysis of a multifunctional redundant gene
family
Roy Zent, Nashville, TN, USA
Integrins, Ilk and the kidney

184
projects
PROJECTS SUPPORTED
BY THE DUTCH CANCER SOCIETY
Principal
investigator
Number of
project Title Started
Aaronson, Neil KWF 2006-3470 Cognitive behavioral therapy (CBT) and physical
exercise for climacteric symptoms in breast cancer
patients experiencing treatment-indiced menopause
Aaronson, Neil KWF 2009-4299 A-CaRe Project 2: Effectiveness of physical exercise
during chemotherapy to improve physical fi rness and
reduce fatigue: A randomized trial
Agami, Reuven KWF 2006-3558 Genetic screens to identify cancer related functions of
human microRNAs
Agami, Reuven KWF 2007-3881 Role of miRNA genes in etiology of malignant germ cell
tumors
Ended/
ends
9/1/2006 11/1/2011
8/1/2009 8/1/2013
11/1/2006 11/1/2010
3/1/2007 3/1/2011
Agami, Reuven KWF 2009-4469 Role of RNA binding proteins in cancer 3/1/2010 3/1/2014
Agami, Reuven KWF 2009-4498 Bromodomain containing proteins in cancer 4/1/2010 4/1/2014
Belderbos, José KWF 2010-4675 Bestralingsdosis verhoging door het geven van een
boost op de longtumor gebaseerd op een PET-scan bij
patiënten met een stadium II of III niet klein-cellig
longcarcinoom
Bergman, André KWF 2009-4356 Investigating the Role of Infl ammation in Prostate
Cancer Development
Bernards, René KWF 2008-4027 Understanding resistance to HER2-targeting therapy in
human breast cancer through functional genetics
Bernards, René KWF 2008-4042 Identifi cation of enzymes involved in regulatory
ubiquitination in TNF a--stimulated signalling by lossof-function
screens
Bernards, René KWF 2009-4337 Identifi cation of genetic modifi ers of sensitivuty to
mTOR pathway inhibition in breast cancer
Bernards, René KWF 2009-4496 Determinats of the response to retinoic acid in
neuroblastoma
Berns, Ton KWF 2008-4253 Designing and testing new intervention therapies for
lung canccer and mesotheliomas
Blank, Christian KWF 2008-3988 Blockade of PD-1/D-L1 interaction to improve T cell
mediated immunotherapy of cancer
Bleiker, Eveline KWF 2008-3209 Psychosocial aspects of genetic testing in families at
high risk of multiple tumors at various sites and ages
Bleiker, Eveline KWF 2008-4016 Identifi cation of psychosocial problems and perceived
need for support in cancer genetics
3/25/2010 3/25/2014
8/1/2009 8/1/2015
11/1/2008 11/1/2012
11/1/2008 11/1/2012
8/1/2009 8/1/2013
1/1/2010 1/1/2014
5/1/2009 5/1/2015
1/1/2008 1/1/2012
11/1/2005 10/31/2010
12/1/2008 3/31/2013

Principal
investigator
Number of
project Title Started
Borst, Jannie KWF 2008-4028 T cell programming at the dendritic cell interface:
impact on anti-tumor immunity
Borst, Jannie KWF 2008-4110 Impact of TRAIL death receptor traffi cking on proapoptotic
signaling
Borst, Piet KWF 2005-3379 A study of drug resistance mechanisms using large
scale RNA interference screens
Borst, Piet KWF 2006-3566 Mechanisms of chemotherapy resistance in
spontaneous tumors of genetically modifi ed mice
185 projects
Ended/
ends
2/1/2008 2/1/2012
3/1/2008 3/1/2012
6/1/2005 6/1/2010
7/1/2006 7/15/2010
Collard, John KWF 2007-3753 The par complex in cell polarity and tumor progression 7/1/2007 7/1/2011
Dalesio, Otilia KWF
Datamanagement
Dannenberg, Jan-
Hermen
KWF Datamanagement 1/1/1982 1/1/2011
KWF 2007-3978 Genetic analysis of class I HDACs in development and
treatment of cancer
Elkhuizen, Paula KWF 2009-4389 Properative accelerated partial breast irradiation
(PAPBI): defi ning radiotherapy sensitivity
Elkhuizen, Paula KWF 2010-4799 Image guided preoperative accelerated partial breast
irradiation (PAPBI): defi ning radiotherapy sensitivity
Haanen, John KWF 2007-3943 HPV 16 E6/E7 DNA prime and E6/E7 long peptide
boost vaccination for multifocal Vulvar Intraepithelial
Neoplasia (VIN)
Jacobs, Heinz KWF 2008-4112 A cancer genome atlas of the activation-induced cytidine
deaminase: novel insights into the pathogenesis and
prognosis of b cell lymphoma
Jacobs, Heinz KWF 2009-4511 Role of the histone methyltransferase Dot1 in gene
expression and leukemic transformation
1/1/2008 1/1/2012
1/1/2010 1/1/2014
7/6/2010 7/6/2015
1/1/2008 1/1/2011
3/1/2008 3/1/2012
1/10/2010 1/10/2014
Jacobs, Jacqueline KWF 2007-3907 Dissecting the telomere damage response 5/1/2008 5/1/2012
Jalink, Kees KWF 2007-3733 TRMP7, a novel regulator of cytoskeletal tension:
implications for cancer progression, invasion and
metastasis
Jalink, Kees KWF 2010-4626 Calcium and phosphoinositides in the control of
podosome formation and tumor cell invasion
Jonkers, Jos KWF 2006-3486 Dissection of the role of E-cadhering loss-of-function in
breast cancer development and metastasis
9/1/2007 9/1/2011
10/1/2010 10/1/2014
2/13/2006 2/12/2011
Jonkers, Jos KWF 2006-3715 KWF 2006-3715 1/1/2007 1/1/2011
Jonkers, Jos KWF 2007-3772 Preclinical validation of chemical inhibitors of poly
(ADP-ribose) polymerase (PARP) in conditional mouse
models for BRCA-associated breast cancer
2/1/2007 2/1/2011

186
projects
Principal
investigator
Number of
project Title Started
Jonkers, Jos KWF 2008-4116 Impact of BRCA muntations on breast tumorigenesis
and treatment response: functional analysist of defi ned
truncation mutants and unclassifi ed variants
Linn, Sabine KWF 2006-3706 Towards patient-tailored systemic therapy in breast
cancer: a combined approached of translational research
and mouse model systems
Linn, Sabine KWF 2009-4435 In kaart brengen van actieve Estrogen Receptor binding
sites bij tamoxifen en aromatase inhibitor resistente
cellijnen en borsttumoren, om zo prodictieve markers
te defi niëren
Moolenaar, Wouter KWF 2005-3383 PIP4K-beta/PtdIns5P/p53: A cellular stress regulated
pathway and its implication in oncogenesis
Ended/
ends
1/1/2008 1/1/2012
1/1/2007 1/1/2013
11/1/2009 11/1/2013
11/1/2005 11/1/2010
Moolenaar, Wouter KWF 2005-3392 KWF 2005-3392 7/1/2005 7/1/2010
Moolenaar, Wouter KWF 2010-4781 Validation of autotaxin, a metastasis-enhancing exophosphodiesterase,
as a therapeutic target
Nederlof, Petra KWF 2007-3749 Specifi c chromosomal imbalance in breast carcinomas
as a marker for breast cancer susceptibility
Neefjes, Jacques KWF 2007-3883 Anti-cancer drugs and their effects on the ubiquitin
cycle
Ovaa, Huib KWF 2010-4781 Validation of autotaxin, a metastasis-enhancing exophosphodiesterase,
as a therapeutic target
8/1/2010 8/1/2014
8/15/2007 8/15/2011
4/1/2007 4/1/2011
8/1/2010 7/1/2014
Peeper, Daniel KWF 2006-3505 KWF 2006-3505 8/15/2006 8/15/2010
Peeper, Daniel KWF 2007-3957 Towards improved melanoma treatment: gene
identifcation, in vivo modeling and drug target
discovery
Peeper, Daniel KWF 2009-4552 Dissecting the essential contribution of Fra-1 to human
tumor cell metastasis
Perrakis, Anastassis KWF 2010-4781 Validation of autotaxin, a metastasis-enhancing exophosphodiesterase,
as a therapeutic target
7/1/2008 7/1/2014
1/1/2010 1/1/2014
8/1/2010 8/1/2014
Remeijer, Peter KWF 2008-4024 Image guided radiotherapy for breast cancer 10/20/08 10/20/2012
Rookus, Matti KWF 2007-3756 Heterogeneity of risk of breast and ovarian cancer in
BRCA1 and BRCA2 mutayion carriers
Rottenberg, Sven KWF 2009-4303 Analysis of tumor recurrence in a mouse model for
hereditary breast cancer: how do tumor-initiating cells
escape eradication by chemotherapy
Schagen, Sanne KWF 2007-3797 Late effects of chemotherapy on brain functioning in
the elderly
Schagen, Sanne KWF 2009-4284 Structural, biochemical and functional indices of
chemotherapy-induced cognitive defi cits in cancer
patients
11/15/2007 11/15/2011
10/1/2009 10/1/2013
7/1/2007 6/30/2011
1/1/2010 1/1/2014

Principal
investigator
Number of
project Title Started
Schagen, Sanne KWF 2010-4876 An online testing approach to assess cognitive problems
associated with cancer and cancer treatment
Schinkel, Alfred KWF 2007-3764 OATP1A/1B (SLCO1A/1B) drug uptake transporters in
anticancer drug pharmacokinetics and toxicity risks;
possible strategies for therapy optimization
Schmidt, Marjanka KWF 2007-3839 Genetics determinants of survival and second breast
cancer development in premenopausal breast cancer
patients
187 projects
Ended/
ends
11/1/2010 10/30/2014
12/1/2007 12/1/2011
1/1/2010 1/1/2012
Schmidt, Marjanka KWF 2009-4363 Breast cancer outcome: genetic destiny 7/1/2009 7/1/2015
Schmidt, Marjanka KWF 2009-4535 The Tissue Issue: towards a uniform consent procedure
for research with excised (cancer) tissue
Schumacher, Ton KWF 2006-3530 Generation of human tumor specifi c T cells with high
affi nity T cell receptors
Schumacher, Ton KWF 2007-3825 SNP-based genome-wide identifi cation of
hematopoiesis-restricted minor histocompatibility
antigens
Schumacher, Ton KWF 2009-4282 Preclinical development of TCR gene therapy for
prostate carcinoma
8/1/2010 8/1/2014
12/1/2006 12/1/2010
7/1/2007 7/1/2011
12/1/2009 12/1/2013
Schumacher, Ton KWF 2009-4581 UV-gevoelig MHC peptide uitwisselbare streptameer 7/1/2009 12/1/2010
Sixma, Titia KWF 2006-3476 Structural and functional analysis of a novel E3
ubiquitin ligase consisting of the polycomb proteins
Bmi1 and Ring 1b (RNF2)
8/1/2006 8/1/2011
Sixma, Titia KWF 2008-4014 Comparative study of Ubiqutin-specifi c proteases 2/1/2008 2/1/2012
Sonke, Jan-Jakob KWF 2005-3378 High precision radiotherapy in the presence of
anatomical changes
Sonke, Jan-Jakob KWF 2006-3545 Imaged guided correction strategies to optimize the
precision of radiotherapy
Sonke, Jan-Jakob KWF 2009-4568 Optimization of stereotactic image guided radiotherapy
for lung cancer through detailed toxicity assessment
Sonnenberg, Arnoud KWF 2008-3996 A mouse stem cell model for skin cancer: Dual role of
the integrin a6B4 in tumor initiation and progression
Sonnenberg, Arnoud KWF 2009-4485 Dissection of the regulation of hemidesmosome
disassembly in tumor cell invasion
Stewart, Fiona KWF 2008-3993 The role of endoglin in induction and repair of
cardiovascular damage after radiation alone or in
combinatione with trastuzumab (Herceptin)
Stewart, Fiona KWF 2009-4480 Modulation of vessel repair to prevent radiation-induced
microvascular damage
11/14/2005 11/14/2010
8/1/2006 8/1/2011
1/1/2010 1/1/2014
8/1/2008 8/1/2012
9/1/2010 9/1/2014
6/16/2008 6/16/2012
1/1/2010 1/1/2014
Tan, Bing KWF KWF Ontwikkelingssamenwerkingsprogramma 1/1/2008 1/1/2012

188
projects
Principal
investigator
Number of
project Title Started
Tan, Bing KWF 2008-4233 Early detection of primary and recurrent
nasopharyngeal carcinoma (NPC) using (anti-)
EBV based tumor markers and options for using
photodynamic therapy (PDT) in the treatment of local
disease
te Riele, Hein KWF 2004-3084 Subtle gene modifi cation to study the role of the
mismatch repair complex MSH2/MSH6 in mutation
avoidance and tumor suppression
te Riele, Hein KWF 2006-3589 Signifi cance of the Fanconi anemia caretaker pathway
in development and treatment of cancer
te Riele, Hein KWF 2007-3790 Role of the G2 restriction point in tumor development
and behavior
te Riele, Hein KWF 2009-4477 Characterization of unclassiefi ed allelic variants of DNA
mismatch repair genes to improve genetic counseling
van Blitterswijk,
Wim
van den Brekel,
Michiel
KWF 2005-3377 Lipid rafts as novel targets for anti-cancer therapy and
induction of apoptosis
KWF 2007-3941 Prediction of local control after radiotherapy in head
and neck cancer
van Driel, Willemien KWF 2006-4176 Phase III randomised clinical trial for stage III ovarian
carcinoma randomising between secondary debulking
surgery with or without hyperthermic intraperiotoneal
chemotherapy (OVHIPEC-1)
van Harten, Wim KWF 2010-4854 A-Care 2: ICT supported patient empowerment, returnto-work,
telerehabilitation and implementation of
Cancer Rehabilitation Programs
van Herk, Marcel KWF 2007-3751 High precision image-guided radiotherapy for bladder
cancer
van Herk, Marcel KWF 2007-3895 Probability based treatment planning with biological
objectives for high-dose high-precision radiotherapy of
prostate cancer
van Leeuwen, Fijs KWF 2009-4344 Targeted CXCR4 with multimodal imaging agents as a
means to improve the effi cacy of breast cancer surgery
via combined pre-, intra- and postoperative imaging
van Leeuwen, Floor KWF 2006-3631 Long-term risk of cancer after ovarian stimulation for in
vitro fertilization
van Leeuwen, Floor KWF 2008-3994 Cardiovascular morbidity and mortality in breast cancer
survivors
van Leeuwen, Fred KWF 2009-4511 Role of the histone methyltransferase Dot1 in gene
expression and leukemic transformation
van Lohuizen,
Maarten
KWF 2006-3476 Structural and functional analysis of a novel E3
ubiquitin ligase consisting of the polycomb proteins
Bmi1 and Ring 1b (RNF2)
Ended/
ends
9/1/2008 9/1/2013
11/15/2004 11/14/2010
10/1/2006 6/1/2011
4/1/2007 4/1/2011
2/1/2010 2/1/2014
2/1/2005 2/1/2010
11/1/2007 11/1/2010
3/14/2007 3/14/2010
9/1/2010 9/1/2014
1/1/2008 1/1/2012
11/1/2007 11/1/2011
9/1/2009 9/1/2015
4/1/2007 4/1/2012
10/27/2008 10/27/2012
9/1/2010 9/1/2014
8/1/2006 8/1/2010

Principal
investigator
van Lohuizen,
Maarten
van Lohuizen,
Maarten
van Lohuizen,
Maarten
Number of
project Title Started
KWF 2007-3803 Cancer stem cells and breast cancer: a polycomb
connection
KWF 2007-3877 Epigenetic regulation by the deubiquitinating enzyme
USP3: impact on genome stability and tumorgenesis
KWF 2010-4757 Deciphering the role of Bmi1 and Ezh2 polycomb group
genes in prostate carcinogenesis and metastatis
van ‘t Veer, Laura KWF 2007-3839 Genetics determinants of survival and second breast
cancer development in premenopausal breast cancer
patients
Verheij, Marcel KWF 2007-3939 Improvement of chemoradiation response in head and
neck cancer by (-)Gossypol (AT-101), a small molecule
inhibitor of Bcl-XL/Bcl-2
Verheij, Marcel KWF 2008-4113 Short-chain sphingolipids for enhanced cellular uptake
of amphiphilic anti-cancer drugs
Vyth-Deese, Florry KWF 2006-3606 Integrated analyses of melanoma-T cell interactions in
situ; relevance for immunotherapy
Wolthuis, Rob KWF 2007-3789 Targeting Mitotic Protein Destruction as an Approach
for Cancer Therapy
Wolthuis, Rob KWF 2008-4135 Divergent Control of Cyclin B1-Cdk1 in Cancer Cells: a
Key Role in Therapy
189 projects
Ended/
ends
4/1/2007 4/1/2011
3/1/2007 3/1/2011
11/1/2010 1/5/2013
1/1/2010 1/1/2011
2/1/2008 2/1/2010
11/1/2008 11/1/2012
7/1/2006 7/1/2011
9/1/2007 9/1/2011
9/1/2008 9/1/2012

190
projects
PROJECTS SUPPORTED
BY THE DUTCH CANCER SOCIETY
Projectleider Titel Sponsor Started
Aaronson, Neil Methods and measures for assessing the HRQL of mid to long
term survivors of testicular and prostate cancer
Ended/
ends
EORTC 5/1/2009 7/31/2011
Aaronson, Neil Development of higher order factor structure for the QLQ-C30 EORTC 7/3/2008 12/31/2012
Aaronson, Neil Onco-Move Paces studie IKA 3/1/2010 1/1/2013
Aaronson, Neil Spoed - erfelijkheidsonderzoek bij borstkanker: invloed
op behandelingskeuzes en welbevinden / Behavioral and
psychosocial effects of rapid genetic counseling and testing in
newly diagnosed breast cancer patients: a multicenter study
Aaronson, Neil A-CaRe. 471242 Stichting Achmea
Gezondheidszorg
Agami, Reuven CBG 3e fase: Identifi cation of cancer-relevant genes using a
functional genetic approach.
Agami, Reuven Dr Josef Steiner Cancer Award 2007 Dr. Josef Steiner
Krebsstiftung
Agami, Reuven Indentifying novel regulatory mechanisms of miRNA functions.
ERC
Agami, Reuven Genome wide search for DNA damage checkpoint functions in
human cells (EURYI-award)
Agami, Reuven Combined miRNA profi ling and genetic screens to identify and
characterize cancer-related miRNAs
Agami, Reuven MicroRNAs and RNA Binding Proteins involved in regulation of
KNA damage responses
Beijersbergen,
Roderick
Beijersbergen,
Roderick
Identifi cation of miRNA’s that control response to Trastuzumab
in Breast Cancer.
St Nuts Ohra 8/1/2007 12/31/2012
1/1/2010 4/1/2011
CBG 1/1/2009 1/1/2014
5/1/2007 5/1/2011
EEG-CEC / EU 10/1/2008 10/1/2013
NWO 2/15/2005 2/14/2010
NWO 10/1/2007 10/1/2012
NWO 3/15/2010 3/15/2013
Inte RNA
Technologies BV
12/1/2009 12/1/2010
CSBC-Beijersbergen NWO-ALW 9/1/2010 9/1/2014
Bernards, René CBG 3e fase: Identifi cation of cancer-relevant genes using a
functional genetic approach.
CBG 1/1/2009 12/31/2013
Bernards, René CGC 2008-2012 CGC 1/1/2010 1/1/2013
Bernards, René Breast Cancer Biomarkers and Functional mediators : harnessing
the New Wealth of Omic Data (Target Breast)
EEG-CEC / EU 2/13/2006 2/12/2010
Bernards, René EG Rubicon Annex I EC EEG-CEC / EU 1/1/2010 6/30/2011
Bernards, René ERC Grant 250043- Functional Genomics Dissecting genetic
dependencies in cancer
EEG-CEC / EU 6/1/2010 6/1/2015
Bernards, René System-based predications of responses to cancer therapy NIH 9/1/2005 2/1/2010

Projectleider Titel Sponsor Started
191 1
Ended/
ends
Bernards, René Spinozapremie NWO 9/26/2005 12/31/2012
Bernards, René CSBC-Bernards NWO-ALW 9/1/2010 9/1/2014
Bernards, René Kinases in cancer (TI Pharma) TI Pharma 7/1/2007 7/1/2011
Berns, Ton CBG 3e fase: Identifi cation of cancer-relevant genes using a
functional genetic approach.
CBG 1/1/2009 12/31/2013
Berns, Ton CGC 2008-2012 CGC 1/1/2008 1/1/2012
Berns, Ton Visiting Professor Programme - Kraft KNAW 8/12/2010 11/1/2011
Berns, Ton Merck Res Coll LKR 25006 Merck 2/1/2006 2/1/2010
Berns, Ton Characterizing oncogenes and tumor suppressors by low noise
expression arry datasets.
NWO 1/1/2009 7/1/2010
Berns, Ton Kinases in Cancer (TI Pharma) TI Pharma 7/1/2007 7/1/2012
Borst, Jannie A novel type of ubiquitination regulates apoptosis signaling by
BH3-only protein Bid.
Borst, Jannie Impact of CD27-CD70 co-stimulation on effector development of
CD4 T cells
NWO 11/1/2008 11/1/2012
NWO-ALW 2/15/2010 2/15/2011
Borst, Jannie TNF ligands in cancer TI Pharma 3/1/2006 7/1/2012
Borst, Piet Molecular mechanisms underlying chemotherapy resistance,
therapeutic escape, effi cacy and toxicity (CHEMORES)
Borst, Piet Characterization of the physiological roles of Multidrug
Resistance Protein (MRP) 1-6 by in vivo screening for their
substrates (TOP subsidie)
Brummelkamp,
Thijn
Brummelkamp,
Thijn
EEG-CEC / EU 2/1/2007 2/1/2012
ZonMw 1/1/2008 1/1/2012
CGC 2008-2012 Junior Group Brummelkamp CGC 12/1/2010 12/31/2012
Vidi 91711316 - Tissue size control and the regulation of Hippo
pathway activity in mammals
Burgers, Sjaak Personalized chemo-radiation of lung and head and neck cancer.
WP5: Image-guided adaptive treatment (evaluation during
treatment). CTMM AIR FORCE 03O-103-01, 04 en 05
Collard, John An integrated concept of tumor metastasis: implications for
therapy.
Dalesio, Otilia “eTEN” Programme - Call identifi er: 05/1 ETEN-029334 -
“TENALEA-ID”
ZonMw 1/1/2011 1/1/2016
CTMM 1/1/2009 1/1/2014
EEG-CEC / EU 4/1/2008 4/1/2012
EEG-CEC / EU 11/1/2006 1/1/2011
Dalesio, Otilia Statistical Center NVALT Clinical Trials in oncology NVALT 5/1/2004 1/1/2011
Dannenberg, Jan-
Hermen
Dannenberg, Jan-
Hermen
The role of class I HDACs in normal physiology, tumorigenesis
and transcriptional regulation
Nieuwe Ontwikkelingen: fi nanciering inhuizing groep
Dannenberg op B4
projects
NWO-ALW 8/1/2007 8/1/2012
SFN 7/1/2010 1/1/2011

192
projects
Projectleider Titel Sponsor Started
de Visser, Karin Functional assessment of CSF-1 receptor signalling in de novo
breast cancer development and metastatis information
de Visser, Karin Anti-cancer effi cacy of a murinized antibody against the CSF-1
receptor in a conditional mouse model for de novo invasive and
metastatic breast cancer
de Visser, Karin The infl ammatory tumor microenvironment and its impact on
breast cancer development and therapy
Elsakkers, Peter PSG Detector LSCA Valorisation
Fund
Elsakkers, Peter weerstandverhoging Ministerie van
VROM
Filippo, Ronald ontwikkeling KNGF-standaard Beweeginterventie oncologie Koninklijk
Nederlands
Genootschap voor
Fysiotherapie
(KNGF)
Fornerod,
Maarten
Ended/
ends
AstraZeneca 11/1/2009 7/1/2011
Roche 1/13/2010 1/13/2011
ZonMw 1/1/2009 1/1/2014
7/1/2010 7/1/2012
12/1/2008 12/1/2013
10/1/2009 8/1/2010
Connecting chromatin to the plasma membrane NWO-ALW 8/15/2007 12/15/2010
Gilhuijs, Kenneth Neoadjuvant drug treatment for breast cancer - responseprediction
and response monitoring (BREAST CARE);
SP (sub-workpackage) 8: Multimodality imaging to monitor and
tailor therapy of breast cancer to individual patients
Gilhuijs, Kenneth CTMM Breast Care WP0: Immalytics workstation for fusing of
images
CTMM 10/1/2008 10/1/2013
CTMM 10/1/2008 10/1/2013
Gilhuijs, Kenneth Image guided intervention and therapy IGIT4health SenterNovem 7/17/2009 1/16/2011
Haanen, John Gentherapie met naakt DNA, mogelijkheden voor deregulering Cogem 8/1/2009 2/1/2010
Haanen, John ZonMW 432-00-001 NWO 9/1/2006 7/1/2012
Hilgers, Frans Development and evaluation of a third generation ProvoxTM voice
prosthesis. (ATOS)
ATOS 6/1/2003 1/1/2013
Innocenti, Metello CGC 2008-2012 junior Group Innocenti CGC 5/1/2009 12/31/2012
Jacobs, Heinz Role of translesion DNA synthesis in immunity and cancer
development.
ZonMw 3/1/2005 3/1/2010
Jacobs, Jacqueline NWO Vidi Jacobs NWO 2/1/2007 2/1/2012
Jalink, Kees MEM-FLIM: Modulated Electron-Multiplied all-solid-state camera
for Fluorescence Lifetime Imaging Microscopy
Jonkers, Jos Genome-wide insertional mutagenesis screens to identify
cancer genes that collaborate with E-cadherin loss in mammary
tumourigenesis
Jonkers, Jos Targeting the P13K network in genetically engineered mouse
models of invasive lobular breast cancer
SenterNovem 3/1/2008 3/1/2012
AICR 10/1/2007 10/1/2010
AstraZeneca 11/1/2008 11/1/2012

Projectleider Titel Sponsor Started
Jonkers, Jos Intervention studies with AZD2281, AZD2461 and AZD7762 in
mouse mammary tumor model
Jonkers, Jos Neoadjuvant drug treatment for breast cancer - response
prediction and response monitoring (BREAST CARE);
SP (sub-workpackage) 4: Molecular imaging of PR- or ER-positive
breast cancers using radiolabeled PR- or ER-antagonists
193 1 projects
Ended/
ends
AstraZeneca 1/1/2010 1/1/2013
CTMM 10/1/2008 10/1/2013
Jonkers, Jos Eurosystem EEG-CEC / EU 3/1/2008 3/1/2012
Jonkers, Jos The role of Polycomb genes in normal stem cell and breast cancer
stem cell self-renewal and maintenance
EEG-CEC / EU 3/1/2009 3/1/2011
Jonkers, Jos Merck Res Coll LKR 25006 Merck 1/1/2010 2/1/2010
Jonkers, Jos CSBC-Jonkers NWO-ALW 9/1/2010 9/1/2014
Jonkers, Jos Kinases in cancer (TI Pharma) TI Pharma 7/1/2007 7/1/2012
Jonkers, Jos Ultra-high throughput analysis of insertional mutations to study
collaborating cancer genes and genetic networks in mouse tumors
Kerkhoven, Ron Deep Sequencing project CBG 2009-2013 (onderdeel van het
CBG project)
Klomp, Houke Roche HQ Neo adjuvant Tarceva: Tumor remission rate with
neoadjuvant erlotinib in operable patients with clinical stage I/II
non-small cell lung cancer (NSCLC) (M06NEL)
ZonMw 7/1/2010 7/1/2011
CBG 1/1/2009 1/1/2013
Roche 7/1/2006 1/1/2011
Klomp, Houke Stichting Vrolijk (NSCLC); aanstelling voor arts-onderzoeker. Vrolijk 8/4/2006 7/1/2010
Linn, Sabine Unrestricted research grant for pilot phase of AFTER study
(N08AFT)
Linn, Sabine Translation of a test for BRCAness in breast cancer into a
standardized test ready to use in daily clinical practice
Linn, Sabine Translation of a BAC array-besed chip into an oligo array-based
with chip to identify hereditary breast cancers with BRCA1
mutatioans and sporadic breat cancers with a defi cient BRCA1
singnaling pathway
AstraZeneca 6/1/2008 6/1/2012
LSCA Validation
Funding
LSCA Valorisation
Fund
Linn, Sabine Clinical evaluation of anti-estrogen resistance in breast cancer Pink Ribbon/A
Sister’s Hope
Linn, Sabine Predictive value of a BRCA2-like array comparative genomic
hybridization classifi er to identify breast cancer patients who
benefi t from DNA double strand break-inducing regimens
Michalides, Rob TI Pharma 3.7: Nuclear receptors in targeted cancer therapy:
improved methods for candidate selection
Moolenaar,
Wouter
CBG 3e fase: Identifi cation of cancer-relevant genes using a
functional genetic approach.
Neefjes, Jacques CBG 3e fase: Identifi cation of cancer-relevant genes using a
functional genetic approach.
Pink Ribbon/A
Sister’s Hope
6/1/2010 6/1/2011
7/1/2010 7/1/2012
11/1/2007 1/31/2012
6/1/2010 6/1/2011
TI Pharma 11/1/2006 1/1/2012
CBG 1/1/2009 1/1/2011
CBG 1/1/2009 12/31/2013

194
projects
Projectleider Titel Sponsor Started
Neefjes, Jacques Systems biology of phagosome formation and maturation -
modulation by intracellular pathogens.
Neefjes, Jacques ERC Advanced Grant Grant Agreement Number 249951 ‘MHC
Class II-omics’ Towards understanding and manipulation of MHC
class II antigen presentation.
Neefjes, Jacques ‘MHC Class II-omics’ Towards understanding and manipulation
of MHC class II antigen presentation
Ended/
ends
EEG-CEC / EU 11/1/2008 11/1/2011
EEG-CEC / EU 9/1/2010 9/1/2015
EEG-CEC / EU 9/1/2010 9/1/2015
Neefjes, Jacques NPC E2.5 Kinome and phosphatasome knock-down libraries NPC 1/1/2009 6/30/2013
Neefjes, Jacques Manipulating the MHC class II system to control immune
responses in autoimmunity (TOP subsidieronde 2006-2007)
Neefjes, Jacques The molecular mechanism of the degradation of large molecular
complexes
NWO 10/1/2007 10/1/2012
NWO 1/1/2010 1/1/2013
Neefjes, Jacques Rubicon Subsidie 2010 - R.M. Spaapen NWO 12/1/2010 12/1/2011
Neefjes, Jacques Using high-throughput screening of chemical libraries to identify
factors involved in cross-presentation
Oldenburg,
Hester
NWO-ALW 5/1/2010 5/1/2011
Werkhervatting en seksualiteit na borstkanker Pink Ribbon 9/1/2008 9/1/2011
Ovaa, Huib Design and use of enhanced T cell antigens and T cell detection
technology for human disease
Ovaa, Huib SPORE in Myeloma: Effect of Proteasome Inhibitors on
Immunoproteasome Activity in Multiple Myeloma ( Career
Development Award)
CTMM 10/1/2010 10/1/2014
Dana Farber Cancer
Institute
6/11/2007 2/1/2010
Ovaa, Huib Construction of Well-defi ned Ubiquitin Conjugates SP3 People EEG-CEC / EU 10/15/2008 10/15/2011
Ovaa, Huib From Receptor to Gene: structures of complexes from signalling
pathways linking immunology, neurobiology and cancer (SPINE 2)
Ovaa, Huib Marie Curie FP7-PEOPLE-2009-IEF “Protein Stabilization by
chemistry: total synthesis of an MHC class I”
Ovaa, Huib Grant Agreement Number 261861 FP7-
INFRASTRUCTURES-2010-1 EU-OPENSCREEN Europenan
Infrastructure of Open Screening Platforms for Chemical BIology
Ovaa, Huib FP7-INFRASTRUCTURES-2010-1 EU-OPENSCREEN Europenan
Infrastructure of Open Screening Platforms for Chemical Biology
Ovaa, Huib Commercial development of ubiquitinated peptides and screening
assays produced by high throughput robotic peptide synthesis
Ovaa, Huib Combinatorial unnatural epitope libraries coupled with MHC
exchangeligand enrichment for rapid defi nition of T-cell vaccine
canidates
EEG-CEC / EU 1/1/2010 7/1/2010
EEG-CEC / EU 10/1/2010 10/1/2012
EEG-CEC / EU 11/1/2010 11/1/2013
EEG-CEC / EU
NGI 1/1/2010 1/1/2012
NGI 8/1/2010 8/1/2011
Ovaa, Huib NPC E2.1 Tyrosine phosphatase inhitors, baits and ABPs NPC 1/1/2009 6/30/2013

Projectleider Titel Sponsor Started
Ovaa, Huib NPC V1 Commercial development of synthetic Nedd8 and SUMO
conjugates
195 1
Ended/
ends
NPC 1/1/2009 7/1/2013
Ovaa, Huib Chemical biology of ubiquitin-like modifi cations NWO 9/1/2005 9/1/2010
Ovaa, Huib Modulation of autotaxin activity by small molecules NWO 11/1/2006 4/1/2010
Ovaa, Huib Development of conditional protein-ligand exchange applied to
immune technology; Onderzoeksprogramma: Integration of
Biosynthesis & Organic Synthesis (IBOS)
Ovaa, Huib How proteoglycan receptors affect antigen cross-presentation and
immunity
Ovaa, Huib Pan- Dub Inhibitors to delineate DUB dependent biology and tools
to study the ubiqultinated proteome
NWO 9/1/2008 9/1/2012
NWO 10/1/2010 10/1/2013
NWO Chemische
Wetenschappen
8/1/2009 8/1/2013
Ovaa, Huib Post-translational transpeptidation and immunity NWO-ALW 10/1/2009 10/1/2012
Ovaa, Huib Large scale production 4-thiolysine to enable commercial
production of ubiquitin and ubiquitin-like conjugates
STW 9/1/2010 3/1/2011
Ovaa, Huib Probing the role of adjuvants in MHC-II antigen presentation Wellcome trust 5/1/2009 10/1/2010
Ovaa, Huib Immunoproteomics: antigen mapping through MHC epitope
exchange reactions
ZonMw 1/1/2009 7/1/2010
Ovaa, Huib Druggable DUBs in trypanosomes ZonMw 8/1/2010 10/1/2011
Peeper, Daniel Synthetic lethality: a novel tactic for selective anticancer drug
target discovery
NWO 6/1/2007 6/1/2012
Peeper, Daniel The role of oncogene-induced senescene in long-latency leukemia Overig 6/30/2010 6/30/2014
Perrakis,
Anastassis
Perrakis,
Anastassis
Perrakis,
Anastassis
Perrakis,
Anastassis
Perrakis,
Anastassis
A multidisciplinary approach to determine the structures of
protein complexes in a model organism.
From Receptor to Gene: structures of complexes from signalling
pathways linking immunology, neurobiology and cancer (SPINE 2)
EEG-CEC / EU 2/1/2005 2/1/2010
EEG-CEC / EU 1/1/2010 7/1/2010
NIH grant 2R01 GM062612-05, EMBL Heidelberg NIH 5/1/2006 5/1/2010
The NKI Protein Facility: Indentifi cation, Production,
Characterization and interactions
NWO 1/1/2009 12/31/2010
Cristallization of Autotaxin 2230 Pfi zer 6/3/2009 6/30/2011
Peters, Peter Public Private Partnership for research into and development of
medicines, vaccines and diagnostic aids in the domain of AIDS,
tuberculosis and malaria
Peters, Peter Understanding prion strains and species barriers and devising
novel diagnostic approaches (strainbarrier)
Peters, Peter Protecting the food chain form prions: shaping European
priorities through basic and applied research (KP 7)
Aeras Global TB
Vaccine Foundation
projects
1/1/2007 12/31/2010
EEG-CEC / EU 11/1/2006 5/1/2010
EEG-CEC / EU 10/1/2009 10/1/2013

196
projects
Projectleider Titel Sponsor Started
Ended/
ends
Peters, Peter FP7 NOTOX Project EEG-CEC / EU 11/1/2010 11/1/2014
Peters, Peter E-cadhering and cadhering-11 and their role in cancer migration”
binnen het SmartMix programma NIMIC.
MIN OCW 1/1/2009 7/1/2013
Peters, Peter Pathways of antigen presentation by CD 1 a, b and c NIH 6/15/2005 2/28/2010
Peters, Peter Pathways of Antigen Presentation by CD1 NIH 7/1/2010 6/30/2011
Peters, Peter HTS&M STW 7/1/2010 7/1/2014
Peters, Peter Financiering Ulrike Zeisse door TU-Delft Tu Delft 1/1/2010 12/31/2010
Rodenhuis, Sjoerd Neoadjuvant drug treatment for breast cancer - response
prediction and response monitoring (BREAST CARE); SP (subworkpackage)
5: Biomarker discovery and validation in patients
Rookus, Matti Plating of 10.000 DNA samples of women tested for BRCA 1/2
mutations
Rookus, Matti Website voor families met een hoog risico op borst- en/of
ovariumkanker
Rottenberg, Sven Neoadjuvant drug treatment for breast cancer - response
prediction and response monitoring (BREAST CARE); SP (subworkpackage)
1: Response biomarker discovery for Cisplatin and
Docetaxel in mouse models of breast cancer
Ruers, Theo Research Collaboration Agreement for Data Collection - Ex-vivo
human tissue study
CTMM 10/1/2008 10/1/2013
BBMRI-NL 9/1/2010 9/1/2011
Pink Ribbon 9/1/2010 9/1/2012
CTMM 10/1/2008 10/1/2013
Philips 10/12/2009 1/12/2010
Ruers, Theo Research collaboration Philips Philips 4/1/2010 6/1/2011
Schagen, Sanne Imaging of cognitive dysfunction in testicular and breast cancer
survivors
Schagen, Sanne Detailed neurocognitive assessment of phophylactic cranial
irradiation for lung cancer patients
Schagen, Sanne Assessment of neuropsychological sequelae of tamoxifen and
exemestane in postmenopausal women with early breast cancer.
TEAM-neuropsychologiestudie
Schellens, Jan Preclinical in vivo testing of AZD1152 to determine the effect of
the ABC-drug transporters P-glycoprotein (Pgp, Mdr1) and BCRP
(Bcrp1, Abcg2) on tissue distribution and clearance of AZD1152.
Schellens, Jan Veiligheid- & kostenbesparingstudie voor DPYD2A genotypering
bij fl uoropyrimidinetherapie. Fonds NutsOhra project
SNO-T-0701-103.
AMC: Beleids OIO 7/1/2010 6/30/2014
Nuts Ohra 7/1/2010 6/30/2014
Pfi zer 6/1/2003 1/1/2009
Astra 9/1/2007 7/1/2011
Fonds NutsOhra 1/1/2008 4/1/2010
Schellens, Jan Merck OncoNet Member Agreement Merck 12/1/2009 12/1/2014
Schellens, Jan Support Roche clinical research associate Candersartan study
M06HER
Schmidt,
Marjanka
Complementatieproject Gene-’environment’ interactions in breast
cancer developments and prognosis
Roche 2/1/2009 1/1/2012
BBMRI-NL 6/15/2010 12/1/2011

Projectleider Titel Sponsor Started
Schmidt,
Marjanka
Complementatieproject Breast cancer risk polymorphisms in
familial, non-BCRA 1/2 breast cancer patients
197 1
Ended/
ends
BBMRI-NL 12/1/2010 12/1/2011
Schoo, Hans Congres : Perspectives of Immunotherapy in GI cancer EORTC 7/1/2009 9/1/2010
Schumacher, Ton Development of conditional protein-ligand exchange applied to
immune technology
Schumacher, Ton Development and clinical evaluation of new strategies for adoptive
cell transfer (ACT) in the treatment of cancer.
Schumacher, Ton Design and use of enhanced T cell antigens and T cell detection
technology for human disease
Schumacher, Ton Attack: Adoptive engineered T-cell targeting to activate cancer
killing
Schumacher, Ton ATTRACT - Advanced Teaching and TRaining for Adoptive Cell
Therapy. FP7. Marie Curie
Schumacher, Ton SPHINX - Spontaneous clearance in Patients acutely infected
with HCV - Immune profi ling, Novel biomarkers and X-omics
approaches
Schumacher, Ton Graft versus Host disease by TCR-engineered T cells: mechanisms
and means for prevention. LSBR. Dossiernr 0804.
Schumacher, Ton Career Development Program. The Leukemia & Lymphoma
Society
ACTS (NWO) 9/1/2008 9/1/2012
Centre for Cancer
Immune Therapy
(CCIT)
1/1/2009 1/1/2014
CTMM 10/1/2010 10/1/2014
EEG-CEC / EU 11/1/2005 11/1/2010
EEG-CEC / EU 10/1/2009 10/1/2013
EEG-CEC / EU 10/15/2010 10/15/2014
Landsteiner
Stichting
Schumacher, Ton Platform For MHC-Exchange Based T-Cell therapy For Melanoma. Melanoma Research
Alliance
1/1/2009 1/1/2012
LLS 7/1/2010 7/1/2013
9/1/2008 9/1/2010
Schumacher, Ton Dissecting metabolic T-Cell dysfunction in cancer NWO 2/1/2009 2/1/2010
Schumacher, Ton T cell receptor gene therapy of metastatic melanoma: a phase I
clinical trial.
ZonMw 4/1/2005 2/1/2013
Schumacher, Ton Intravital imaging and computational modeling of skin immunity ZonMw 3/1/2011 1/1/2015
Sixma, Titia CBG 3e fase: Identifi cation of cancer-relevant genes using a
functional genetic approach.
Sixma, Titia MCRTN Ubiregulators: Signal Transduction by Ubiquitination, a
Matter of Location
Sixma, Titia Neurotransmitter Cys-loop recptors: structure, function and
disease.
Sixma, Titia Mismatch2model-Characterization and quantitative modeling of
DNA mismatch repair and its role in the maintenance of genomic
stability and cancer avoidance.
projects
CBG 1/1/2009 1/1/2014
EEG-CEC / EU 1/1/2007 1/1/2011
EEG-CEC / EU 2/1/2008 2/1/2012
EEG-CEC / EU 11/1/2008 5/1/2013
Sixma, Titia EG Rubicon Annex I EC EEG-CEC / EU 1/1/2010 6/30/2011
Sixma, Titia The balance of ubiquitin conjugation and deconjugation EEG-CEC / EU 2/1/2010 2/1/2015

198
projects
projects
Projectleider Titel Sponsor Started
Sixma, Titia The regulatory role of Ubl domains in ubiquitin specifi c protease
function
Sixma, Titia New approaches for Ligand-Gated Ion Channel (LGIC) drug
discovery. TI Pharma D2-103
Sonke, Jan-Jakob Neoadjuvant drug treatment for breast cancer - response
prediction and response monitoring (BREAST CARE); SP (subworkpackage)
9: Image guided radiation therapy
Sonke, Jan-Jakob HYPERImage: hybrid PET-MT system for concurrent ultrasensitive
imaging
Sonnenberg,
Arnoud
Sonnenberg,
Arnoud
Sonnenberg,
Arnoud
Function of tetraspanin-integrin complex Cd151- a3B1 in
development and maintenance of the glomerular fi ltration barrier.
NSN project.
Controlling hemidesmosome dynamics by phosphorylation of
residues on the integrin b4 subunit.
NWO Chemische
Wetenschappen
Ended/
ends
10/1/2010 10/1/2015
TI Pharma 1/1/2007 7/1/2011
CTMM 10/1/2008 10/1/2013
EEG-CEC / EU 4/1/2008 4/1/2011
nierstichting 10/1/2008 10/1/2012
NWO-ALW 3/1/2007 4/1/2011
De titel van het project komt nog wanneer het contract klaar is. STW 6/1/2009 6/1/2012
Stewart, Fiona Cardiorisk - The mechanisms of cardiovascular risks after low
radiation doses
Stroom, Joep Accurate target defi nition of non-small cell lung tumors using
pathology-validated PET and CT imaging for the optimization of
radiotion treatment.
EEG-CEC / EU 2/1/2008 7/1/2011
Maastro 9/1/2006 12/31/2010
Tan, Bing The cost-effectiveness study/ZonMw project Biolitec-NKI Biolitec 12/1/2008 1/1/2012
Tan, Bing A multi-centre cost-effectiveness evaluation of a novel treatment
option in the Netherlands: Photo Dynamic Therapy with
temoporfi ne for the treatment of advanced incurable head and
neck cancers, for whom prior conventional treatments have failed.
te Riele, Hein Modifi ers of tumor development in Lynch syndrome MAAG LEVER
DARM
te Riele, Hein Subtle is the oligo: in vitro and in vivo gene modifi cation by singlestranded
DNA oligonucleotides Horizon 050-71-051
te Riele, Hein A novel procedure for enzymatic production of low-complexity
RNAI Libraries to identify coding- and non-coding turmor
suppressor genes
Valdes Olmos,
Renato
Mammography with molecular imaging (MAMMI) Specifi c
Targeted project
ZonMw 3/1/2009 3/1/2012
2/1/2010 2/1/2012
NWO 12/1/2006 12/1/2011
NWO-ALW 8/1/2009 8/1/2012
EEG-CEC / EU 1/1/2007 1/1/2011
van Boven, Hester Neoadjuvante registratiestudie BOOG Astra 1/1/2006 1/1/2013
van de Vijver,
Marc
Neoadjuvant drug treatment for breast cancer - response
prediction and response monitoring (BREAST CARE); SP (subworkpackage)
7: Identifi cation and validation of predictive markers
for drug response in metastatic breast cancer
CTMM 10/1/2008 10/1/2013

Projectleider Titel Sponsor Started
van den Brekel,
Michiel
van der Poel,
Henk
199 1 projects
Ended/
ends
Fanconi anemia pathway defects in head and neck cancer. Verwelius BV 7/1/2010 7/1/2013
Prostate Cancer Molecular Medicine (PCCM) CTMM 12/1/2009 12/1/2014
van Herk, Marcel CTMM AIR FORCE WP3: Development and validation of PET-CT
imaging tools: software.
van Herk, Marcel CTMM AIR FORCE WP6: Data integration “Oncology Research
Workstation” (ORW)
van Herk, Marcel EOS EOS / voorheen
Philips
van Leeuwen,
Fijs
van Leeuwen,
Fijs
van Leeuwen,
Fijs
van Leeuwen,
Flora
van Leeuwen,
Flora
van Leeuwen,
Flora
van Leeuwen,
Flora
van Leeuwen,
Flora
van Leeuwen,
Flora
van Leeuwen,
Flora
van Leeuwen,
Flora
van Leeuwen,
Flora
van Leeuwen,
Flora
van Leeuwen,
Flora
Neoadjuvant drug treatment for breast cancer-response-prediction
and response monitoring (BREAST CARE). SP (sub-workpackage)
3: Improved assays for analysis of multidrug resistance transporter
activity
Intra-operative Multi-Spectral Imaging Systems for radical tumor
resection (MUSIS)
HYPERImage: Hybrid PET-MR system for concurrent ultrasensitive
imaging
CTMM 9/1/2008 9/1/2013
CTMM 12/1/2008 7/1/2013
5/1/2000 5/1/2010
CTMM 10/1/2008 10/1/2013
CTMM 4/1/2010 4/1/2014
EEG-CEC / EU 4/1/2008 4/1/2011
Heart-breast and heart-Hodgkin case-control studies CTSU 11/1/2009 1/1/2012
Collaborative Oncological Gene-environment Study EEG-CEC / EU 5/1/2009 5/1/2013
Bijdrage LVN Project BETER LVN 7/1/2010 7/1/2011
Risk and prognosis of uterine corpus cancer after tamoxifen
treatment for breast
Prediction Model: Breast cancer in women irradiated for a
pediatric malignancy.
Shift work and risk of breast cancer, a prospective cohort study
among Dutch nurses
Van Vlissingenfonds: polikliniek overlevers Hodgkin Vlissingen
Lymfoomfonds
HEBON research facility for individuals tested for BRCA 1/2
mutations
Cardiovascular disease (CVD) after breast cancer and CVD after
Hodgkin case-control studies
EG GENE-Rad-Risk: Radiation exposures at an early age: impact of
genotype on breast cancer risk.
Screening van overlevenden van Hodgking Lymfoom Lymfklierkanker
Vereniging
projects
NIH 9/25/2007 9/1/2010
NIH 4/1/2010 1/31/2011
Universiteit Utrecht 9/1/2010 9/1/2014
8/1/2009 8/1/2012
ZonMw 10/23/2009 10/23/2013
CTSU 1/1/2010 1/1/2012
EEG-CEC / EU 6/1/2005 11/30/2010
7/1/2010 7/1/2011

200
projects
Projectleider Titel Sponsor Started
van Leeuwen,
Flora
van Leeuwen,
Flora
van Leeuwen,
Flora
van Leeuwen,
Fred
van Leeuwen,
Fred
van Leeuwen,
Fred
van Lohuizen,
Maarten
van Lohuizen,
Maarten
van Lohuizen,
Maarten
van Lohuizen,
Maarten
van Lohuizen,
Maarten
van Lohuizen,
Maarten
van Lohuizen,
Maarten
van Lohuizen,
Maarten
van Lohuizen,
Maarten
van Sandick,
Johanna
van Sandick,
Johanna
Voorlichting over en screening op mammacarcinoom bij jonge
vrouwen die met radfi otherapie zijn behandeld voor Hodgkin
lymfoom
Cardiovascular morbidity in testicular cancer survivors:
case-control study of risk factors and assessment of
pharmacogenomic determinants of toxicity
Van Vlissingenfonds: polikliniek overlevenden
Hodgkin Lymfoom
Ended/
ends
Pink Ribbon 12/1/3010 11/30/2014
UMCG 2/18/2005 7/1/2010
Vlissingen
Lymfoomfonds
8/1/2009 8/1/2012
NPC T1.4 Chromatin dynamics and epigenetic changes NPC 1/1/2009 7/1/2013
The role of histone methylation in silencing and transcriptional
memory
A multifunctional genome-wide screen to unravel the roles
of the conserved epigenetic regulator Dot1 in chromosome
organization (ECHO subsidieronde 2006-2007)
CBG 3e fase: Identifi cation of cancer-relevant genes using a
functional genetic approach
NWO 2/1/2005 1/19/2011
NWO 11/16/2007 9/1/2010
CBG 1/1/2009 1/1/2014
CGC 2008-2012 CGC 10/1/2010 1/1/2013
Platforms for biomedical discovery with human ES cells. EEG-CEC / EU 8/1/2006 8/1/2010
Eurosystem EEG-CEC / EU 3/1/2008 3/1/2012
Functional genetic characterization of a mouse model of Glioma
(FCGCMOG)
EEG-CEC / EU 8/1/2010 8/1/2012
Merck LKR #40778 PO X21000363. Merck 6/2/2006 2/1/2012
NPC T4.2 Properties of pluripotency in mouse ESCs and iPS NPC 1/1/2009 6/30/2013
Genome-wide Spatial Profi ling of Polycomb Domains in
Drosophila melanogaster
Maintenance of genome integrity by histone (de)ubiquitinating
enzymes.
NWO-ALW 12/1/2007 12/1/2010
NWO-ALW 11/1/2008 11/1/2013
Slokdarmkankeronderzoek Stichting Vrolijk Vrolijk 1/1/2008 5/1/2013
Lab slokdarmkankeronderzoek Stichting Vrolijk Vrolijk 9/1/2009 1/1/2011
van Steensel, Bas Multi-gene chromatin domains NGI 12/1/2006 12/1/2010
van Steensel, Bas Chromatin Protein Discovery Project NGI 2/1/2008 2/1/2012

Projectleider Titel Sponsor Started
van Steensel, Bas Whole-genome analysis of the regulation of chromatin structure
and gene expression
201 2
Ended/
ends
NWO 3/1/2005 3/1/2010
van Steensel, Bas Nuclear lamina genome interactions in mammalian cells NWO-ALW 7/1/2009 7/1/2014
van ‘t Veer, Laura BBMRI NL Project CP45 BBMRI-NL 10/1/2010 10/1/2011
van ‘t Veer, Laura CGC 2008-2012 CGC 1/1/2010 1/1/2013
van ‘t Veer, Laura Translating molecular knowlegde into early breast cancer
management: building on the BIG (Breast International Group)
network for improved treatment tailoring. TRANSBIG
EEG-CEC / EU 3/1/2004 3/1/2011
van ‘t Veer, Laura Collaborative Oncological Gene-environment Study EEG-CEC / EU 5/1/2009 5/1/2013
van ‘t Veer, Laura Breast Cancer Somatic Genetics Study Grant agreement no.:
242006
EEG-CEC / EU 7/1/2010 7/1/2014
van ‘t Veer, Laura Predicting drug responses in breast cancer. TI Pharma T3-108 TI Pharma 7/1/2007 7/1/2011
Verhoef, Koen 371 BAC CGH BRCA1 classifi er for predicting response CGC 10/1/2010 1/1/2012
Wessels, Lodewyk Pathway-based tumour characterisation & alignment of cell
panels against tumour samples
Wessels, Lodewyk Neoadjuvant drug treatment for breast cancer - responseprediction
and response monitoring (breast care);
SP (sub-workpackage) 10: Biomarkers informatics
AstraZeneca 2/1/2010 2/1/2012
CTMM 10/1/2008 10/1/2013
Wessels, Lodewyk Collaborative Oncological Gene-environment Study EEG-CEC / EU 5/1/2009 5/1/2013
Wessels, Lodewyk NBIC / BioAssist / HTHC Programmeur NBIC 1/1/2008 1/1/2011
Wessels, Lodewyk NBIC-II BioRange BR4.9 NBIC 10/1/2009 12/31/2012
Wessels, Lodewyk NCSB AddOn projecten CGC en CMSB NGI 11/1/2010 11/1/2013
Wessels, Lodewyk CSBC-Wessels NWO-ALW 9/1/2010 9/1/2014
Wessels, Lodewyk Signaling pathways and gene regulatory networks responsible for
th17 cell differentiation
Wolthuis, Rob Developing Cell Cycle Profi les to Classify Responses and
Regulators
projects
ZonMw 5/1/2010 5/1/2013
H.F.S.P.O. 10/1/2010 10/1/2013
Wolthuis, Rob Coordination of cell division by regulated protein destruction. NWO-ALW 1/1/2007 12/31/2011

202
personnel index
PERSONNEL INDEX
Aalbers, Arend 147
Aaronson, Neil 92
Aarts, Marieke 61
Abdallah, Abdallah 34
Abrao Possik, Patricia 83
Achachah, Mohamed 100
Ackerstaff, Annemiek 148
Adriaansz, Sandra 115
Agami, Reuven 47
Aguilar, Helena 75
Ait Moha, Daoud 100
Ajouaou, Abderrahim 100
Akthar, Waseem 79
Albers, Harald 32
Alderden, Carolien 115
Alderliesten, Maaike 24
Alderliesten, Tanja 124
Aleman, Berthe 88, 124
Alendar, Andrej 81
Alexi, Xanthippi 31
Amendola, Mario 49
Amore, Alessia 32
Angelopoulos, Nicos 71
Anirudhan, Gireesh 83
Appelman, Jolanda 162
Appels, Marije 114
Arens, Robin 162
Argenzio, Elisabetta 24
Ariotti, Slivia 57
Aukema, Tjeerd 99, 147
Baank, Saskia 99
Baars, Danny 162
Baars, Joke 114
Baars, Philippe 99
Baas, Paul 38, 114
Baas-Vrancken Peeters, Marie-Jeanne 147
Badhai, Jitendra 81
Bakker, Jeroen 29
Bakker, Martine 99
Bakker, Sandra 114
Bakker, Sietske 61
Balague Ponz, Olga 99
Balm, Alfons 148
Banishki, Nikola 63
Barbier, Nathalie 162
Bartelink, Harry 124
Batteau, Lukas 101
Beaudry, Jean Bernard 79
Beekman, Chantal 99, 101
Beelen, Karin 67, 114
Beers-Bauhuis, Carolien 99
Begg, Adrian 36
Beijersbergen, Roderick 75
Beijnen, Jos 43, 114
Belderbos, José 94, 124
Bendle, Gavin 55
Benraadt, Tinie 162
Bentin Toaldo, Cristiane 31
Bergman, André 114
Berkers, Celia 32
Bernards, René 73
Berns, Anton 81
Berns, Katrien 73
Besnard, Peter 100
Bessels, Frauwkje 162
Betgen, Anja 125
Bex, Axel 148
Bies, Laura 55
Biewenga, Petra 148
Bijos, Dominika 49
Bin Ali, Rahmen 81
Blank, Christian 60, 114
Blanken, Roel 115
Bleiker, Eveline 96, 100
Blitz, Johanna 81
Bloemendal, Karen 147
Bloemer, Martine 147
Bloemers, Monique 124
Blom, Marie-José 88
Blom, Marleen 79
Boekel, Naomi 88
Boekhout, Annelies 115
Boekhout, Michiel 77
Boele, Florien 94
Boer, Mandy 85
Boerrigter-Barendsen, Lucie 100
Boessenkool-Pape, Juliet 88
Bonarius, Marja 114
Bonfrer, Hans 99
Boogerd, Willem 94, 114
Boon, Ute 65
Boot, Henk 114
Borgemeester, Maarten 148
Borst, Gerben 124
Borst, Jannie 53
Borst, Piet 63
Bos, Arnold 47
Bos, Melanie 114
Boshuizen, Rogier 114
Bosma, Astrid 41, 73
Boss, David 114
Bot, Ilja 114
Botma, Henk 162
Boucher, Audi 162
Boutmy-de Lange, Majella 100
Bouwman, Peter 65
Braaf, Linde 41
Brandsma, Dieta 114
Brandwijk, Kim 41
Braumuller, Tanya 65
Braunschweig, Ulrich 49
Broeks, Annegien 41
Brohet, Richard 88
Brood, Monique 148
Brouwer, Oscar 99
Brouwers, Elke 114
Bruin, Natascha 99
Bruin, Sjoerd 41, 147
Bucholtz, Karina 115
Buckle, Tessa 99, 101
Buikhuisen, Wieneke 114
Buitelaar, Dirk 149
Buning-Kager, Marian 99
Bunschoten, Anton 99, 100
Burgers, Sjaak 114
Burylo, Artur 43
Caillat, Christophe 20
Canisius, Sander 71
Carreno, Monique 162
Cats, Annemieke 96, 114
Chen, Chun 124
Chen, Wei 101
Chin, Patrick 99, 100
Ciampricotti, Metamia 65
Citterio, Elisabetta 79
Claassen, Jolien 162
Clapham, Renee 148
Clerici, Marcello 18
Clijsters, Linda 77
Collard, John 26
Cooke, Saskia 162
Coquet, Jonathan 53
Cornelissen, Paulien 79
Cornelissen, Sten 41
Courrech Staal, Ewout 147
Dalesio, Otilia 162
Damen, Eugène 124
Dannenberg, Jan-Hermen 52
de Backer, Ralph 114
de Boer, Jan Paul 114
de Boer, Roel 124
de Bois, Josien 125
de Groot, Dirk-Jan 22
de Haas, Marcel 63
de Jong, Annemieke 32
de Jong, Daphne 99
de Jong, Gerda 162
de Jong, Johann 71
de Jong, Monique 36, 124
de Jong, Rianne 125
de Jong, Trees 57
de Lange, Judith 88
de Leeuw, Renée 31
de Lint, Klaas 75
de Punder, Karin 34
de Ridder, Jeroen 71
de Ronde, Jorma 45, 71
de Ruiter, Michiel 94
de Visser, Karin 65
de Vreeze, Ronald 147
de Vries, Evert 53

de Vries, Hilda 81
de Vries, Nienke 79
de Vries, Sandra 61
de Vries, Sebastiaan 162
de Waal, Marjolijn 162
Deenen, Maarten 43, 115
Dekker, Marleen 61
Dekker, Rob 61
Delzenne-Goette, Elly 61
Devriese, Lot 43, 115
Dewit, Luc 124
Didraga, Mihaela 41
Diemer, Frederiek 88
Dirac, Annette 73
Donker, Mila 147
Doodeman, Barry 125
Doornebal, Chris 65
Dorlo, Thomas 115
Douma, Sirith 83
Doumont, Gilles 65
Drost, Brigitte 149
Drost, Jarno 47
Drost, Rinske 65
Dubbelman, Anne Charlotte 115
Dubbelman, Ria 115
Dufournij, Brigitte 162
Duijts, Saskia 92
Duppen, Joop 125
Durmus, Selvi 69
Efthymiou, Katina 149
Eijzenga, Willem 92, 96
Eilers, Marlou 162
Ekhart, Corinne 115
Ekkebus, Reggy 32
El Atmioui, Dris 32
El Oualid, Farid 32
Elias, Sjoerd 41
Elkhuizen, Paula 124
Elkon, Rani 47
Ellenbroek, Saskia 26
Elouarrat, Dalila 24
Elshof, Lotte 101
Emmering, Jasper 99
Engelhardt, Ellen 88
Evers, Danny 147
Fabius, Armida 75
Faesen, Alex 18
Feenstra, Christel 99
Fernandez Salcedo, Ernesto 162
Filion, Guillaume 49
Fish, Alex 18
Fles, Renske 148
Floot, Ben 38
Foekema, Joke 115
Frederix, Geert 43, 115
Frijns, Evelyne 22
Gadiot, Jules 60
Gallenne, Tristan 83
Galovic, Magda 27
Gargiulo, Gaetano 79
Garstka, Gosia 29
Gasparini, Alessia 124
Gebretensae, Abadi 115
Gehring, Karin 92
Genest, Paul-André 86
Gennissen, Annemiek 73
Gerlach, Carmen 55
Gerritsen, Bram 71, 75
Gerritsma, Miranda 92
Geumann, Ulf 53
Geurink, Paul 32
Geurts, Tom 148
Geutjes, Ernst-Jan 73
Gilhuijs, Kenneth 100
Godsave, Sue 34
Goey, Andrew 115
Gomez Benito, Maria 47
Gomez, Raquel 57
Graafl and, Niels 148
Greig, Kylie 83
Grernrum, Wipawadee 73
Groen, Wim 98
Groenendijk, Floris 73
Groot, Harmke 88
Groothuizen, Flora 18
Guislain, Aurelie 60
Gundy, Chad 92, 94, 96
Guyader, Charlotte 63
Haanen, John 57, 114
Haas, Rick 124
Hage, J Joris 149
Hagenaars, Christiane 162
Hahn, Christoph 149
Hahn, Daniela 96, 100
Halonen, Pasi 75
Hameed, Dharjath 32
Hamming-Vrieze, Olga 124
Haramis, Anna-Pavlina 87
Haringhuizen, Annebeth 114
Harms, Emmy 115
Harrick, Mieke 124
Hau, Song-Hieng 162
Hau, Tissee 65
Hauptmann, Michael 71
Haussman, Jens 20
He, Youji 41
Heemsbergen, Wilma 71, 124
Heemskerk, Bianca 57
Heidebrecht, Tatjana 20
Heideman, Richard 52, 59
Heijens, Claudia 115
Helgason, Helgi 115
Henneman, Linda 28
Hennink, Roos 162
Heynen, Guus 73
Hibbert, Rick 18
Hiemstra, Annelies 162
Hijmans, Marielle 73
Hilarius, Doranne 92
Hilgers, Frans 148
Hilkens, John 85
Hilkmann, Henk 32
Hillebrand, Michel 115
Hoebers, Frank 124
Hoefnagel, Cornelis 99
Hoekstra, Paula 162
Hofl and, Ingrid 36
Hogenbirk, Marc 59
Hogervorst, Frans
Hogervorst, Frans 41, 88, 99, 100
Holland, Carlijn 148
Hollenstein, Andreas 55
Hollmann, Birgit 124
Holstege, Henne 65
Holtkamp, Marjo 115
Hölzel, Michael 73
Hömig, Cornelia 83
Hompes, Daphne 147
Honnef, Joeri 125
Hoogenboom, Martijn 99
Hooijkaas, Anna 60
Hoopman, Rianne 92
Hoppes, Rieuwert 32
Horenblas, Simon 148
Houben, Anna 24
Houben, Diane 34
Houlleberghs, Hellen 61
Hoving, Saske 38, 100
Huang, Sidong 73
Huijbers, Ivo 81
Huissoon, Sandra 149
Huitema, Alwin 114
Hulshoff, Lenie 149
Hulsman, Danielle 79
Hummel, MJM 98
Iden, Sandra 26
Ikink, Gerjon 85
Innocenti, Metello 27
Iskit, Sedef 83
Isogai, Tadamoto 27
Iusuf, Dilek 69
Ivanov, Eduard 88, 162
Jacobi, Irene 148
Jacobs, Heinz 59
Jacobs, Jacqueline 86
Jalink, Kees 28
Jan, Sabrina 63
Jansen, Edwin 124
Jansen, Hans 34
Jansen, Robert 63
Janssen, Esther 88
Janssen, Lennert 29
Janssen, Luuk 148
Janssens, Tine 115
Jaspers, Janneke 65
Jeanson, Kiki 88
Jenal, Mathias 47
Jibodh, Aarti 162
Jong, Eefje 115
Jongsma, Maikel 24
Jongsma, Marlieke 29
Jonker, Marcel 125
Jonkers, Irene 162
203
personnel index

204
personnel index
Jonkers, Jos 45, 65
Jonkman, Joop 162
Joosten, Krista 20
Joosten, Robbie 20
Jorritsma, Annelies 57
Kaiser, Andrew 60
Kalisvaart, Robin 125
Kant, Josien 162
Kaplon, Joanna 83
Karakullukcu, Baris 148
Kasiem, Mobien 100
Kedde, Martijn 47
Kedziora, Kasia 28
Keessen, Marianne 115
Keizer, Ron 115
Kemperman, Myrle 94
Kenter, Gemma 148
Kersbergen, Ariena 63
Kerst, Martijn 114
Kersten, Kim 88
Kerver, Emile 114
Ketema, Mirjam 22
Kind, Jop 49
Klarenbeek, Jeffrey 28
Klarenbeek, Sjoerd 65
Klaver, Yvonne 147
Klijn, Christiaan 65, 71
Klinkenberg, Astrid 94
Klomp, Houke 147
Klop, Martin 148
Klous, Marjolein 114
Klümpen, Heinz Josef 115
Kluijt, Irma 96, 100
Knegjens, Joost 124
Knol, Cora 88
Knols, Ruud 92
Koenen, Annemieke 115
Kolmschate, Lies 162
Kolodziej, Katy 49
Koning, Gerben 40
Kool, Jaap 79
Koolen, Stijn 115
Koopman-Kroon, Ciska 115
Koops, Wim 100
Koornstra, Rutger 67
Koppelmans, Vincent 94
Koppens, Martijn 79
Korse, Tiny 99
Koudijs, Marco 65
Kranenburg, Andor 81
Krap, Menno 148
Kreeft, Annemarijn 148
Kreft, Maaike 22
Kregel, Eva 65
Krewinkel, Han 124
Krijger, Peter 59
Krimpenfort, Paul 81
Kristel, Petra 45
Kröger, Robert 100
Krumpochova, Petra 63
Kuenen, Marianne 88
Kuenen, Marianne 92, 94
Kuijl, Coen 29
Kuijpers, Wilma 98
Kuiken, Johan 75
Kuikman, Ingrid 22
Kuil, Joeri 99, 100
Kuiper, Maria 115
Kujala, Pekka 34
Kumar, Prasanth 73
Kunst, Peter 114
Kvistborg, Pia 148
Kwint, Margriet 125
Kwon, Min-chul 81
Lambooij, Jan Paul 81
Lammens, Chantal 92, 96
Lancini, Cesare 79
Lange, Charlotte 100
Lange, Jan 148
Lankheet, Nienke 115
Laval, Severine 22
le Sage, Carlos 47
Lebesque, Joos 124
Leestemaker, Yves 32
Leijen, Suzanne 43, 115
Lenain, Christelle 83
Leunis, Nicoline 148
Léveillé, Nicolas 47
Lieftink, Cor 75
Lieshout, Michiel 148
Linders, Dorothé 99
Linn, Sabine 114
Linn, Sabine 41, 67, 94, 114
Linnemann, Carsten 55
Lips, Esther 41, 45
Liu, Zhen 27
Lodder, Wouter 148
Lohuis, Peter 148
Loo, Claudette 100
Lopez-Yurda, Marta 71
Lorist, Lisette 162
Lubsen-Brandsma, Lottie 148
Lucas, Luc 115
Lukas, Anne 114
Lutkenhaus, Lotte 101
Maas, Chiel 53
Maas, Marnix 125
Maatje, Marlies 148
Mager, Alet 114
Mahn, Marianne 162
Majewski, Ian 73
Mallo, Henk 115
Manders, Peggy 88
Mandjes, Ingrid 162
Mans, Anton 125
Marchetti, Serena 43, 114
Margadant, Coert 22
Matas, Elisa 24
Mattiroli, Francesca 18
Meijer, Richard 148
Meijerman, Irma 43
Meijnen, Philip 124
Meinhardt, Wim 148
Meissl, Katrin 83
Melis, Jacoline 92
Melo, Carlos 47
Mencarelli, Angelo 125
Menendez, Victoria 29
Menning, Sanne 94
Merkx, Remco 32
Merqui-Roelvink, Marja 115
Mertens, Sander 26
Meuleman, Wouter 49, 71
Meulenaar, Jelte 115
Mexner, Vanessa 125
Mezzadra, Riccardo 55
Michalak, Ewa 65
Michalides, Rob 31
Mijnheer, Ben 124
Mikolajewska, Izabela 41
Milojkovic, Bojana 162
Minderhoud, Tom 125
Minkema, Danny 125
Mittempergher, Lorenza 41
Modder, Carla 162
Moes, Johannes 115
Mooij, Thea 88
Mook, Stella 41, 124, 147
Moolenaar, Wouter 24
Moonen, Luc 124
Morris, Ben 75
Mulder, Ina 88
Mulder, Lennart 41, 45
Mullenders, Jasper 73
Muller, Pietje 162
Muller, Saar 99, 100, 148
Muusers, Rick 99
Mylvaganan, Chelvi 99
Nacerdine, Karim 79
Nagtegaal, Tanja 96
Naik, Shalin 55
Nan-Offeringa, Lianda 115
Navran, Arash 124
Nederlof, Petra 41, 45, 99, 100
Neefjes, Jacques 29
Nieuweboer-Krobotova, Inka 149
Nieuwenhuis, Lotte 147
Nieweg, Omgo 147
Nijkamp, Wouter 75
Nijwening, Jeroen 75
Nol, Annemarie 115
Nooijen, Willem 99
Nowee, Marlies 124
Nuijen, Bastiaan 114
Nuijten, Elvira 162
Numan, Rachel 147
Nunnink, Chantal 148
Ogink, Janneke 77
Oldenburg, Hester 92, 147
Olszewska, Agnieszka 125
Onderwater, Suzanne 115
Ong, Nico 34
Oosterhuis, Koen 57

Oosterkamp, Rianne 73
Opdam, Mark 67
Oude Vrielink, Joachim 47
Ouwens, Gabey 88
Ovaa, Huib 32
Paape, Anita 101
Pagie, Ludo 49
Pameijer, Frank 100
Pang, Baoxu 29
Panneman, Carmen 125
Pasca, Edoardo 100
Paul, Petra 29
Paulus van Pauwvliet, Cecile 162
Pawlitzky, Inka 79
Pecharroman Gallego, Raul 125
Peeper, Daniel 83
Peeters, Koen 147
Pelders, Saskia 88
Pengel, Kenneth 101, 125
Peperzak, Victor 53
Peric-Hupkes, Daniel 49
Perrakis, Anastassis 20
Peters, Carolien 125
Peters, Peter 34
Peulen, Heike 124
Peuscher, Marieke 86
Piek-den Hartog, Marianne 147
Pierson, Jason 34
Pieterse, Mark 65
Pijpe, Anouk 88
Pindyurin, Alexey 49
Ploeger, Lennert 125
Plug, Rob 100
Pluim, Dick 43
Pluister, Yvonne 99
Poller, Birk 69
Pool, Bert 99
Poppema, Boelo 114
Ports, Michael 22
Pos, Floris 124
Postel, Ruben 22
Prahallad, Anirudh 73
Pramana, Jimmy 148
Prasetyanti, Pramudita 65
Prevoo, Warner 100
Pritchard, Colin 81
Pronk, Loes 162
Proost, Natalie 81
Pruntel, Roelof 100
Qi, Wen 101
Quaak, Susanne 57
Quispel, Josine 114
Srámek, Michael 149
Rasch, Coen 124
Rebers, Suzanne 88
Rehorst, Harriet 162
Reichgelt, Babs 124
Reijnders, Vera 162
Reinders, Anneke 162
Relyveld, Germaine 149
Remeijer, Peter 124
Retèl, Valesca 98
Ridderbos, Jan-Nico 41
Rietbergen, Daphne 99
Rit, Simon 125
Roberts, Daniel 162
Rodenhuis, Sjoerd 45, 114
Rodenko, Boris 32
Roef, Marc 99
Rohr, Jan 55
Romer, Philip 100
Ronday, May 149
Roodbergen, Rita 94
Rookus, Matti 88
Rooswinkel, Rogier 53
Rooze, Lyandra 99
Rosado, Arantxa 49
Rosenberg, Efraim 100
Rosing, Hilde 114
Roskam, Marielle 162
Rossi, Maddalena 101, 125
Rottenberg, Sven 63
Rozendaal, Roel 125
Rucktooa, Prakash 18
Ruers, Theo 147
Ruijs, Marielle 100
Ruijter-Schippers, Henrique 100
Russell, Nicola 38, 88, 124
Rutgers, Emiel 88, 92, 147
Ryan, Martin 162
Sachs, Norman 22
Sadaka, Charlotte 29
Sahtoe, Danny 18
Salomon, Izhar 29
Salverda, Govert 124
Sanders, Marloes 98
Sani, Musa 34
Sapthu, Sunny 63
Saveur, Lisette 115
Scanu, Tiziana 29
Schaake, Eva 147
Schaapveld, Michael 88
Schaefer, Henning 47
Schaefer, Ronny 26
Schagen, Sanne 94
Scharpfenecker, Marion 38
Scheenstra, Alize 125
Scheenstra, Renske 148
Scheerman, Esther 100
Schellens, Jan 43, 114
Schilder, Christien 94
Schinkel, Alfred 69
Schippers-Gillissen, Carla 100
Schlicker, Andreas 71
Schmidt, Marjanka 41, 88
Schmitz, Alexander 101
Schmitz, Annemarie 101
Schneider, Christoph 124
Schol, Joke 115
Schot, Margaret 115
Schotte, Remko 55
Schouten, Philip 67, 115
Schrier, Mariette 47
Schrijver, Lieske 88
Schumacher, Ton 55
Schutte, Peter 149
Schwandt, Noortje 148
Secades, Pablo 22
Seemann, Ingar 38, 100
Seigers, Riejanne 94
Serresi, Michela 79
Seto, Azusa 18
Severson, Tesa 67
Shu, Jenny 55
Sidharta, Grace 96
Siedschlag, Christian 101
Sinaasappel, Michiel 99, 100
Sivro-Prndelj, Ferida 99
Sixma, Titia 18
Smeele, Ludi 148
Smit, Judith 18
Smit, Marjon 83
Snoek, Margriet 81
Sol, Wendy 63
Sombroek, Cherita 88
Sommeijer, Dirkje 114
Sonke, Gabe 92, 114
Sonke, Jan-Jakob 124
Sonneborn, Mariska 99
Sonnenberg, Arnoud 22
Soueidan, Hayssam 71
Spaan, Mandy 88
Sparmann, Anke 79
Spliethoff, Jarich 147
Spreeuw, Hanno 125
Staiger, Christine 71
Steeghs, Neeltje 43, 114
Steuten, L 98
Stewart, Fiona 38
Stokkel, Marcel 99
Storm, Dea 162
Stornaiuolo, Mariano 18
Stroom, Joep 124
Stuiver, Martijn 92, 147
Stulemeijer, Iris 51
Stuurman, Rik 115
Sun, Chong 73
Sutherland, Kate 81
Swellengrebel, Maurits 115
Sznajder, Beata 162
Taal, Babs 114
Talhout, Wendy 49
Tan, Bing 148
Tanger, Ellen 79
Te Poele, Johannes 38
Te Riele, Hein 61
Teertstra, Jelle 100
ten Cate, Julia 149
ten Hoeve, Jelle 71
ter Brugge, Petra 45, 66
ter Riet, Bas 77
Terweij, Marit 51
Tesselaar, Margot 114
205
personnel index

206
personnel index
Teunissen, Bas 115
Theunissen, Noortje 148
Thijssen, Bas 115
Tibben, Matthijs 115
Tielen, Ivon 100
Tielenburg, René 125
Tilgenkamp, Ria 162
Timmer-Arents, Linda 98
Timmers, Adriaan 148
Tjin, Esther 57
Toebes, Mireille 55
Tolhuis, Bas 79
Tomasoa, Brenda 124
Topolnjak, Rajko 125
Torres Acosta, Alex 162
Tripathi, Pankaj 63
Tulner, Linda 115
Uno, Naoki 83
Urbanus, Jos 55
Uren, Anthony 81
Uyterlinde, Wilma 115
Valdés Olmos, Renato 99
Valkenet, Ludy 163
van ’t Veer, Laura 41, 88, 100
van As-Brooks, Corina 148
van Bemmel, Joke 49
van Berkel, Peter 98
van Beurden, Marc 92, 147, 148
van Boven, Hester 99
van Buuren, Marit 55
van Coevorden, Frits 147
van de Ahé, Fina 81
van de Kasteele, Willeke 57
van de Kooij, Bert 53
van de Noll, Ruud 115
van de Poel, Henk 148
van de Steeg, Evita 69
van de Velde, Sophie 96
van de Velde, Tony 163
van de Ven, Marieke 65
van de Vijver, Marc 100
van de Wetering, Koen 63
van de Wiel, Bart 100
van Deemter, Liesbeth 63
van den Belt-Dusebout, Sandra 88
van den Berg, Joost 57, 115
van den Berk, Paul 59
van den Boer, Cindy 148
van den Brekel, Michiel 147, 148
van den Broek, Sandra 88
van den Ende, Merel 94
van den Haak, Marjolijn 163
van den Heuvel, Michel 114
van den Hoorn, Tineke 29
van den Hoven, Jolanda 115
van der Berg, Marieke 149
van der Burg, Eline 45, 65
van der Donk, Emile 163
van der Gulden, Hanneke 65
van der Hage, Jos 147
van der Heijden, Ingrid 65
van der Heijden, Iris 57
van der Heijden, Michiel 73
van der Horst, Gerda 53
van der Kammen, Rob 27
van der Kant, Rik 29
van der Kolk, Lizet 100
van der Leij, Femke 124
van der Maas, Martin 57
van der Molen, Lisette 148
van der Sar, Jana 115
van der Steeg, Evita 115
van der Torre, Jaco 86
van der Veen, Wietze 149
van der Velden, Sophie 100
van der Velden, Yme 87
van der Vlies, Dalith 148
van der Wal, Anja 61
van der Weide, Marchien 114
van der Wel, Nicole 34
van Deventer, Sjoerd 29
van Dijk, Pim 18
van Dongen, Miranda 100
van Driel, Willemien 148
van Dyk, Ewald 71
van Eggermond, Anja 88
van Esch, Anita 69
van Gerwen, Suzan 100
van Gijn, Roel 114
van Haaften, Gijs 47
van Harten, Wim 98
van Hasselt, Coen 115
van Heijst, Jeroen 55
van Hell, Albert 40
van Herk, Marcel 124
van Hien, Richard 41
van Huizum, Martine 149
van Kasteren, Sander 32
van Kouwenhove, Marieke 47
van Leeuwen, Fijs 38, 99, 100
van Leeuwen, Flora 41, 88
van Leeuwen, Fred 51
van Leeuwen, Marieke 92
van Lent, Wineke 98
van Lohuizen, Maarten 79
van Luenen, Henri 63
van Maanen, Peter 148
van Miltenburg, Martine 65
van Monsjou, Hester 148
van Montfort, Erwin 81
van Mourik, Anke 125
van Netten, Gabry 163
van Nimwegen, Rianne 88
van Noord, Janet 86
van Pel, Renée 99
van Rens, Anja 96, 100
van Riel, Helma 26
van Rooij, Nienke 55
van Rossum-Schornagel, Quirine 114
van Sandick, Johanna 147
van Son, Rob 148
van Steensel, Bas 49
van Strien, Teun 114
van Tellingen, Olaf 94
van Tinteren, Harm 162
van Triest, Baukelien 40, 124
van Turnhout, Arjen 149
van Velthuysen, Loes 100
van Vliet, Mariska 115
van Vliet-Vroegindeweij, Corine 124
van Vugt, Huub 79
van Waardenberg, Wil 163
van Waart, Hanna 92
van Welsem, Tibor 51
van Werkhoven, Erik 162
van Winden, Annemieke 115
van Zeijl, Leonie 24
van Zon, Maaike 34
Vanhoutvin, Steven 163
Vanneste, Ben 124
Vargas, Mark 18
Veenstra, Hidde 147
Vendel, Brian 124
Vens, Conchia 36, 40
Veraar, Elise 53
Vergouwe, Ingeborg 149
Verhagen, Caroline 36, 148
Verheij, Marcel 40, 124
Verheus, Martijn 88
Verhoef, Senno 41, 88, 92, 96, 100
Verhoeven, Els 79
Verloop, Janneke 88
Vermeeren, Lenka 99
Vermeulen, Eric 88, 92
Verschueren, Karijn 124
Verwaal, Vic 147
Verwijs, Manon 36
Verwoerd, Desiree 31
Verzijlbergen, Kitty 51
Vidal Rodriguez, Jordi 71, 75
Vincent, Andrew 162
Visser, Daan 28
Visser, Dick 115
Visser, Nils 38
Vissers, Joep 79
Vlaming, Hanneke 51
Voets, Erik 77
Vogel, Celia 83
Vogel, Wouter 99, 124
Vollebergh, Marieke 67
von Castelmur, Eleonora 20
von Meyenfeldt, Erik 147
Voncken, Francine 124
Vormer, Tinke 61
Vos, Matthijn 34
Vreeswijk, Sandra 125
Vrijaldenhoven, Suzan 114
Vroonland, Colinda 99
Vyth-Dreese, Florry 57
Wagenaar, Els 69
Wals, Anneke 163
Wals, Kim 32
Wang, Liqin (Bruce) 87

Weevers, Marion 94
Wendling, Markus 125
Wesseling, Jelle 45, 100
Wessels, Lodewijk 45, 71
Wessels, Ronnie 147
Westerman, Bart 79
Wever, Carolien 149
Wever, Lidwina 163
Wevers, Marijke 92, 96
Wielders, Camiel 61
Wielders, Eva 61
Wientjens, Ellen 65
Wieringa-Ariaens, Aafke 100
Wigbout, Gea 100
Wijdeven, Ruud 29
Wijnands, Yvonne 163
Wildeman, Maarten 148
Willemse, Els 163
Wilting, Roel 52
Winterwerp, Herrie 18
Wit, Niek 59
Witte, Marnix 125
Witteveen, Thelma 124
Wittkämper, Frits 124
Woerdeman, Leonie 96, 149
Wojciechowicz-Grzadka, Kamila 61
Wolthuis, Rob 77
Wondergem, Marloes 148
Wouters, Michel 147
Wreesmann, Volkert 148
Wu, Amy 29
Xiao, Yanling 53
Xu, Guotai 63
Yanover, Eva 52
Zandbergen, Jeroen 163
Zander, Serge 63
Zerp, Shuraila 40
Zevenhoven, John 81
Zijp, Lambert 125
Zimmerman, Marion 115
Zlotorynski, Eitan 47
Zupan-Kajcovski, Biljana 149
Zuur, Charlotte 148
Zuurmond, Kirsten 101
207
personnel index

THE
NETHERLANDS
CANCER
INSTITUTE
SCIENTIFIC
ANNUAL
REPORT 2010
The Netherlands Cancer Institute
Antoni van Leeuwenhoek Hospital
Plesmanlaan 121
1066 CX Amsterdam
The Netherlands