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

THE
NETHERLANDS
CANCER
INSTITUTE
SCIENTIFIC
ANNUAL
REPORT 2009

SCIENTIFIC ANNUAL REPORT 2009
3

4
Patron HM The Queen Beatrix

SCIENTIFIC ANNUAL REPORT 2009
ThE NEThERLANdS CANCER INSTITUTE
CANCER RESEARCh LAbORATORy ANd CANCER hOSPITAL
www.nki.nl
5

6 COPyRIGhT
Scientific Annual Report 2009
Illustrations and unpublished data in these reports may not be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 12 1
1066 CX Amsterdam
The Netherlands
Phone +3 1 20 5 12 9 1 1 1
Fax +3 1 20 6 17 2625
ISSN 1387-86 1 1
COLOFON
Coordinators
Suzanne Corsetto
Henri Van Luenen
Monique Duyndam
Photograph HM The Queen Beatrix
Enquiry’s/permission:
Rijksvoorlichtingdienst, 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
Plesmanlaan 12 1
1066 CX Amsterdam
The Netherlands
Printed by Thieme Amsterdam

CONTENTS
Board Members 8
Research Divisions 9
Introduction 11
Education in Oncology 16
Division of Biochemistry 18
Division of Cell Biology I 22
Division of Cell Biology II 32
Division of Experimental Therapy 39
Division of Gene Regulation 49
Division of Division of Immunology 56
Division of Molecular Biology 65
Division of Molecular Carcinogenesis 77
Division of Molecular Genetics 83
Division of Psychosocial Research and Epidemiology 93
Division of Diagnostic Oncology 104
Division of Medical Oncology 119
Division of Radiotherapy 132
Division of Surgical Oncology 155
Biometrics Department 169
Clinical Trials 174
Invited Speakers 191
Projects 194
Personnel Index 209
7

8board members
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
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
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 Blijkaar (from December 2009)
MJ Van Mourik
FH Schröder
D Sinninghe Damsté (until June 2009)
MC Smeets
EH Swaab (from December 2009)
PC Van der Vliet
GP Vooijs (until June 2009)
scientific advisory council
AJM Berns, chairman
JJ Neefjes, secretary
S Rodenhuis
M Verheij
J Borst
B Van Steensel

esearcH divisions
Biochemistry
Titia Sixma (head)
Anastassis Perrakis
Franciska Manuputty (office manager)
Cell Biology I
Arnoud Sonnenberg (head)
Wim van Blitterswijk
John Collard
Metello Innocenti
Kees Jalink
Wouter Moolenaar
Ed Roos
Patty Lagerweij (office manager)
Cell Biology II
Jacques Neefjes (head)
Rob Michalides
Huib Ovaa
Peter Peters
Marieke van der Velde (office manager)
Experimental Therapy
Laura van ’t Veer (head)
Adrian Begg
Jan Schellens
Fiona Stewart
Marcel Verheij
Jelle Wesseling
Thea Eggenhuizen (office manager)
Gene Regulation
Reuven Agami (head)
Maarten Fornerod
Fred van Leeuwen
Bas van Steensel
Suzanne Corsetto (office manager)
Immunology
Jannie Borst (head)
Christian Blank
John Haanen
Heinz Jacobs
Ton Schumacher
Florry Vyth-Dreese
José Overwater (office manager)
Molecular Biology
Hein Te Riele (head)
Piet Borst (honorary staff member)
Jos Jonkers
Sabine Linn
Alfred Schinkel
Lodewyk Wessels
Tom de Knegt (office manager)
Linda Römer (secretary)
Molecular Carcinogenesis
René Bernards (head)
Roderick Beijersbergen
Rob Wolthuis
Franciska Manuputty (office manager)
Molecular Genetics
Maarten van Lohuizen (head)
Anton Berns
Jan-Hermen Dannenberg
Jacqueline Jacobs
Anna Haramis
John Hilkens
Paul Krimpenfort
Daniel Peeper
Margriet Snoek
Erica Delwel (office manager)
Marie Anne van Halem (secretary)
Psychosocial Research and Epidemiology
Neil Aaronson (head)
Eveline Bleiker
Wim van Harten
Flora van Leeuwen
Matti Rookus
Sanne Schagen
Yvonne Driessen-Ruwaard
(office 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
Efraim Rosenberg
Marielle Ruijs
Michiel Sinaasappel
Ferida Sivro
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
Willem Boogerd
Henk Boot
Dieta Brandsma
Wieneke Buikhuisen
Sjaak Burgers
Annemieke Cats
Jan Paul de Boer
Leo Gualtherie van Weezel
Alwin Huitema
Martijn Kerst
Sabine Linn
Anne Lukas
Sjoerd Rodenhuis
Jan Schellens
Marianne Smits
Gabe Sonke
Babs Taal
Margot Tesselaar
Michel van den Heuvel
Marchien van der Weide
Mariëlle de Kwant (secretary)
Karin van Leuveren (secretary)
Radiotherapy
Marcel Verheij (head)
Berthe Aleman
Harry Bartelink
José Belderbos
Monique Bloemers
Eugene Damen
Roel de Boer
Luc Dewit
Paula Elkhuizen
Rick Haas
9
researcH divisions

10
researcH divisions
Olga Hamming-Vrieze
Wilma Heemsbergen
Frank Hoebers
Edwin Jansen
Han Krewinkel
Joos Lebesque
Ben Mijnheer
Luc Moonen
Arash Navran
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
Corine van Vliet
Marieke van Zwienen
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
Michiel van den Brekel
Omgo Nieweg
Peter Schutte
Peter Lohuis
Simon Horenblas
Vic Verwaal
Wietze van der Veen
Willemien van Driel
Wim Meinhardt
Annemieke Hoogland
(office manager)
Biometrics department
Otilia Dalesio
Financial Administration
Frieda Boekweit
General Facilities, ICT and Personnel Department
Eric de Wilde
General Research Coordination
Jacques Neefjes, deputy scientific director
Henri van Luenen, director of operations

Director of Research Ton Berns
introduction
I am pleased to present our Scientific Annual Report 2009.
It provides an overview of the scientific 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 organisation. 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.
Our clinical activities again exhibited growth, made possible
by the expansion of our operating facilities with a sixth
operating theatre, specifically designed for minimal invasive
surgery. A new temporary outpatient clinic has been built and
will be operational in January 2010. A collaboration
agreement was signed with the Technical University Twente
to initiate translation of technical innovations into clinical
practice and to participate in the teaching program of
Technical Medicine. We will establish satellite radiotherapy
treatment centres near two other hospitals since further
expansion of our radiotherapy department is not possible on
location. Also, the plan to establish a proton therapy centre
together with the Erasmus University, the University Medical
Centre Leiden, and the Delft Technical University, is steadily
progressing. We made progress in establishing collaborations
between European cancer centres. We filed an application to
the EU to implement high throughput diagnostic platforms
for genomics/proteomics assays. We need such platforms to
refine diagnosis and to identify biomarkers that predict
responses to therapy. Ultimately this should result in more
effective combination therapies. This is an integral part of our
plans to advance personalised medicine. We have filled most
of the vacancies for our nursing staff and for our pathologists.
However, we will need to recruit more personnel in both
these areas to meet our ambition of growth for the hospital
and to further strengthening our molecular pathology as a
critically important connection between lab and clinic.
The transition to a new reimbursement system for health
care has enabled us to further expand our clinical activities.
We concluded 2009 with a small profit for the hospital.
11
introduction
Also our research expenditure remained within budget in
2009. The renovation of the laboratories in the old research
building has been commissioned and we expect to move into
the renovated labs in the beginning of 2011. Also the
planning for the construction of a new animal facility
progressed according plan and we will put out a tender for its
construction in the course of 2010. We expect to balance the
budget for both the hospital and the research in 2010 as well,
however, the situation for 2011 is much more uncertain due
to the current economic crisis.
In the beginning of 2009 we had our second 2-yearly staff
retreat. It focussed on how we could further promote
interactions between research lab and clinic. One of the
recommendations was to establish a Translational Research
Board, consisting of clinicians and basic researchers who will
evaluate and provide input for the clinical trials that are
conducted in the Institute and stimulate translational
research collaborations. The Board has been installed in July
and meets monthly. The second outcome of the retreat was to
strengthen our molecular pathology as it serves as a critical
interface between lab and clinic especially as novel techniques
like parallel sequencing and proteomics bring high
throughput analysis of tumour samples within reach.
The other suggestion was to arrange an introductory program
for newly appointed basic researchers in the clinic. It is
expected that this will come into effect in early 2010.
In October 2009 an international site visit of the Institute
took place under supervision of the Royal Netherlands
Academy of Sciences. This visit was the first of what will be a
quinquennial evaluation meant to inform the core funding
agencies, the Dutch Cancer Society and the Ministry of
Health, about the quality, output and international standing
of the Institute. The opinion of the site visit team was very
positive, positioning the Institute at the forefront of
international cancer research with specific appreciation for
the leading position in the area of radiotherapy, high
throughput screens, mouse models of cancer, biomarkers and
mechanisms of drug resistance. The leading role in the
development of array-based diagnostic tests for breast cancer
was mentioned as an example of the high benefit of longterm
investment in molecular pathology. The site visit
committee also noted that it was a remarkable
accomplishment that the NKI had improved its international
position to such an extent in the past 8 years, while core
funding has remained the same – and therefore effectively
had decreased – (see Table 1). The site visit team concluded
that the Institute will be unable to retain its prominent
position unless core funding is increased substantially.
Accomplishing this will be an important task for the Board of
Directors in the coming year. The challenge is particularly
significant in view of the relatively low level of governmental
funding available for research in the Netherlands and the
apparent reluctance of the Dutch government to augment
investments in research.
Highlights
Our institute continues to have a strong scientific output;
2009 was again a productive year. It is always difficult 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

12
introduction
table 1
Core research funding NKI-AVL from the Dutch Cancer Society and the Ministry of Health in the period 2001-2009 in million
Euro’s.
Year 2001 2002 2003 2004 2005 2006 2007 2008 2009
Dutch Cancer Society 8.7 8.7 9.1 8.9 8.6 8.3 8.8 9.3 10.2
Ministry of Health 9.4 9.9 10.1 9.1 9.3 9.4 9.5 9.9 10.2
Total* 18.1 18.6 19.2 18.0 17.9 17.7 18.3 19.4 20.4
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 2009.
Institute. A more complete and detailed account of specific
projects can be found in the reports of individual group
leaders in this SAR and on the website (www.nki.nl).
Michael Hölzel and Sidong Huang, working in the group of
René Bernards (Division of Molecular Carcinogenesis),
identified a mechanism of resistance to retinoic acid (RA) in
neuroblastoma. This is a clinically relevant question, as this
drug is used with only limited success to treat aggressive
forms of this disease. Using a genetic approach, they
identified cross talk between the NF1-RAS-MEK signaling
cascade and RA signaling as a major cause of RA resistance
in neuroblastoma. Activation of the NF1-RAS-MEK cascade
suppresses expression of the Retinoic Acid Receptor
co-activator ZNF423, which they found to be essential for
transcriptional activation by this receptor. They demonstrated
that inhibition of MEK signaling with small molecules
restores responsiveness to RA, providing a potentially
powerful combination therapy to overcome RA resistance,
a phenotype seen in over half of primary neuroblastomas.
Mirjam Epping, working in the group of René Bernards,
identified a function for TSPYL5, a gene of unknown function
that is frequently amplified in breast cancer. She found that
TSPYL5 interacts with USP7, the de-ubiquitinating enzyme
for p53. Expression of TSPYL5 prevents USP7 from removing
ubiquitin residues from p53, leading to increased proteasomemediated
degradation of p53. Consequently, p53 function is
suppressed by TSPYL5 overexpression in breast cancer.
She received the prestigious AvL-award 2009 for her work.
In collaboration with Marleen Kok and Sabine Linn (Division
of Molecular Biology) and Goran Landberg and colleagues
(University of Lund, Sweden), Rob Michalides (Division of
Cell Biology II) found that phosphorylation of the Estrogen
Receptor at Serine 305 predicted a poor tamoxifen response
in breast cancer patients. Using PAK-1 as a additional marker,
they could identify 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 a benefit from treatment with tamoxifen.
The results indicate that resistance to tamoxifen is largely
determined by the phosphorylation state of two serine
residues in Era. These will be further tested as a predictive
marker for breast cancer treatment.
The Neefjes lab explained how cholesterol controls transport
of late endosomes where transmembrane signaling should be
terminated. They showed how an ER protein VAP contacts a
late endosomal Rab7-RILP-ORP1L complex to remove the
associated dynein motor. This novel mechanism explains the
position of lysosomes in patients with so-called stapling
diseases.
Valeria De Marco in the group of Tassos Perrakis (Division
of Biochemistry) showed that the Geminin: Cdt1 complex,
an inhibitor of the formation of the pre-replication complex,
is necessary to be assembled prior to DNA replication in
eukaryotic cells. With a crystal structure of a human truncated
Geminin/Cdt1 complex and X-ray solution scattering
experiments (SAXS) it was shown that the complex can exist
both as a heterotrimer and a heterohexamer under
physiological conditions. This led to the suggestion of a
‘conformational switch’ where the same protein complex in
two different functional states can act as ‘inhibitory’ or
‘permissive’ with respect to DNA replication licensing.
Sine Hadrup in the group of Ton Schumacher (Division
of Immunology) developed a technology to analyse in parallel
T cell responses against many different tumour antigens.
This technology opens the possibility to perform highthroughput
analysis of disease- or therapy-induced tumourspecific
T cell responses in patient samples. Jeroen van Heijst
in the group of Ton Schumacher used a genetic tagging
strategy to determine how the magnitude of CD8+ T cell
responses is regulated. His data show that recruitment of
antigen-specific T cells is near constant under a variety of
conditions, and that the strength of cytotoxic T cell responses
is therefore almost exclusively determined by the clonal burst
that each recruited cell undergoes.
In a genome-wide screen for new cancer genes, Daniel
Peeper and colleagues (Division of Molecular Genetics)
previously identified the neurotrophic receptor tyrosine
kinase TrkB. Marjon Smit and Thos Geiger in the Peeper
group now found that the oncogenic properties of TrkB are
accompanied by a strong morphological transformation of
cells, resembling epithelial- mesenchymal transition (EMT).
This required induction of Twist and Snail and suppression
of E-cadherin. Depletion of either transcription factor blocked
TrkB-induced EMT and growth of tumour xenografts.
Epistasis experiments indicated that Twist acts upstream
from Snail. These results demonstrate that TrkB signaling

activates a Twist-Snail axis that is critically involved in EMT
and tumorigenesis.
Using functional genetic screens, the group of Reuven Agami
(Division of Gene Regulation) identified in the past several
oncogenic and potentially tumour suppressor miRNAs.
This year they extended these observations by identifying miR
192 and miR 194 as regulators of the Period genes and the
circadian clock in humans, and miR-26 as an oncogene in
glioblastomas. Furthermore, a new layer of regulation was
uncovered whereby RNA binding proteins regulate the
miRNAs. They showed previously that RNA binding proteins
can inhibit the accessibility of miRNAs to their targets. New
work shows that RNA binding proteins can also stimulate
miRNA activity by modulating RNA structure. Thus, miRNA
levels in the cell do not necessarily correlate with their activity.
They developed now bioinformatics algorithms to deduce
miRNA activity from mRNA expression arrays.
The group of Maarten Fornerod studies chromatin
interactions with nuclear pore complex components.
They showed that apart from their role in chromatin
organisation at the nuclear envelop, the nucleoporins inside
the nucleoplasm predominantly interacted with
transcriptionally active genes away from the core complex
thereby stimulating their expression.
The group of Fred van Leeuwen studies gene regulation by
epigenetic mechanisms. They investigated in yeast histone
H3 lysine 79 methylation by the methyltransferase DOT1 and
how H3-Lys79 methylation affects chromatin structure and
function. They showed that in contrast to other
methyltransferases, DOT1 acts in a non-processive manner
and H3-Lys79 methylation serves as an anti-binding signal.
In addition, they developed a Recombination-Induced Tag
Exchange method to track old and new proteins. They showed
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.
To gain insight into the role of chromatin proteins in
determining chromatin structure and gene regulation, the
group of Bas van Steensel uses DamID, an in vivo approach
they developed to detect protein-chromatin interactions.
In the past they have successfully mapped lamin association
with repressed chromatin. Now, they have generated a
genome-wide high-resolution binding map for the linker
histone H1 and H3.3 in Drosophila cells. They showed that
the H3.3 protein counteracts the association of H1 in
transcription start sites of active genes, providing a
mechanism to keep diverse genomic sites in an open
chromatin conformation. Additionally, they have embarked
on a Chromatin Protein Discovery Project with the aim of
generating genome-wide binding maps for a large set of novel
chromatin proteins.
Using combination knockout mice, Robert van Waterschoot
in the group of Alfred Schinkel (Division of Molecular
Biology) demonstrated that two of the most important drugdetoxifying
proteins, the multidrug transporter P-glycoprotein
and the multispecific drug-metabolizing enzyme Cytochrome
13
introduction
P450 3A, can cooperate together in a highly efficient manner
to decrease the availability, and therefore the toxicity, but also
potentially the therapeutic efficacy of anticancer drugs.
The group of Jan Schellens (Division of Medical Oncology)
participated in a phase I clinical trial with the oral PARP-1
inhibitor olaparib. Breast and ovarian cancer patients with
germ-line mutations in BRCA1 or BRCA2 genes that were
treated in this study, showed impressive response rates
despite failure of previous treatments, illustrating that in the
tumours with a homologous recombination deficiency,
suppression of single strand DNA break repair mechanism
can be very beneficial for patients: high response rate with
low toxicity.
In August 2009, the Division of Radiotherapy introduced
Volumetric Modulated Arc Therapy (VMAT), a further
refinement of existing advanced technologies such as IMRT.
During VMAT the target is continuously irradiated while the
source of the beam is rotated around the patient in single or
multiple arcs, while simultaneously controlling gantry
position, gantry speed, MLC leaves, collimator angle and dose
rate. A unique feature developed at the department consists
of a continuous dosimetric verification of VMAT, maximizing
the safe delivery of the radiation. They demonstrated that for
early stage lung cancer, prostate, rectal and head and neck
cancer VMAT provides a highly conformal dose distribution
delivered in significantly less time than with current
techniques.
The long-term impact of pathologic characteristics for stage
I and II invasive breast cancer patients treated with breast
conserving therapy (BCT) demonstrated that young age and
high-grade invasive ductal cancer were the most important
risk factors for local relapse, while margin status had no
significant influence. Fortunately, a boost dose of 16 Gy
significantly reduced the negative effects of both young age
and high-grade invasive cancer.
Quality of research
The quality of research can be monitored in several ways.
Our scientific productivity as assessed by objective
bibliometric parameters (citations and impact of scientific
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. An independent externally conducted
bibliometric analysis, in which we were benchmarked with
other cancer centres, confirmed the prominent position of the
NKI. This was independently confirmed by the international
site visit team (see above).
While a high citation score is gratifying, it is only one
measure of scientific productivity. The quality of our work
should also be gauged by the invitations of our staff to present
at prestigious scientific meetings, awards obtained by our
staff. We score high on all these accounts. We are also
successful in securing grant support, e.g. René Bernards and
Titia Sixma both obtained a prestigious ERC grant. Fijs van
Leeuwen and André Bergman received KWF personal career
grants. The quality of research of the groups in each division
is evaluated approximately every 5 years by an external site
visit team. In November 2009 the Division of Surgical

14
introduction
table 2
Short-term citations and impact of scientific 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 5463 13.8 2590
2008 445 2553
2009 517 3122
As from 1997 publication year of articles is the criterion, instead of Scientific Report-year listing.
From 2008 the online publication data is used as criteria for the year of publication.
* Citations in the two years after publication, excluding self-citations.
** 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.
Oncology was evaluated. The reviewers were positive about
the activities of the division but also had a number of
suggestions for further improvements that will be discussed
with members of the division in 2010.
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 2009, 28 staff members
had professorships at one of the Dutch Universities. Reuven
Agami was appointed as Professor of microRNA and
pathogenesis at the Erasmus Medical Centre in Rotterdam.
Laura van ‘t Veer was elected as EMBO member. Piet Borst,
Harry Bartelink, René Bernards and I were elected as fellows
of the European Academy of Cancer Sciences. Jannie Borst
was elected Van Loghem lecturer 2009 by the Dutch Society
for Immunology (NVVI), which is a career award for key
contributions to the field of immunology. Peter Peters was
elected to present the Robert Feulgen lecture 2009.
There were substantial changes in our clinical staff mostly
due by retirements. Fortunately, we have been able to recruit
excellent successors. Huib Ovaa and Jan-Jacob Sonke were
appointed as tenured staff members in the Division of Cell
Biology II and Radiotherapy, respectively. Metello Innocenti
joined the Division of Cell Biology I as tenured staff member
from the University of Frankfurt and Thijn Brummelkamp
will join the Division of Biochemistry in the course of 2010
from the Whitehead Institute. Neil Aaronson stepped down
as head of the Division of Psychosocial Oncology and
Epidemiology and was succeeded by Floor van Leewen.
John Haanen was appointed as head of the Division of
Medical Oncology.
Staff of the NKI-AVL fulfilled numerous functions in national
and international organisations, on boards of scientific
journals, as members of study sections, and as organisers or
co-organisers of scientific meetings, workshops and
congresses.
outlook and acknowledgements
We are confident that the NKI-AVL will continue to flourish,
even in the face of a worldwide economic crisis which may
force us to be even more frugal than we already are.
The recent site visit has confirmed that our Institute is truly
a Centre of Excellence that deserves more generous core
support than it currently receives. Collectively, we have the
scientific skills and expertise, as well as the drive and
motivation to effectively complement the core resources with
substantial external grant support. But we need to increase
our core funding to maintain and upgrade our infrastructure.
Together with the Dutch Cancer Society we have founded a
Research Fund to specifically support research at the NKI.
This fund will also enable us to further increase our visibility
to industry and private sponsors as an organisation worth
investing in. Real breakthroughs in cancer treatments are
most likely to come from the combination of basic
understanding of the mechanisms underlying cancer
development and the identification of the drivers that can
serve as target for therapy in individual tumours. In this
regard, state of the art molecular tumour typing using
genomic and proteomic techniques combined with robotic
screens to identify pathways that act in a synthetic lethal
fashion with the oncogenic lesions offer promising
perspectives. This is an area in which the NKI has a strong
record. I am convinced 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

sufficiently large scale. This requires substantial investments
in molecular pathology. 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 significant sponsor of our research; the Ministry
of Health, which provides a substantial core grant to the
15
introduction
Institute and has provided the funds for a new hospital and
for the renovation of our research facilities; and, last but not
least, the many individuals who provide us with financial,
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 are inspired by
this report.
Ton Berns
Director of Research

16
education in oncoloGy
education in oncoloGy
In the Netherlands Cancer Institute, students are trained at
many different levels, from Master and PhD to post-doctoral
level and from medical workers to technical personnel or
scientists. Research and clinical staff as well as 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 affiliation
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
J Borst (chair and dean Master students), H te Riele (general
affairs), R Beijersbergen (Master course), J Hilkens,
J Collard (HLO students and publicity), Titia Sixma (dean
PhD students) and F Balm (clinical teaching). P Peters
functions outside the education committee as dean for the
post-docs.
master students
The program in Experimental Oncology attracts Master
students of all national universities. 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 2009, 34 Dutch university
students and 3 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) (13), the University of Amsterdam (UvA)
(7) and Utrecht University (8), but also from Leiden (2),
Nijmegen (2), Wageningen (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 significant
contributions to research progress of the PhD students and
post-docs who supervise them. The core element of
theoretical training is the course in Experimental Oncology,
given twice yearly (Table 1). This course is compulsory for
NKI Master students who do an internship, 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.
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
2009, the institute had ~130 PhD students registered with
the OOA. Of these, 20 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 scientific 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 field 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 1-2 times per year in these
opportunities to exchange views with experts in the field.
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 first year and presentations in subsequent year, 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 significantly to interaction
between different research groups.
In addition, senior graduate students can participate in a
joint retreat with other cancer institutes in Europe. In 2009,
this event was hosted by CrUK in London, with participants
from British CrUK institutes, the Italian IFOM and the
table 1 – Courses in Experimental Oncology
Epidemiology F van Leeuwen
Surgery E Rutgers
Radiodiagnostics R Valdés Olmos,
M Sinaasappel
Pathology D de Jong
Molecular diagnostics** R Kerkhoven, S Linn
Drug therapy S Rodenhuis
Radiotherapy* M Verheij
DNA damage response
and apoptosis** A Begg, J Borst
Signal transduction** W Moolenaar
Cell cycle** R Bernards, D Peeper
Cell Division R Wolthuis
Cell senescence and
genomic instability** R Beijersbergen, H te Riele
Cell adhesion** E Roos
Tumor microenvironment de Visser
Mouse models of cancer** J Jonkers
Immunology and
immunotherapy** T Schumacher, J Haanen
Analysis of protein structure T Sixma
Rational drug development
and drug delivery** J Beijnen, A Schinkel
* including tour
** including tutorial

table 2 – OOA graduate student courses 2009
9-12 March The Macroscopic and Pathologic Anatomy of
the mouse
WH Lamers, CJF van Noorden
May English Writing and Presenting
A Bless
14-16 April Epigenetics
R Steenbergen, T vd Elsen, J Kooter
18-29 May Signal transduction
W Moolenaar and A Sonnenberg
Spring Stem cells and Cancer
JP Medema
5 - 9 October In the Footsteps of Antoni van Leeuwenhoek,
basic course
P Peters, C van Noorden, K Jalink, E Reits et al
14-16 October Annual Graduate Student Retreat
T Sixma
29 October Tumor microenvironment
- 6 November E Roos en K de Visser
November English Writing and Presenting
A Bless
Spanish CNIO contributing to a program of scientific
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. In 2009 we have introduced a mid-term review.
After two years of PhD research the student, supervisor and
committee evaluate the state of the project and the likelihood
of achieving a PhD within a reasonable time frame. This
meeting can be used to redefine 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. The council also mediates
communication between the graduate students and research
manager or board of directors.
Post-docs
Post-docs in the Netherlands Cancer 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, postdocs in the biological sciences, medicine and
technology have a preference for working in an institute or
university and indeed, many of our post-docs go on to
careers in science.
There are many alternative options available for an interesting
career after a successful postdoc period. The Postdoc Career
Development Initiative (PDCI) was initiated based on the
post-doc platform at the NKI-AVL. This nationwide activity
offers a variety of perspectives for careers for post-doctoral
fellows. We also assist in improving skills that will increase
career choices, both within and outside academia. The
activities we have offered can be visited at www.pcdi.nl.
17
education in oncoloGy
The annual postdoc retreat was again our main event; a
three-day, intensive skills training and career development
conference. On top of the retreat we have organized other
one-day activities, such as company visits, laboratory tours,
and hands-on career development and networking activities
for life sciences professionals. Under the leadership of Peter
Peters the retreats have been attended by more then 1000
postdocs over the last 10 years.
retreat 2009
On 24 - 26 June 2009, eighty postdocs and final year PhD
students in Life Sciences gathered to reflect on their careers.
Surrounded by key players from academia, policymakers
and entrepreneurs from industry, burning questions could
finally be asked: ‘What are my career perspectives?’, ‘What
are my options if I were to leave academia?’, ‘What are my
chances and how can I improve them?’,’ Is the outlook really
as grim as some of us may think?’.
No, by no means! This was made very clear during the
postdoc retreat. By their profession, postdocs have developed
skills that can make them attractive candidates for a variety
of positions. Many are however unaware of their own skills.
The first day, themed ‘Exploring Your Possibilities’, therefore
aimed at discovering valuable hidden talents and what one
actually enjoys doing. On the other hand, some vital skills
too often remain undeveloped in academia. What if you
don’t know how to sell your work, convince people, negotiate
effectively or manage people? Would you then become a
good principal investigator? And what if you were to leave
academia, how far would you get with a strong publication
record alone? It is obvious that there is still a lot of work to
do besides knowing and using the skills you already have.
Day two of the retreat was therefore a starting point to
‘Increase Your Skills’, where participants got trained in
workshops, focused on essential transferable skills.
Throughout the retreat, examples of successful careers were
illustrated by our invited speakers, from hardcore academics
(Hidde Ploegh – professor at MIT) to people who completely
left research behind to pursue their passion (Alexander
Griekspoor – CEO Mekentosj). Two important messages
were clear: First, ‘Make sure you choose what you like and
go for it!’ and secondly, ‘While you only focus on pipetting,
your next ideal job is not going to find you – increase your
skills and be pro-active!’.
Before the participants went home, PCDI arranged them an
afternoon of networking with representatives of potential
professions within, but also outside academia, mostly expostdocs.
This was appreciated from both sides. For
postdocs, usually only exposed to academics from their
department, it was a unique chance to meet and ask
questions to external people at a rather informal way. The
need for this was revealed by the PCDI questionnaire,
completed prior to the retreat: a majority of the participants
agreed that the bio-business is an invisible phenomenon for
postdocs. Ignorance may lead to missing out major career
opportunities! For bio-businesses, the networking afternoon
was a unique opportunity to present themselves to potential
employees. Answering questions about required skills,
experiences and ‘cultural differences’ to open-minded
postdocs may potentially lead to more suitable, better
prepared candidates in future.

18
biochemistry
Division head, group leader Titia Sixma
titia sixma PhD Group leader
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
Annet reumer 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
Hibbert RG, Mattiroli F, Sixma TK.
Structural aspects of multi-domain RING/
Ubox E3 ligases in DNA repair. DNA
Repair (Amst). 2009;8:525-35
Kasheverov IE, Zhmak MN, Fish A,
Rucktooa P, Khruschov AY, Osipov AV,
Ziganshin RH, D’Hoedt D, Bertrand D,
Sixma TK, Smit AB, Tsetlin VI. Interaction
of alpha-conotoxin ImII and its analogs
with nicotinic receptors and acetylcholinebinding
proteins: additional binding sites
on Torpedo receptor. J Neurochem.
2009;111:934-44
Knipscheer P, Klug H, Sixma TK,
Pichler A. Preparation of sumoylated
substrates for biochemical analysis.
Methods Mol Biol. 2009;497:201-10
DiVisioN oF biochemistry
strUctUrAL bioLoGy
Development of cancer is generally due to errors that occur in cellular pathways.
Structural biology can help to understand these errors at the atomic level, by studying
the molecules involved. We use X-ray crystallography as a tool to provide threedimensional
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.
In the past year we have finalized our native mass spectrometry analysis of the MutS
protein in complex with DNA, performed in collabroration with the group of Albert
Heck in Utrecht. Interestingly in the fine structure the different nucleotide states
could be recognized. Careful deconvolution of the peaks by Tassos Perrakis and Serge
Cohen, made it possible to define which nucleotides were bound.
The MutS protein follows a nucleotide binding cycle that is dependent on the presence
of magnesium. We studied the role of magnesium in this cycle. In this analysis it
became clear that magnesium is most important for the high affinity step of the
reaction cycle.
Our detailed analyses of the steps following mismatch recognition are building up to
a model that reveals the details of the complex ATPase cycle of the mismatch repair
proteins.
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 of so 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 to develop 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 flexible
manner. Although the overall mechanism has been defined, the atomic details have
been lacking and the specificity determining factors are unclear. We study several
E2/E3 complexes involved in conjugation of ubiquitin and the related modifier
SUMO as well as proteins involved in de-ubiquitination.
We characterized the activity of the Triad1 E3 ligase and revealed that it can make K63
linked chains in conjunction with Ubc13/Mms2, in agreement with data from the lab
of Bert van der Reijden (Nijmegen) (figure 1). This activity is inhibited in the
presence of excess E3-ligase (Marteijn et al, 2009). In unanchored chain assays we
could see no activity of Triad1 with UbcH7, but in the presence of a putative target we
did observe mono ubiquitination of the target.

In a collaboration with Rolf Boelens in Utrecht we performed a series of structural
studies on domains of the Rad18/Rad6 complex. A high resolution crystal structure
of the Ring domain and the complex between Rad6 and the secondary interaction
domain on Rad18, called R6BD, revealed a number of unexpected aspects about this
E2/E3 combination that may have significance for their cellular function in DNA
damage tolerance (Hibbert, Huang et al, in preparation).
Figure 1: E3 ligase Triad can promote formation of K63 linked Ubiquitin chains, but acts autoinhibitory at
high concentrations (Marteijn et al, 2009).
Nicotinic Acetylcholine receptor homolog AchbP In 2008/2009 several new
structures of bacterial homologs of the nicotinic acetylcholine receptor appeared in
the literature, but since these channels do not respond to nicotinic agonists or
antagonists, the use of Acetylcholine Binding Proteins (AChBP) as tools for
understanding the ligand binding in pentameric ligand-gated ion-channels continues
to remain relevant. AChBP has strong sequence similarity to the a-subunits of the
nicotinic acetylcholine receptor ligand-binding domain and our AChBP crystal
structures are the established high-resolution model for the ligand-binding domains
in this class of ion channels.
We have used in silico analysis of novel ligands excecuted in collaboration with the
group of Iwan de Esch to find novel binding modules for AChBP. Several of these
also interact with nicotinic acetylcholine receptors. A number variants have been
tested in structure analysis. Some compounds reveal a variable binding mode, that
was not predicted in the computational screens (Ulens et al, 2009); others show a
more defined binding orientation. Binding studies using isothermal calorimetry and
surface plasmon resonance provide more insight in the details of ligand binding.
These were compared to a novel apo structure of AChBP, the first structure without
ligands in the binding site. A previous structure was published without ligands, but
electron density is observable in these sites, indicating the presence of unspecified
ligands. Here we noticed that in the absence of ligands the C-loop is flexible (figure 2).
Structural studies were also performed on a series of reference compounds. These
are currently being placed in the context of the thermodynamic binding parameters.
A careful comparison of the available crystal structures and complexes of the AChBPs
reveal where this model protein is most useful (Rucktooa et al, 2009).
Conotoxins are small peptide toxins that bind with high specificity and affinity to
different ion channel subtypes. In collaboration with the Tsetlin group in Moscow we
studied details of the ImII conotoxin binding to AChBP, showing that on the binding
protein the interaction with the conotoxin competes with known ligands. From other
data it appears that there may be secondary sites on the nicotinic receptor itself
(Kasheverov et al, 2009).
Publications (continued)
19
biochemistry
Marteijn JA, van der Meer LT, Smit JJ,
Noordermeer SM, Wissink W, Jansen P,
Swarts HG, Hibbert RG, de Witte T,
Sixma TK, Jansen JH, van der Reijden BA.
The ubiquitin ligase Triad1 inhibits
myelopoiesis through UbcH7 and Ubc13
interacting domains. Leukemia.
2009;23:1480-9
Rucktooa P, Smit AB, Sixma TK. Insight in
nAChR subtype selectivity from AChBP
crystal structures. Biochem Pharmacol.
2009;78:777-87
Ulens C, Akdemir A, Jongejan A,
van Elk R, Bertrand S, Perrakis A, Leurs R,
Smit AB, Sixma TK, Bertrand D,
de Esch IJ. Use of acetylcholine binding
protein in the search for novel alpha7
nicotinic receptor ligands. In silico docking,
pharmacological screening, and X-ray
analysis. J Med Chem. 2009;52:2372-83
Figure 2: The apo structure of Acetylcholine
Binding Protein.

20
biochemistry
biochemistry
Group leader Anastassis Perrakis
Anastassis Perrakis PhD Group leader
christophe caillat PhD Post-doc
eleonora von castelmur PhD Post-doc
Patrick celie PhD Post-doc
serge cohen PhD Post-doc
Valeria de marco PhD Post-doc
Dene Littler PhD Post-doc
Krista Joosten PhD Post-doc
robbie Joosten PhD Post-doc
eirini mitsiki PhD Post-doc
Jens haussman msc PhD Student
evangelos christodoulou Technical staff
tatjana heidebrecht Technical staff
Publications
De Marco V, Gillespie PJ, Li A,
Karantzelis N, Christodoulou E,
Klompmaker R, et al. Quaternary structure
of the human Cdt1-Geminin complex
regulates DNA replication licensing. Proc
Natl Acad Sci U S A. 2009;106:19807-12
Celie PH, Toebes M, Rodenko B, Ovaa H,
Perrakis A, Schumacher TN. UV-induced
ligand exchange in MHC class I protein
crystals. J Am Chem Soc.
2009;131:12298-304
Rodenko B, Toebes M, Celie PH,
Perrakis A, Schumacher TN, Ovaa H.
Class I major histocompatibility complexes
loaded by a periodate trigger. J Am Chem
Soc. 2009;131:12305-13
Mooij WT, Cohen SX, Joosten K,
Murshudov GN, Perrakis A. ‘Conditional
Restraints’: Restraining the Free Atoms in
ARP/wARP. Structure. 2009;17:183-9
Mooij WT, Mitsiki E, Perrakis A.
ProteinCCD: enabling the design of protein
truncation constructs for expression and
crystallization experiments. Nucleic Acids
Res. 2009;37:W402-5
strUctUrAL bioLoGy
We continue to divide our interest between the work in the DNA replication license
and in spindle assembly checkpoint proteins, while we pursue our in-house
collaborations, and our interest in computation methods for structural biology.
DNA replication licensing and the role of Geminin complex We were able to
conclude our work on the function of the Geminin:Cdt1 complex, a regulator of the
formation of the pre-replication complex, which is necessary to be assembled prior to
DNA replication in eukaryotic cells. Based on our crystal structure of a human
truncated Geminin: Cdt1 complex and X-ray solution scattering experiments (SAXS)
we demonstrated that the complex can exist both as a heterotrimer and a
heterohexamer under physiological conditions. We suggested a ‘conformational
switch’ where the same protein complex in two different functional states can act
as ‘inhibitory’ or ‘permissive’ with respect to DNA replication licensing (figure 3)
(De Marco et al, 2009).
Figure 3: A model for the permissive and inhibitory switch of the Geminin-Cdt1 complex.
The tCtd1-tGeminin complex is depicted as a cartoon of structural elements, while additional
components of the replication license are depicted as geometrical schemes.
A new twist on our research is the discovery of a homologue of Geminin, Idas (in
Greek mythology, Idas is the cousin of the two Gemini) which seems to act as an
inhibitor of Geminin in specific cell types. Biophysical and structural investigations
of Idas are underway.
structural studies of proteins involved in mitotic progression Our highthroughput
approach towards characterising different proteins involved in mitotic
progression has resulted to an additional structure, that of the regulatory domain of
Mps1 kinase. We have also analyzed the previously determined structure of the DNAbinding
domain of the FoxM1 transcription factor, that we determined last year
(Littler et al, submitted for publication).
FoxM1 is a member of the Forkhead family of transcription factors that is implicated
in a cell’s proliferation potential and in cancer. FoxM1 binds promoter regions with a
preference for tandem repeats of a consensus ‘TAAACA’ recognition sequence.
FoxM1 adopts the winged-helix fold, typical of the Forkhead family. Neither ‘wing’ of
the fold however, makes significant contacts with the DNA, while the second,
C-terminal, wing adopts an unusual ordered conformation across the back of the
molecule. The lack of standard DNA-’wing’ interactions may be a reason for FoxM1’s
relatively low affinity, compared to other Forkhead proteins. Their role is maybe
undertaken by other parts of FoxM1 that directly interact with the target DNA, or
alternatively, higher affinity for the consensus sequence is mediated by interaction
with other binding partners. Finally, we were unable to show clear preference for
tandem consensus sites recognition in DNA-binding, transcription activation and
bioinformatics analysis; we concluded that FoxM1’s moniker, ‘Trident’, is not
supported by our data. Our goal is now to determine the structure of full-length
FoxM1 and understand how phosphorylation regulates transcriptional activity.

The MPS1 (MonoPolar Spindle 1) family of kinases was originally shown to be a dual
specificity 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 sufficient 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. Mutagenesis studies to elucidate the
function of this novel domain are ongoing.
structural studies of Autotaxin Autotaxin (NPP2) hydrolyzes circulating
lysophosphatidylcholine. Engineering of a human cell line (HEK293 derivative) that
is stably expressing glycosylation mutants of rat autotaxin led to crystallization
success, and crystals diffracting to high resolution. Structure determination efforts
are close to fruition.
structural studies of JbP1 The JBP1 protein, discovered by the Piet Borst group,
binds to DNA that contains base J (J-DNA). It has been shown to be essential for
survival in many major protozoal human pathogens such as T. brucei (sleeping
sickness), T. cruzi (Chagas’ disease) and Leishmania species (various types of
Leishmaniasis). We have demonstrated with biochemical experiments and site
directed mutagenesis, that a single aspartate residue is solely responsible for
conferring specificity of JBP1 towards J-DNA (figure 4). Structural experiments
are ongoing.
structural studies of mhc class i molecules Major histocompatibility complex
(MHC) class I molecules associate with a variety of peptide ligands and present these
ligands on the cell surface for recognition by cytotoxic T cells. Together with the
groups of Ton Schumacher (Division of Immunology) and Huib Ovaa (Division of
Cell Biology) we have generated a protocol for the in crystallo exchange of a photocleavable
Se-Met labelled peptide bound to a class I molecule, with a new peptide,
alleviating the need to refold and crystallize every new class I - peptide complex.
This year we determined a few additional structures, including a complex with a new
chemically cleavable peptide (Celie et al, 2009, Rodenko et al, 2009).
methods for high throughput structural biology The technical developments for
Structural Biology have now been transferred to the Protein Facility, which will foster
new developments while providing many services to the NKI and the Netherlands
and International community.
methods for X-ray crystallography Over a decade has passed since we developed
the ARP/wARP software, a new method, which was first to automate building of
three-dimensional atomic models of proteins in the field of X-ray crystallography.
Our developments culminated in ARP/wARP, which inherently functions as an expert
system that gathers information about protein structure, to allow the building of new
models. As we gain more information about protein structures, we aim now to
provide tools for the final stages in model building (Mooij et al, 2009). These are
now the most challenging and time consuming steps in the process of
crystallographic structure determination.
The crystallographic models in the Protein Data Bank (PDB) were deposited there
over several decades, and were created using the 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 refine PDB models for which experimental data are available.
This year, we have started extending the scope of PDB_REDO by providing it with
tools allowing it not only to optimize but also to rebuild protein models, and make
them available to a user community of tens of thousands of biologists, chemists,
bioinformaticians and drug designers.
Publications (continued)
21
biochemistry
Repanas K, Fuentes G, Cohen SX,
Bonvin AM, Perrakis A. Insights into the
DNA cleavage mechanism of human
LINE-1 retrotransposon endonuclease.
Proteins. 2009;74:917-28
Ulens C, Akdemir A, Jongejan A, van Elk R,
Bertrand S, Perrakis A, et al. Use of
acetylcholine binding protein in the search
for novel alpha7 nicotinic receptor ligands.
In silico docking, pharmacological screening,
and X-ray analysis. J Med Chem.
2009;52:2372-83
Hilge M, Aelen J, Foarce A, Perrakis A,
Vuister GW. Ca2+ regulation in the
Na+/Ca2+ exchanger features a dual
electrostatic switch mechanism. Proc Natl
Acad Sci U S A. 2009;106:14333-8
Figure 4: DNA binding of JBP1 and a single
point mutant (D525A) to J-DNA and the
same sequence without the J (T-DNA), as
measured by fluorescence polarization
anisotropy.
While JBP1 binds J-DNA with very high
affinity and T-DNA with an affinity about
four to five orders of magnitude lower, the
D525A mutants recognizes both substrates
with very similar (low) affinity and shows
no preference for J-DNA.

22
MoleCUlAR CARCinogeneSiS
Cell Biology i
Division head, group leader Arnoud Sonnenberg
Arnoud Sonnenberg PhD Group leader
Michael Ports PhD Post-doc
Ruben Postel PhD Post-doc
Pedro Rifes PhD Post-doc
Pablo Secades 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
Margadant C, Raymond K, Kreft M,
Sachs N, Jansen H, Sonnenberg A.
Integrin a3b1 inhibits directional migration
and wound re-epithelialization in the skin.
J Cell Sci 2009;122:278-88
de Pereda JM, Lillo MP, and Sonnenberg A.
Structural basis of the interaction between
integrin a6b4 and plectin at the
hemidesmosomes. EMBO J 2009;8:1180-90
Figure 1: Blistering phenotype of the
zebrafish Frem2 mutant: (A,B) Tail fin of a
wild-type (A) and Frem2 mutant zebrafish
embryo (B). Black arrow marks the
epidermal blister. (C,D) Electron
microscopic sections of the tail fin from a
wild-type (C) and frem2 mutant embryo.
Black arrows mark the position of the
basement membrane. Red arrows mark the
gap between the epidermal cells (EC) and
the basement membrane. ECM,
extracellular matrix.
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 specifically interested in understanding the significance of and
characterizing the interactions that takes 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 filament system and the nucleus. We wish to understand
how the interaction between the different components of this intermediate filamentnucleus
connection is regulated during cellular processes, such as cell division and
differentiation, and define the role of nesprin-3 in mechanotransduction. The
physiological roles of these proteins and their interaction are studied in model
organisms, including mice and zebrafish.
Modulating podocyte adhesion Modulation of cell adhesion can be achieved in a
variety of ways ranging from the expression of integrins with different affinities for
their ligands to their activation by binding to intracellular molecules, leading to an
increase in avidity, i.e. the collective binding of single integrins referred to as integrin
clustering. The tetraspanin CD151 is thought to increase integrin-mediated adhesion
strengthening, but the mechanism by which this occurs is entirely unclear. Deletion
or mutation of this tetraspanin can cause focal segmental glomerulosclerosis leading
to kidney failure as we and others have previously shown in mouse models and
human disease. We now have evidence that CD151 does neither alter integrin affinity
or their expression levels, leaving integrin clustering as the most likely mechanism of
increasing their adhesive strength. An assumption we are currently testing.
We furthermore discovered that podocytes lacking CD151 loosen their cell-cell contacts
at far lower shear stress than wild-type cells possibly explaining why in mouse models
proteinuria is observed before abnormalities at the podocyte-basement membrane
interface. As interference with the contractile Rho-ROCK-myosin axis can prevent the
cell-cell-contact disruption in vitro, we intend to test drugs blocking this pathway in
vivo hopefully delaying onset of CD151KO induced nephropathy.
nesprin-3 in zebrafish Like mice, zebrafish express two isoforms, nesprin-3a and
nesprin-3b. As opposed to mice, in which nesprin-3b lacks the complete first spectrin
repeat, zebrafish nesprin-3b only lacks seven amino acids in this domain.
Immunoprecipitation data as well as co-localization studies in vitro showed that these
seven amino acids are crucial for binding to plectin and its recruitment to the nuclear
envelope (NE). Both nesprin-3 isoforms are expressed in the epidermis and the skeletal
muscle during embryonic development, as demonstrated by in situ hybridization.
To further analyze the localization of nesprin-3, we have generated a polyclonal
antibody recognizing the zebrafish variant of this protein. In addition to nesprin-3
staining at the NE in cells in the epidermis and skeletal muscle, we also observed a
specific staining of the myotendinous junctions, which suggests that there might be
other, as yet unidentified, isoforms of nesprin-3, not associated with the NE. We are
currently looking into the function of nesprin-3 by means of morpholino-mediated
knock-down and knock-out zebrafish.
Zebrafish as a model for the blistering disease fraser syndrome From a forward
genetic ENU mutagenesis screen in zebrafish we isolated mutants that develop
embryonic epidermal blisters. Some of these mutants map at the Fras1, Frem1 and
Frem2 loci. Fras/Frem proteins play a critical role in epidermal-mesenchymal
interaction during embryonic development and in humans, loss of their function
results in the embryonic blistering disorder Fraser syndrome (FS). In FS, epidermal
adhesion defects cause large blisters during embryonic development resulting in
various defects, e.g. cryptophthalmos (fused skin covering the eyes). We identified, in
one of the epidermal blistering mutants, a missense mutation in one of five calcium-

inding Calx-b domains of Frem2 that leads to an Ala2115Glu amino acid substitution.
The Ala2115 residue in the Calx-b domain of Frem2 is conserved in many species and
it is also conserved in the Calx-b domains of other unrelated proteins. Furthermore,
the Ala2115 is situated in close proximity of the calcium binding pocket and
substitution by glutamic acid is likely to affect the binding of calcium. Interestingly,
mutations in the human Calx-b domain of Frem2 have been linked to FS, suggesting
that the Calx-b domain is essential for the function of Frem2. We aim to identify the
function of the Calx-b domain in Frem2 and reveal the mechanism by which the
Fras/Frem complex establishes embryonic epidermal adhesion.
expression of the orphan protein Plet-1 during trichilemmal differentiation of
anagen hair follicles We have previously generated and selected the rat monoclonal
antibody 33A10 based on its reaction pattern in the developing mouse mammary
gland. Further analysis revealed that 33A10 also reacts with cells in the hair follicle, a
highly regenerative system of different cell lineages undergoing specific programs of
terminal differentiation. 33A10 reactivity is confined to the membrane of the most
differentiated cells of the outer root sheath (ORS) and to the terminally differentiated
companion layer cells of the isthmus region, which contact the cuticle. Moreover, the
33A10-defined antigen was shown to accumulate during ORS cell differentiation in
culture, suggesting that the protein recognized by 33A10 is expressed during
trichilemmal differentiation. Expression profiling of several cell lines and transient
transfection experiments revealed that the antigen recognized by 33A10 is identical to
the orphan protein, Placenta-expressed transcript (Plet)-1. Further characterization of
Plet-1 showed it to be a glycosylphosphatidylinositol (GPI)-anchored glycoprotein
with N-linked carbohydrates. Knockdown of Plet-1 protein levels in ORS keratinocytes
decreased migration in a wound-scratch assay, while it increased adhesion to
Collagen I and IV. Interestingly, expression of Plet-1 during wound healing is induced
at the leading-edge of migrating keratinocytes that are in close proximity to the eschar,
suggesting that Plet-1 may contribute to keratinocyte interactions with inert tissues.
Kindlin-1 regulates inside-out signaling of ß1 integrins and adhesion
strengthening Loss-of-function mutations of kindlin-1 cause the skin blistering
disorder Kindler syndrome (KS). Although the kindlin family members have been
firmly established as modulators of integrin activity, the exact role of kindlin-1 is
poorly understood. KS patients show microblistering along the dermal-epidermal
junction, multiplication of the basement membrane, and occasionally also
progressive hair loss. We have previously described a similar phenotype for mice that
lack the integrin a3b1 in the epidermis, suggesting a link between a3b1 and kindlin-1.
We are investigating kindlin-1 functions using human keratinocytes derived from KS
patients. Laminin-332 synthesis and deposition, as well as the formation of
hemidesmosome-like structures are not impaired. However, there is hardly any
clustering or activation of b1-containing integrins in the patient’s cells. In accordance,
adhesion to and cell spreading over different b1 integrin substrates do not occur, and
cytoskeletal organization and focal adhesion (FA) assembly are disturbed.
Reconstitution with kindlin-1 restores adhesion and cell spreading. Furthermore, it
induces reorganization of the actin cytoskeleton, FA assembly, and phosphorylation
of FA proteins. Treatment of kindlin-1-deficient cells with the b1 integrin-activating
antibody TS2/16 also rescues adhesion, cell spreading, cytoskeletal organization, FA
assembly, and integrin signaling to paxillin and FAK, indicating that kindlin-1 is not
per se required for outside-in integrin signaling or integrin-mediated cytoskeletal
organization. However, kindlin-1 expressing cells are much more resistant to shear
stress than knockout cells, and this is dependent on the integrity of the actin
cytoskeleton. We conclude that kindlin-1 regulates inside-out but not outside-in
signaling of b1 integrins. In addition, kindlin-1 reinforces the integrin connection to
the actin cytoskeleton and thus regulates adhesion strengthening, providing
resistance against mechanical stress.
Publications (continued)
23
Cell Biology i
Alonso-García N, Inglés-Prieto A,
Sonnenberg A, De Pereda JM. Structure of
the Calx-beta domain of the integrin beta4
subunit: insight into function and cationindependent
stability. Acta Crystallogr D
Biol Crystallogr 2009;65:858-71
Groot AJ, Khattabi ME, Sachs N,
Van der Groep P, Van der Wall E,
Van Diest PJ, Sonnenberg A, Verrips CT,
Vooijs M. Reverse proteomic antibody
screening identifies anti-adhesive VHH
targeting VLA-3. Mol Immunol
2009;46:2022-8
Buijsrogge JJ, De Jong MC,
Kloosterhuis GJ, Vermeer MH, Koster J,
Sonnenberg A, Jonkman MF, Pas HH.
Anti-plectin autoantibodies in subepidermal
blistering diseases. Br J Dermatol
2009;161:762-71
de Pereda JM, Ortega E, Alonso-García N,
Gómez-Hernández M, Sonnenberg A.
Advances and perspectives of the
architecture of hemidesmosomes: Lessons
from structural biology. Cell Adh Migr
2009;3:361-4
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 2009 (in press)
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 α-actinin. Exp Cell Res
2009 (in press)
Margadant C, Sonnenberg A. Integrin-
TGF-b crosstalk in fibrosis, cancer and
wound healing. EMBO Rep 2009 (in press)
Raymond K, Richter A, Kreft M, Frijns E,
Janssen H, Slijper M, Pretzel-Wunder S,
Langbein L, Sonnenberg A. Expression of
the orphan protein Plet-1 during
trichilemmal differentation of Anagen hair
follicles. J Invest Dermatol 2009 (in press)

24
Cell Biology i
Group leader Wouter Moolenaar
Wouter Moolenaar PhD Group leader
elisabetta Argenzio PhD Post-doc
Maikel Jongsma PhD Post-doc
Bas Ponsioen 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. LPA receptor signaling has
been implicated in wound healing, embryonic development and cancer. LPA is
produced by a secreted lysophospholipase D (lysoPLD), named autotaxin (ATX), an
autocrine motility factor for tumor cells and strongly implicated in tumor progression
and angiogenesis. Our current research focuses on the characterization of novel LPA
receptors as well as the regulation of ATX and its in vivo functions. Since ATX is
found overexpressed in various cancers, whilst LPA promotes tumor progression in
mouse models, ATX qualifies as an attractive target for therapy. Using high-throughput
screening of chemical compound libraries, we have identified and validated novel
small-molecule inhibitors of ATX (collaboration H. Ovaa). Our work should lead to
novel ways of interfering with LPA receptor signalling and with inappropriate LPA
production in the tumor-stroma microenvironment.
lPA receptor signaling
The LPA-RhoA signaling pathway: identification of CLIC4 as a new player
LPA receptors strongly couple to the G(13)-RhoA pathway to regulate the actin
cytoskeleton. Through live-cell imaging studies, we discovered 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 vascular tubulogenesis. However, its function(s) and regulation remain unclear.
CLIC4 recruitment is strictly dependent on the LPA-induced G(13)-RhoA pathway.
Membrane-targeted CLIC4 does not enter the plasma membrane, nor does it
modulate transmembrane chloride currents. CLIC4 translocation depends on
conserved residues, whose equivalents are critical for the enzymatic function of
GSTs. We conclude that membrane-targeted CLIC4 does not function as an inducible
chloride channel but rather as a RhoA-regulated transferase that catalyses a yet
unknown chemical reaction. Through yeast two-hybrid screening, we have identified
novel binding partners of CLIC4 that shed new light on its possible function(s) in
LPA signalling.
Figure 2: Rapid recruitment of GFP-CLIC4 to the plasma membrane in response to RhoA-activating
receptor agonists (TRP or LPA). Note that CLIC4 recruitment is accompanied by RhoA-mediated cell
contraction.
Novel LPA receptors: LPA5 and P2Y5
LPA serves as a chemoattractant for many cell types, both normal and malignant.
It does so by acting on classic ‘Edg-family’ receptors (termed LPA1-3). We recently
discovered an exception to that rule in that LPA5, a novel ‘non-Edg’ family receptor,
mediates a strong inhibition of melanoma cell chemotaxis. LPA5 stimulation leads to
a persistent rise in cAMP, which may explain the inhibitory action of this LPA
receptor. Thus, selective agonists of LPA5 may prove useful to counteract the
migration of metastatic melanoma cells.
Another novel LPA receptor, termed P2Y5 or LPA6 (encoded by P2RY5), couples
specifically to RhoA activation and. Strikingly, P2Y5 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 Paul Krimpenfort and Margriet Snoek, Division of Molecular Genetics).
Autotaxin, a secreted lysophospholipase D implicated in tumor progression
Autotaxin (ATX) is a unique, secreted lysophospholipase D that produces LPA from
lysophosphatidylcholine. Overexpression of ATX promotes tumorigenesis and tumor
aggressiveness in experimental animals. Through genetic studies in mice, we
previously have established that ATX is essential for vascular development. To
identify small-molecule inhibitors of ATX, we have performed high-throughput
screening of chemical compound libraries (collaboration Huib Ovaa and
collaborators). Several positive hits were obtained that inhibited ATX activity as well
as ATX-mediated tumor cell migration. Among these hits are several new chemical
structures that have been optimized to reach IC(50) values in the nanomolar range.
Specificity, selectivity and toxicity of these pharmacological inhibitors are being
further evaluated in cell-based and animal studies. (see the report by Huib Ovaa).
Strikingly, an optimized 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.
Finally, our collaborator Anastassis Perrakis (Division of Biochemistry) has made
significant progress in solving the crystal structure of ATX. These structural studies
should help to better understand how ATX activity is regulated and to optimize our
small-molecule inhibitors.
ATX isoforms
The so-called ‘melanoma-derived’ isoform of ATX, termed ATXm, contains a 52-residue
polybasic insert of unknown function in the catalytic domain. We find that the ATXm
insert is cleaved by the protease furin, yet proteolytic cleavage does not affect the
catalytic activity of ATXm. It appears that ATXm cleavage occurs at or near the cell
surface, while both cleavage products remain covalenty linked to keep ATXm
functionally intact. The polybasic insert confers increased affinity for heparin on
ATXm, but proteolytic cleavage did not alter heparin binding affinity. We hypothesize
that cleavage of ATXm may serve to modulate the interaction of ATXm with an as-yetunidentified
binding partner. We are currently exploring this scenario
Publications
25
Cell Biology i
Ponsioen B, van Zeijl L, Langeslag M,
Berryman M, Littler D, Jalink K,
Moolenaar WH. Spatiotemporal regulation
of chloride intracellular channel protein
CLIC4 by RhoA. Mol Biol Cell.
2009;20:4664-72
Jonkers J, Moolenaar WH. Mammary
tumorigenesis through LPA receptor
signaling. Cancer Cell. 2009;15:457-9
Pamuklar Z, Federico L, Liu S,
Umezu-Goto M, Dong A,
Panchatcharam M, Fulerson Z,
Berdyshev E, Natarajan V, Fang X,
van Meeteren LA, Moolenaar WH,
Mills GB, Morris AJ, Smyth SS.
Autotaxin/lysopholipase D and
lysophosphatidic acid regulate murine
hemostasis and thrombosis. J Biol Chem.
2009;284:7385-94
Jalink K, Moolenaar WH. G proteincoupled
receptors: the inside story.
BioEssays. 2009 (in press)

26
Cell Biology i
Group leader John Collard
John Collard PhD Group leader
Sandra iden PhD Post-doc
Saskia ellenbroek MSc PhD student
Sander Mertens MSc PhD student
Helma van Riel Technical staff
Ronny Schaefer Technical staff
genetiC ContRol of invASion AnD MetAStASiS
The aim of our research is to identify and characterize genes that play an essential
role in invasion and metastasis of tumor cells. 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 identified different invasioninducing
genes by retroviral insertional mutagenesis in combination with in vitro
selection of invasive T-lymphoma cells. Of these, the invasion-inducing Tiam1 gene
encodes an activator (GEF) for the Rho-like GTPase Rac. Rho family proteins, which
include Cdc42, Rac 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 promotes E-cadherin-based cell-cell adhesions
whereas knock down of Tiam1 leads to loss of cell-cell adhesions and epithelialmesenchymal
transition in MDCKII cells. Furthermore, Tiam1 associates with Par3
of the Par polarity complex and thereby regulates polarity processes in various cell
types and different contexts. In keratinocytes, 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 to junctional adhesion molecule A (JAM-A), which functions as a scaffold
to initiate epithelial cell polarity. In the absence of cell-cell adhesions, epithelial cells
develop front-rear polarization and migrate in a persistent fashion involving Tiam1-
Par complex signaling. Tiam1 in conjunction with the Par complex also influences
directional protrusion outgrowth in astrocytes. In T lymphocytes, Tiam1-Par signaling
is required for chemokine- and S1P-induced Rac activation and subsequent directional
cell migration. Tiam1-deficient 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 efficient crawling of T-cells on
endothelial cells.
tumorigenicity in tiam1-deficient mice Tiam1-deficient (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 Tiam1 function in tumorigenesis induced by other oncogenic signaling
pathways and in different cell types. Tiam1 is a Wnt-responsive gene and promotes
b-catenin/TCF-induced intestinal tumor formation. Mammary tumor formation and
progression 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. As polarity
proteins also play an important role in processes related to different aspects of cancer,
we are currently analyzing the function of polarity proteins in tumorigenesis.

Figure 3: Mammary tumor formation induced by MMTV-c-neu – but not MMTV-c-myc - is delayed in
Tiam1-deficient mice (black diamonds) when compared to wild type mice (open circles)
opposing functions of Rac1 and Rac3 Rac1 and Rac3 are highly homologous
proteins of the Rho GTPase family. Rac1 is ubiquitously expressed whereas Rac3 is
primarily expressed in the brain. We found opposing functions for Rac1 and Rac3 in
neuronal cells. Knock-down of Rac1 leads to decreased cell-matrix adhesions and cell
rounding in neuronal cells whereas knock-down of Rac3 stimulates cell adhesion and
neuritogenesis. Both Rac1 and Rac3 interact with Git1, a multifunctional ArfGAP
protein which regulates cell-matrix adhesion and endocytosis. In contrast to Rac1, the
Rac3/Git1 interaction is not mediated by bPix and Rac3 severely attenuates Git1/
paxillin interaction required for cell spreading. Moreover, Arf6 activity is strongly
reduced in Rac3 expressing cells. Our data suggests that Rac3 and Rac1 oppose each
other by differently modulating Git1 function.
Publications
27
Cell Biology i
Strumane K, Rygiel TP, Van der Valk M
and Collard JG. Tiam1-deficiency impairs
mammary tumor formation in MMTV-cneu
but not in MMTV-c-myc mice. J Cancer
Res Clin Oncol 2009;135:69-80
Gérard A, van der Kammen RA, Janssen H,
Ellenbroek SI, Collard JG. The Rac
activator Tiam1 controls efficient T-cell
trafficking and route of trans-endothelial
migration. Blood 2009;113:6138-47
Hajdo-Milasinovic A, Moneva Z,
Collard JG. Rac3 inhibits adhesion and
differentiation of neuronal cells by
modifying Git1 downstream signaling.
J Cell Sci 2009;122:2127-36

28
Cell Biology i
Group leader Metello Innocenti
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
Figure 4: Filopodia form in the absence of
the Arp2/3 complex. RNAi was used to
downregulate the expression of the Arp2/3
complex in Hela cells. After serum
starvation, the cells were stimulated with
EGF, fixed and stained with rhodaminephalloidin
to specifically detect F-actin.
ACtin DynAMiCS in CAnCeR CellS
The polymerization of actin monomers into filaments 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 efficiently form filaments.
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
filaments 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/ruffles are the primary organelles of motility for
cells adopting mesenchymal-type movement. WAVE proteins confine 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/
ruffles to advance. WASP proteins are instead implicated in endocytosis and
trafficking: 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 filopodia, finger-like extensions of the plasma membrane. At variance from
lamellipodia, the role of filopodia in cell migration is still debated. mDia2 also
regulates vesicle trafficking, which provides supplies to the leading edge of crawling
cells.
Regulation of WAve2- and n-WASP-based complexes The NPF activity of
WAVE2 and N-WASP, two ubiquitous members of the WASP/WAVE-family
proteins, is controlled by protein-protein and protein-lipid interaction. WAVE- and
N-WASP-based core complexes have been shown to spatially and temporally restrict
Arp2/3-dependent actin polymerization. However, these core complexes 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 specificity to both WAVE and N-WASP 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
filopodia are likely to be fundamental in completing both developmental and
homeostatic programs, little is known about their formation and functions in cell
migration. Do filopodia 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 modified cell line that allows us
to induce filopodium 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 filopodium-regulatory proteins and reveal the filopodium protein
signature, respectively.

BioPHySiCS of Cell SignAling
Our lab employs biophysical techniques to study cell signaling events with high spatial
and temporal resolution. Electrophysiological and advanced imaging 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, a key regulator of podosomes, controls 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 (ECM). 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. FN van Leeuwen (Nijmegen), we
identified the atypical ‘channel-kinase’ TRPM7 as novel component of the
invadosome ring. TRPM7 is a transmembrane ion channel fused to a protein kinase
domain which functions as a mechanosensor and regulator of local Ca 2+ entry.
Strikingly, forced expression of TRPM7 in neuroblastoma cells is sufficient to induce
podosome formation. PIP 2-hydrolizing receptor agonists trigger TRPM7-mediated
Ca 2+ influx and further promote podosome formation by local inhibition of myosin II
function. Thus, TRPM7 may function as a master regulator of invadosomes under
the control of GPCR signals.
tRPM7 and associated proteins predict breast cancer metastasis We recently
found that TRPM7 confers a highly invasive phenotype on otherwise non-invasive
neuroblastoma cells. Moreover, mRNA expression profiling 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. These findings
provide strong support for a role of TRPM7 in tumor cell dissemination. Mass
spectrometry analysis of TRPM7 immuno-complexes identified some 40 proteins
implicated in cytoskeletal regulation, cell adhesion and -migration. The large majority
of these proteins localizes to podosomes. 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 find that TRPM7 medi ates
local Ca 2+ influx to specifically activate PLCd1 in podosomes, leading to PIP2
hydrolysis and sustaining the TRPM7 open-channel state. TIRF imaging studies
confirm that podosomes function as distinct Ca 2+ microenvironments.
Regulation of the invadosome 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+ -influx dependent feedback loop in the
activation of TRPM7. We have also started analyzing localization and role of novel
invadosome components identified in the mass spec screen, both by imaging of GFP
chimera (e.g., of drebrin, a-actinin 4, myosin IIa, b and c, and SIPA1) and by RNAimediated
knockdown or overexpression. Using FRAP experiments, we found that
whereas podosomes are stable cell adhesion structures that live on average for >15
minutes, the individual GFP-tagged components are highly dynamic, exchanging
with the cytosolic pool on average about once every 10 s. Currently we address the
regulation of this rapid exchange. Furthermore, we study the podosome complex by
probing for FRET between GFP- and mRFP-tagged constituent proteins.
As we have identified a host of novel and known invadosome components in the
TRPM7 interactome, we have started to analyze these proteins in the human breast
carcinoma cDNA arrays. Strikingly, out of ~30 analyzed candidates, 13 individual
protein components correlate significantly with poor outcome. We will focus on these
candidates and investigate their role in migration in vitro as well as in nude mice
(collaboration with Linda Henneman and Ed Roos in our division).
Group leader Kees Jalink
Kees Jalink PhD Group leader
Michiel langeslag PhD Post-doc
Bas Ponsioen MSc PhD student
Daan visser MSc PhD student
Jeffrey Klarenbeek MSc Technical staff
Publications
29
Cell Biology i
Jalink K, Van Rheenen J, FilterFRET:
quantitative imaging of FRET by sensitized
emission. In: FRET and FLIM methods.
Editor: Th Gadella, Elsevier, New York
2009;33:289-349
Ponsioen B, van Zeijl L, Langeslag M,
Berryman M, Littler D, Jalink K,
Moolenaar WH. Spatiotemporal regulation
of chloride intracellular channel protein
CLIC4 by RhoA. Mol Biol Cell
2009;20:4664-72
Zwart W, Rondaij M, Jalink K, Sharp ZD,
Mancini MA, Neefjes J, Michalides R.
Resistance to antiestrogen arzoxifene is
mediated by overexpression of cyclin D1.
Mol Endocrinol 2009;23:1335-45
De Groot T, Lee K, Langeslag M, Xi Q,
Jalink K, Bindels RJ, Hoenderop JG.
Parathyroid hormone activates TRPV5 via
PKA-dependent phosphorylation. J Am Soc
Nephrol 2009;20:1693-704
Ponsioen B, Gloerich M, Ritsma L,
Rehmann H, Bos JL, Jalink K. Direct
spatial control of Epac1 by cyclic AMP. Mol
Cell Biol. 2009;29:2521-31
Ponsioen B. Imaging the translocations of
Clic4 and Epac1. Leiden University, 2009
Goedhart J, Van Weeren L, Hink MA,
Vischer NOE, Jalink K, Gadella Jr.TWJ,
Bright Cyan Fluorescent Protein variants
identified by Fluorescence Lifetime
Screening. Nature Methods 2009 (in press)
Jalink K, Moolenaar WH. G proteincoupled
receptors: the inside story. BioEssays
2009 (in press)

30
Cell Biology i
Group leader Wim Van Blitterswijk
Wim van Blitterswijk PhD Group leader
Marcel verheij MD PhD Group leader
Maaike Alderliesten PhD Post-doc
Albert van Hell PhD Post-doc
Shuraila Zerp BSc Technical staff
Publications
Van Blitterswijk WJ, Klarenbeek JB,
van der Luit AH, Alderliesten MC,
van Lummel M, Verheij M. CD95/Fas
downregulation in lymphoma cells through
acquired alkyl-lysophospholipid resistance;
partial role of associated sphingomyelin
deficiency. Biochem J 2010;425:225-34
Zerp SF, Stoter R, Kuipers G, Yang D,
Lippman ME, Van Blitterswijk WJ,
Bartelink H, Rooswinkel R, Lafleur V,
Verheij M. AT-101, a small molecule
inhibitor of anti-apoptotic Bcl-2 family
members, activates the SAPK/JNK
pathway and enhances radiation-induced
apoptosis. Radiat Oncol 2009 (in press)
Baldanzi G, Alchera E, Imarisio C,
Gaggianesi M, Dal Ponte C, Nitti P,
Domenicotti C, van Blitterswijk WJ,
Albano E, Graziani A, Carini R. Negative
regulation of diacylglycerol kinase £
mediates adenosine-dependent hepathocyte
preconditioning. Cell Death Differ 2010
(in press)
Figure 5: GC-modified liposomal
doxorubicin (LDox-GC, 10 mg/kg) reduce
the size of an invasive lobular mouse
mammary carcinoma, in comparison to
conventional LDox or free Dox. Liposomes
were injected on day 0 and tumor volume
was measured in time.
liPiD MetABoliSM in SignAl tRAnSDUCtion
Membrane lipids have both structural and signaling functions. We focus on
diacylglycerol (DAG) as physiological activator of protein kinase C (PKC) and
substrate of DAG kinase, on inositol phospholipids, as mediators in survival
signaling, and on sphingolipids that self-associate and cluster with cholesterol in
dynamic microdomains known as lipid rafts, facilitating apoptosis induction. In this
context we study mechanisms of tumor cell sensitivity and (cross-) resistance to
various apoptotic stimuli, and how exogenous short-chain sphingolipids enhance the
anti-cancer action of doxorubicin and related amphiphilic drugs in vitro and in vivo.
Role of diacylglycerol kinase u in egf receptor signaling Epidermal growth
factor receptor (EGFR) activation is negatively regulated by PKC signaling, which
involves EGFR phosphorylation at Thr654. The PKC activator DAG can be removed
by DAG kinase (DGK) activity. We were the first to clone DGKu and found, in A431
and HEK293 cells, that this isozyme translocated from the cytosol to the plasma
membrane, where it bound the activated EGFR, became tyrosine-phosphorylated and
then moved into EGFR-containing intracellular vesicles. This translocation was
dependent on both activation of EGFR and PKC signaling. The PKC-mediated
changes in EGFR phosphorylation were attenuated by overexpression of DGKu and
augmented by siRNA-induced DGKu downregulation. Our data indicate that DGKu
translocation and activation is regulated by the concerted activity of EGFR and PKC
and that DGu attenuates PKC-mediated Thr654 phosphorylation that is linked to
desensitisation of EGFR signaling (collaboration with N Divecha, Manchester, UK).
Apoptosis sensitivity to anti-tumor alkylphospholipids (APls) Synthetic APLs
such as edelfosine (1-O-octadecyl-2-O-methyl-glycero-3-phosphocholine) and perifosine
(a piperidine analog) induce apoptosis in tumor cells, leaving normal cells relatively
unaffected. APLs are used as anti-cancer agents in the clinic and enhance radiation-
and chemotherapy-induced cell death. APLs incorporate in cell membranes, are
internalized by raft-dependent endocytosis and/or through a lipid transporter. They
then interfere with phospholipid biosynthesis/ signaling and the Akt/PKB survival
pathway.
S49 lymphoma cells can develop resistance to APL (S49 AR cells), which involves
upregulation of the PI3-kinase/PKB survival signaling and is accompanied by crossresistance
to other apoptotic stimuli such as DNA damage and Fas/CD95 ligation.
Resistant cells were defective in raft-mediated endocytosis, in sphingomyelin
synthesis (SMS1), and in SHIP1, a PIP 3 5-phosphatase. Phosphoinositide levels
changed accordingly. We investigate the mechanism underlying the cross-resistance
to multiple apoptotic stress stimuli and the connection between SMS1 and SHIP1
deficiency.
For clinical applications of APLs, see the report by Marcel Verheij, Division of
Radiotherapy.
Short-chain sphingolipids enhance doxorubicin uptake by tumor cells
Doxorubicin (Dox) is often administered as a liposomal formulation (Caelyx ® ), from
which it is gradually released into the interstitial space and then taken up by tumor
cells. We discovered that certain short-chain sphingolipids such as N-octanoylglucosylceramide
(GC), co-formulated in the liposomes, accelerate the cellular uptake
and accumulation of Dox in various tumor cell lines, but not in normal cardiac
myoblasts. We found significantly enhanced anti-tumor efficacy of these GC-modified
liposomes in an A431 xenograft tumor model. Increased drug delivery to tumor cells
was also observed by intravital microscopy in mice with a B16 melanoma implanted
in a dorsal skin flap window chamber (collaboration with G. Koning, Erasmus MC,
Rotterdam). We further tested this GC-enrichment in a more clinically relevant
tumor model, a spontaneous E-cadherin- and p53-deficient fast-growing invasive
lobular mouse mammary carcinoma. In this model the GC-enriched Dox liposomes
showed superior anti-tumor activity compared to conventional liposomes, and led to
prolonged survival of the mice without adverse effects. This new liposomal
formulation of Dox has been patented and seems promising for clinical application.

MeCHAniSMS of MetAStASiS
the chemokine receptor CXCR4 in carcinoma metastasis We showed previously
that CXCR4, the receptor for the chemokine SDF-1 (CXCL12), is required for liver
and lung metastasis of CT26 colon carcinoma cells. CXCR4 is not involved in
invasion from the blood since CXCR4-deficient cells colonized these tissues to a
similar extent. In fact, the control CXCR4-proficient CT26 cells did not express
CXCR4 in vitro and therefore had no CXCR4 when they were injected. They
upregulated CXCR4 in vivo after 7 days, i.e. long after invasion of the tissues, whereas
the CXCR4-deficient cells obviously did not. We next observed that the CXCR4deficient
cells proliferated initially at the same rate as controls but stopped growth
when micrometastases were less than 1000 cells in size (< 0.1 mm). In contrast, after
s.c. injection of >100,000 cells, CXCR4-deficient cells formed large tumors, similarly
as controls. This suggested that CXCR4 is particularly important for outgrowth of
single cells, as occurs during metastasis formation. To show this, we s.c. injected cells
in Matrigel. This solidified immediately so single cells remained dispersed and
subsequently grew into multiple tumors. Again, we found that growth of CXCR4deficient
tumors stopped before they reached a size of ~1000 cells, confirming the
notion, and also showing that the role of CXCR4 is independent from the lung and
liver microenvironment. We had generated the CXCR4-deficient cells by expressing a
CXCL12-KDEL ‘intrakine’ that traps CXCR4 in the ER by binding to both the ERresident
KDEL-receptor and newly synthesized CXCR4. The latter is thus trapped in
the ER and prevented from reaching the cell surface. However, the intrakine also traps
CXCR7, the more recently discovered second CXCL12 receptor. In addition CXCR7
binds CXCL11. By selective downregulation of CXCR7 using a CXCL11-KDEL
intrakine, we have demonstrated that CXCR7 is not required for outgrowth of
metastases. As a control, we used a CXCL10-KDEL intrakine, because CXCL11 and
CXCL10 both bind CXCR3. In addition to the colon carcinoma, we found that CXCR4
was also necessary for outgrowth of KEP1 mammary carcinoma, a mouse model for
human invasive lobular mammary carcinoma.
Surprisingly, the CXCR4 ligand CXCL12 is not involved, as demonstrated with
inhibitors and the K1R-CXCL12 antagonist. In fact, CXCL12 is not present in the
Matrigel and not produced by the tumor cells. We are presently investigating
whether MIF (Macrophage migration Inhibitory Factor) is the relevant alternative
ligand. In contrast to CXCL12, MIF is produced by the tumor cells and expression is
increased in vivo. MIF was recently shown to bind to a complex of CXCR4 and CD74,
a previously identified MIF-receptor. MIF-CD74 interaction was also shown to protect
ischemic heart tissue from nutrient deprivation by activating AMPK (AMP-activated
protein kinase).
Our current hypothesis is that CXCR4 is essential for resistance against glucose
deprivation, and that this depends on MIF-1, that acts by binding to a CD74-CXCR4
complex, resulting in the activation of AMPK. Angiogenesis is induced by tumors
only when they are larger than 1 mm and contain more than a million cells.
Therefore, metastases that are smaller than this have no blood supply and have to
adapt to hypoxia and glucose deprivation. Indeed, we found that the AMPK activator
AICAR enhanced survival and proliferation of the CT26 and KEP carcinoma cells at
glucose concentrations between 0 and 5 mM, whereas it had no effect at 25 mM, the
glucose concentration in DMEM medium. We are presently testing the effect of
constitutively active and dominant-negative mutants of AMPK, expressed in these
carcinoma cells, on outgrowth of metastases and resistance to glucose deprivation.
Group leader Ed Roos
31
Cell Biology i
ed Roos PhD Group leader
linda Henneman MSc Post-doc
yvonne Wijnands Technical staff
Magdalena Król Guest
Figure 6: Hypothesis. Tumor cells produce
and secrete the cytokine MIF that binds to a
CXCR4-CD74 complex, which leads to the
activation of AMPK. This allows cells to
adapt to glucose deprivation and thus to
survive and proliferate when blood supply is
still limited, before the onset of angiogenesis
which occurs only when metastases have
grown much larger.

32
CeLL BIoLoGy II
Division head, group leader Jacques Neefjes
Jacques Neefjes PhD Group leader
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
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
Ruud Wijdeven MSc PhD student
Lennert Janssen BSc Technical staff
Publications
Benlalam H, Jalil A, Hasmim M, Pang B,
Tamouza R, Mitterrand M, Godet Y,
Lamerant N, Robert C, Avril MF, Neefjes J,
Tursz T, Mami-Chouaib F, Kieda C,
Chouaib S. Gap junction communication
between autologous endothelial and tumor
cells induce cross-recognition and
elimination by specific CTL. J Immunol
2009;182:2654-64
Kuijl C, Neefjes J. New insight into the
everlasting host-pathogen arms race.
Nat Immunol 2009;10:808-9
Pang B, Neijssen J, Qiao X, Janssen L,
Janssen H, Lippuner C, Neefjes J. Direct
antigen presentation and gap junction
mediated cross-presentation during
apoptosis. J Immunol 2009;183:1083-90
Rocha N, Kuijl C, van der Kant R,
Janssen L, Houben D, Janssen H,
Zwart W, Neefjes J. Cholesterol sensor
ORP1L contacts the ER protein VAP to
control Rab7-RILP-p150 Glued and late
endosome positioning. J Cell Biol
2009;185:1209-25
CeLL BIoLoGy II
ANTIGeN PReSeNTATIoN By MHC MoLeCuLeS
AND THe CoNTRoL of eNDoCyToSIS
Our aim is to understand how the immune system detects infections and tumors and
how to control this system. The immune system is able to recognize and eliminate
tumors but tumors will counteract this system. We are studying both arms of this
response; at the level of control of immune activation and at the level of factors
secreted by tumor cells and peripheral tissue controlling the immune system. Since
antigen presentation processes involve the endosomal pathway, we are also studying
how endocytosis is controlled. This is important as signaling through transmembrane
proteins is controlled during endocytosis and manipulating this system may affect
growth signaling as in tumor cells. We use a large variety of state-of-the-art
technologies like single cell biochemistry, chemistry and high-throughput screening.
Degrading the degradation machinery; the proteasome The proteasome is a very
large and stable intracellular protease critical in antigen presentation by MHC class I
molecules but also in the control of cell division and cancer. How the proteasome is
constructed is reasonably clear but how it is degraded is obscure. In collaboration
with the yeast group of Fred van Leeuwen, we have established a system where the
fluorescent label on proteasomes can be switched genetically to distinguish old from
new proteins on the basis of color. To determine how proteins are degraded, we have
crossed in yeast mutant strains to identify the cell biological pathways controlling
degradation of the proteasome. We have developed techniques to visualize the effects
of the various yeast mutants on proteasome distribution and half-life. Therefore we
use the liquid handling robot and the microarray printer to ‘print’ the different yeast
strains on glass coverslips, such that yeast can be visualized at high resolution by
light microscopy. We have coupled this to a confocal microscope with autofocus
options and a high precision step table. This technology will be used for the (yeast)
genome-wide analysis for factors controlling the ‘life’ of proteasomes, which is
entirely unknown. As the proteasome acts as an example of a large stable protein
complex (like the ribosome, nuclear pore and others) the factors identified may be
tested for other cases as well. We will obviously use the data extracted from the yeast
studies to define pathways and to search for human homologues. These human
homologues can be tested by siRNA or shRNA technology for function in controlling
the life of proteasomes and for effects on (tumor) cell growth and differentiation.
Gap junctions, apoptosis and cross-presentation The proteasome generates
peptides for MHC class I molecules and the cell ensures that these are presented on
the same cell. However, peptides can be presented by antigen presenting cells such as
dendritic cells when made by other cells or in a soluble extracellular form. This process
is called cross-presentation. We have shown that peptides can move from one cell to
its neighbor when connected by so-called gap junctions. During apoptosis this may
not occur and we have studied the cell biology of apoptosis in more detail. We show
that many processes continue when the cell is ‘in apoptosis’ including direct antigen
presentation and cross-presentation after gap junction transfer of peptides. Gap
junctions are broken when cells in apoptosis become apoptotic bodies. Whether every
apoptotic body from a single cell is identical in content and in ability to deliver antigens
for cross-presentation, is unclear. We have made fluorescent cell lines with various
compartments marked and follow these during apoptosis. We will FACS-sort the
apoptotic bodies and test them biochemically and for their immunological response.
Genome-wide screen for factors in MHC class II antigen presentation:
pathway analysis and more MHC class II molecules present fragments of antigens
acquired in the endosomal pathway. This process is critical for the induction of
specific antibody responses and for activation of specific cytotoxic T cells. We aimed
at identifying new factors controlling MHC class II expression and peptide loading
using a FACS-based genome-wide siRNA screen. After 100.000 FACS analyses and

cross-correlation with microarray data from a series of primary human immune cells,
some 233 hits were identified as controlling MHC class II expression and 45 as
controlling peptide loading of MHC class II molecules.
To determine the function of these hits, we developed secondary high-throughput
screens. To identify the hits involved in controlling transcription of MHC class II
molecules, they were silenced by siRNA and expression of the transcription master
regulator CIITA, MHC class II, the invariant chain and MHC class I (for locus
specific effects) were determined by qPCR. This yielded some 9 factors that either
controlled CIITA, the locus or MHC class II transcription. To identify the relationship
between these hits, they were silenced and analyzed by qPCR as well. The data were
analyzed and interpreted yielding three transcriptional feedback networks controlling
MHC class II expression.
To identify the hits acting on the cell biology of MHC class II, all hits were silenced
again and the effect on MHC class II, early endosomes and Golgi was determined by
microscopy. The images were analyzed by Cell Profiler and the parameters derived
from this analysis subclustered in a series of potential networks. Data base analyses
confirmed various networks controlling MHC class II expression.
We have subsequently combined expression and functional (siRNA) data sets to
predict factors controlling MHC class II transport in activated dendritic cells. We
could validate three new factors controlling this process. A series of other factors have
been defined including new GTPases and factors secreted by peripheral tissues. In
addition, we are combining chemical library screens using a strategy identical to that
used for siRNA screening. Combining these data sets should allow prediction of
potential targets for the leads identified which may yield novel target-lead
combinations for manipulation of antigen presentation by MHC class II molecules.
endosomes and MHC class II antigen presentation MHC class II molecules
meet peptides for antigen presentation somewhere in the endocytic pathway.
How endosomes move and fuse is only partly understood but is an essential process
in cell biology as also many growth factor receptors use this pathway for signal
tranduction. We have identified how the dynein motor protein binds to endosomes
(through the Rab7-RILP motor receptor) and how this interaction is controlled by
another intracellular compartment, the ER. The cholesterol-sensor ORP1L exists in
two states corresponding to late endosomal cholesterol levels. Under cholesterol-low
conditions, ORP1L exposes a domain recognized by the ER protein VAP that then
removes the dynein motor from Rab7-RILP resulting in positioning of these vesicles
in the cell periphery. This interaction is prevented at high levels of endosomal
cholesterol, stabilizing the dynein motor which results in endosome clustering at the
minus end of microtubules. These data explains how cholesterol affects interactions
between the ER and late endosomes to control the positioning of the late endosomes.
Rab7-RILP appears to be a critical regulator for many processes on late endosomes.
We have identified a link between minus-end transport and fusion of late endosomes
where both dynein motors and the HOPS complex (that control tethering of vesicles
preceding fusion) use the receptor RILP. This is currently studied in more detail.
expanding the PKB/Akt1 pathway using bacterial infection systems We have
shown that Salmonella activates the PKB/Akt1 pathway for intracellular survival.
Various other kinases were activated as well. These kinases are almost all involved in
cancer and we now use an infection model to study these kinase networks. We have
identified (using siRNA) the phosphatases involved in intracellular survival of
Salmonella and are currently testing where the phosphatases act in the kinase
pathway identified before. Therefore we silence phosphatases in cells that express the
activated form of the various kinases. This should reveal whether the phosphatases
are up- or downstream of the activated kinases. Simultaneously, Huib Ovaa’s group
constructed a chemical phosphatase-inhibitor library which was used in the same
model screening system. This yielded two compounds inhibiting Salmonella
infection. We are currently combining the siRNA data with the chemical library data
to identify new target-lead combinations that may affect the PKB/Akt pathway. Along
similar lines, we are studying how Akt1 controls intracellular survival. We have
identified Rab9 and 14 as two GTPases critical in this process and have - using yeast
two hybrid - identified the effector protein which we are now studying in more detail.
Publications (continued)
33
CeLL BIoLoGy II
Souwer Y, Chamuleau ME,
van de Loosdrecht AA, Tolosa E,
Jorritsma T, Muris JJ,
Dinnissen-van Poppel MJ, Snel SN,
van de Corp, Ossenkoppele GJ, Meijer CJ,
Neefjes JJ, van Ham SM. Detection of
aberrant transcription of major
histocompatibility complex class II antigen
presentation genes in chronic lymphocytic
leukaemia identifies HLA-DOA mRNA as
a prognostic factor for survival.
Br J Haematol 2009;145:334-43
Souwer Y, Griekspoor A, Jorritsma T,
de Wit J, Janssen H, Neefjes J,
van Ham SM. B cell receptor-mediated
internalization of salmonella: a novel
pathway for autonomous B cell activation
and antibody production. J Immunol
2009;182:7473-81
Souwer Y. MHC class II antigen
presentation by B cells in health and disease.
Leiden University, 2009
Kedde M. New RNA playgrounds. Leiden
University, 2009
Figure 1: Genes identified in a genome-wide
RNAi screen affect the localization of MHC
class II molecules in human dendritic cells.
When silenced (lower right) in immature
human dendritic cells (imDC, upper left)
MHC class II disappears from intracellular
vesicles and redistributes to the plasma
membrane. This phenotyope resembles the
localization of MHC class II in mature
dendritic cells (upper right).
Scale bar = 10 mm

34
CeLL BIoLoGy II
Group leader Rob Michalides
Rob Michalides PhD Group leader
Wilbert Zwart PhD Postdoc
Renée de Leeuw MSc PhD student
Cristiane Bentin Toaldo Technical staff
Desiree Verwoerd Technical staff
Publications
Holm C, Kok M, Michalides R, Fles R,
Koornstra RHT, Wesseling J,
Michael Hauptmann M, Neefjes J,
Peterse JL, Stål O, Landberg G, Linn SC.
Phosphorylation of the Oestrogen
Receptor a at Serine 305 and Prediction
of Tamoxifen Resistance in Breast Cancer.
J.Path 2009;217:372-379
Zwart W, Rondaij M, Jalink K,
David Sharp D, Mancini D, Neefjes J,
Michalides R. Resistance to Antiestrogen
Arzoxifene is mediated by overexpression of
cyclin D1. Molecular Endocrinology
2009;23:1335-1354
Carraz M, Zwart W, Phan T, Michalides R,
Brunsveld L. Pertubation of Estrogen
Receptor a Localization with Synthetic
Nona-Arginine LXXLL-peptide Coactivator
Binding Inhibitors. Chemistry and Biology
2009;16:702-711
Kok M, Holm-Wigerup C, Hauptmann M,
Michalides R, Stål O, Linn S, Landberg G.
Estrogen Receptor a Phosphorylation at
Serine-118 and Tamoxifen response in
breast cancer. J.Natl Cancer Inst.
2009;101:1-5
Zwart W. Shedding light on anti-estrogen
resistance and antigen presentation through
biophysical techniques, Thesis University of
Leiden, 2009
Zwart W, De Leeuw R, Rondaij M,
Neefjes J, Mancini M, 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, 2009 (in press)
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 is still sensitive to other
endocrine treatments. The clinical benefits of clarifying pathways underlying
resistance can therefore be profound. We aim to get 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.
eRa and eRb and their response to estradiol and tamoxifen The two variants of
the human Estrogen Receptor, ERa and ERb, differ in their response to estradiol and
tamoxifen, which is determined by the ability of their N-terminal AF-1 domains to
interact with co-factors and by their hinge regions. Through a collaborative action of
both AF-1 and the hinge region, ERa can induce transcription in the presence of its
antagonist tamoxifen, a capacity which is lacking in ERb.
We described in a collaborative study with Luc Brunsveld (Technical University
Eindhoven), a novel way to evaluate the binding between ERa and its cofactors in
vivo, making use of a specific nona-arginine tag on the cofactor fragment that directs
the whole complex after binding, to the nucleoli.
Modifications in eRa associated with resistance to anti-estrogens
Combinations of phosphorylation of specific sites on ERa and overexpression of
cofactors are responsible for resistance to particular anti-estrogens. Resistance to
Fulvestrant requires phosphorylation of Serine 305 by PKA in combination with
phosphorylation of Serine 118 by MAPkinase or overexpression of SRC-1 and cyclin
D1, whereas resistance to arzoxifene is specifically influenced by overexpression of
cyclin D1 as co-factor of ER. This is highly relevant, since these anti-estrogens are
currently in use in the clinic as alternatives for treatment with tamoxifen.
In contrast to ERa, phosphorylation of ERb by either Protein Kinase A or
MAPkinase, did not result in resistance to tamoxifen.
Properties of eRa that is phosphorylated at Serine 305 by Protein kinase A
Protein stability studies indicated that ERa phosphorylated at Serine 305 by Protein
kinase A is more rapidly degraded in the presence of tamoxifen than the wild-type
ERa. Since a transcriptionally active ERa is less stable than a silent ERa, this finding
supports our previous findings that phosphorylation of ERa at Serine 305 by PKA
renders the ERa transcriptionally active in the presence of tamoxifen.
In a collaboration with Pamgene (Den Bosch), we found that ERa that is
phosphorylated at Serine 305 by Protein kinase A recruits cofactors more efficiently
in the presence of tamoxifen than the non-phosphorylated form of ERa. This, again,
supports the mechanism of this form of tamoxifen resistance.
Clinical relevance of phosphorylation of eRa In collaboration with Marleen Kok
and Sabine Linn (Department of Molecular Biology) and Goran Landberg and
colleagues (University of Lund), we found that a specific 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. In addition, we found that a combined measurement of two independent
markers, phosphorylation of the Estrogen Receptor at Serine 305 by Protein kinase A
and PAK-1, identified approximately 60% of all tamoxifen resistant cases of ERpositive
breast cancer.
The quite opposite has been found for phosphorylation of Serine 118 in ERα, which is
associated with a benefit from treatment with tamoxifen. Phosphorylation of Serine
118 in ERa is associated with an activated ERa.
These results indicate that resistance to tamoxifen is largely determined by two
opposite effects of phosphorylation of ERa. They also indicate that application of the
laboratory findings of how modulation of ERa and its cofactors affects sensitivity
towards anti-estrogens will lead to a tailoring of endocrine treatment of breast cancer
patients.

CHeMISTRy AND BIoLoGy of uBIQuITIN-MeDIATeD
PRoTeoLySIS AND ANTIGeN PReSeNTATIoN
The ubiquitin proteasome system and MHC class I antigen presentation T
lymphocytes serve to recognize and eliminate cells that express foreign (e.g. virusderived
or tumor-specific) proteins. The recognition of antigen presenting cells
(APCs) is based on the binding of the T cell receptor to Major Histocompatibility
Complexes (MHCs) that have bound a peptide derived from a foreign protein. The
MHC class I pathway presents endogenously antigens, e.g. viral protein fragments,
produced by cytosolic proteolysis, sequestered within the infected cell. Recognition of
pMHC class I complexes by cytotoxic T cells leads to death of the antigen-expressing
cell. The proteolytic cascades that produce MHC class I epitopes start with
proteasomal degradation.
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 remodelling and Ub recycling. Because of this complexity, tools that allow detailed
studies of the effects of ubiquitination status on protein turnover are urgently needed.
Figure 2: Fluorescent probes can be used to study the distribution and activity of proteasomes in cells.
A). Confocal images showing MelJuSo cells expressing b7-RFP or b1i-GFP incubated with different
probes. From left to right: localization of GFP/RFP-labeled proteasome subunits, localization of probe,
overlay showing colocalization of probe and proteasome. B) CLSM images showing MelJuSo cells
expressing b7-RFP or b1i-GFP pulsed with MG132 (1 h) and chased with read or green fluorescent probes
(1 h). From left to right: localization of GFP/RFP-labeled proteasome subunits after MG132 treatment,
localization of probe after MG132 treatment, overlay. Arrows indicate unstained nucleoli.
Activity profiling with chemical probes Major progress has been made in
understanding cancer on a genetic level but this knowledge is not easily translated
into new drugs. Genetic methods generally do not assign protein function or enzyme
activity, which are often in turn regulated by protein-protein interactions and posttranslational
mechanisms. Post translational modifications and proteolytic processing
modulate protein function, localization, half-life, and protein expression. The study of
protein function is limited by current techniques. Commonly used techniques to study
proteins include radiolabelling of newly synthesized polypeptides, use of antibodies
that may not be specific for the full repertoire of post-translational modifications, and
determination of transcript levels with similar limitations. No single ideal technique
Group leader Huib Ovaa
Huib ovaa PhD Group leader
Alessia Amore PhD Post-doc
farid el oualid PhD Post-doc
Boris Rodenko PhD Post-doc
Anitha Shanmugham PhD Post-doc
Sander Van Kasteren PhD Post-doc
Harald Albers MSc PhD student
Celia Berkers MSc PhD student
Annemieke De Jong MSc PhD student
Reggy ekkebus MSc PhD student
Rieuwert Hoppes MSc PhD student
Henk Hilkmann Ing Technical staff
Karianne Schuurman MSc Research
assistant
Publications
35
CeLL BIoLoGy II
Celie PH, Toebes M, Rodenko B, Ovaa H,
Perrakis A, Schumacher TN. UV-induced
ligand exchange in MHC class I protein
crystals. J Am Chem Soc 2009;131:12298-304
Crawford LJ, Windrum P, Magill L, Melo JV,
McCallum L, McMullin MF, et al.
Proteasome proteolytic profile is linked to
Bcr-Abl expression. Exp Hematol
2009;37:357-66
Hadrup SR, Toebes M, Rodenko B,
Bakker AH, Egan DA, Ovaa H, et al. Highthroughput
T-cell epitope discovery through
MHC peptide exchange. Methods Mol Biol
2009;524:383-405
Middeldorp J, Kamphuis W, Sluijs JA,
Achoui D, Leenaars CH, Feenstra MG, et
al. Intermediate filament transcription in
astrocytes is repressed by proteasome
inhibition. FASEB J 2009;23:2710-26
Ovaa H, van Leeuwen F. Chemical biology
approaches to probe the proteome.
Chembiochem 2008;9:2913-9
Rodenko B, Toebes M, Celie PH,
Perrakis A, Schumacher TN, Ovaa H.
Class I major histocompatibility complexes
loaded by a periodate trigger. J Am Chem
Soc 2009;131:12305-13

36
CeLL BIoLoGy II
Publications (continued)
Ruckrich T, Kraus M, Gogel J, Beck A,
Ovaa H, Verdoes M, et al.
Characterization of the ubiquitinproteasome
system in bortezomib-adapted
cells. Leukemia 2009;23:1098-105
Toebes M, Rodenko B, Ovaa H,
Schumacher TN. Generation of Peptide
MHC class I monomers and multimers
through ligand exchange. Curr Protoc
Immunol 2009;Chapter 18
Figure 3: MHC class I-mediated immunity:
antigen processing, -loading -presentation
and the T cell kill. A. The proteasome is
responsible for protein degradation assisted
by other (endo)-proteolytic activities
affording epitopes that can be loaded into
MHC class I complexes which may result in
a T cell-mediated kill of the antigen
presenting cell.
Figure 4: MHC exchange. A. Principle of
MHC class I-exchange and exchange-based
screening assay based on fluorescence
anisotropy (FP). B. A modified influenza A
epitope is shown, designed to disintegrate
upon UV exposure.
currently exists and methods that determine protein function, enzyme activity and
receptor function directly have to be developed. This is the primary aim of our lab,
while we focus on targeted cytosolic proteolysis and antigen presentation.
Our lab aims to develop tools to profile cellular enzymatic activities associated with
post-translational modification of proteins with ubiquitin and we study proteasome
activity, antigen production and antigen presentation by designing tools that inferfere
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 leading to 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 centred around one central theme: chemistry and biology of targeted
cytosolic proteolysis and antigen presentation, and currently divided into three main
topics:
(1) ubiquitin chemistry and biochemistry
(2) proteasome action
(3) MHC class I antigen presentation
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 fluorescent proteasome activity reporters we have analyzed
proteasome activity in mice. We were able to monitor pharmacological inhibition in
vivo and we were able to visualize active proteasomes in (primary) human cells both
by confocal microscopy and flow 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 specific 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 efficiently 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°C). Importantly, when UV-mediated cleavage is performed in the presence of
another MHC binding peptide, an efficient ligand exchange reaction results in a class
I molecule complexed with the epitope of choice. Ligands that disintegrate on
command have now been identified for various different human MHC alleles (i.e.
HLA-A2 and -A1, -A3, -A11 and –B7), 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 fluorescence polarization assays.
Improvements and development of new technologies to profile 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.

NANoBIoLoGy
The cellular nanocosm in normal and cancer cells is made up of numerous types of
macromolecular complexes or biological nanomachines. These form functional
modules that are organized into complex subcellular networks and are altered in
malignancy. 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 fixatives, 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. X-ray and NMR data can be ‘docked’ or fitted 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 in our laboratory for electron tomography. 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 have 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 film. 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 film. This results in specimen
instability during image acquisition and subsequent difficulty 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 film using electrostatic charging. The ribbon of vitreous cryosections
remains in place during transfer and storage and is devoid of stamping
related artefacts. We have illustrated these improvements by exploring the structure
of putative cellular 80S ribosomes within 50nm, vitreous cryo-sections of
Saccharomyces cerevisiae.
Future challenges for the next year include visualizing individual conformations
(imposed by drugs) of GFP tagged ribosomes and using volumetric classification
algorithms based on maximum likelihood statistical approach. Our initial attempt at
understanding the spatial arrangements of the ribosome in situ is exciting and hints
at the possibilities of imaging the vast majority of other biological machines in which
the cellular spatial arrangement and molecular interactions that still remain elusive.
One specific example that we now address is the transmembrane protein conduit
machinery, the SecY/Sec61 system, which is attached to the ribosome and
responsible for translocating nascent proteins into the lumen of the endoplasmic
reticulum. With new technologies being developed by us and others a resolution of
2.5 nm is approaching, allowing template matching the X-ray crystal structure with
the ultimate goal to explain the structure of our averaged density maps.
Recombinant BCG and its translocation In collaboration with the group of
Michael Brenner (Harvard, Boston) we published that after prolonged infection in
macrophages and dendritic cells, M. tuberculosis translocates from phago-lysosomes
to the cytosol (Cell. 2007;129:1287-98). The BCG vaccine strain failed to translocate
from the phago-lysosome. The translocation into the cytosol was totally unexpected
and against textbook knowledge. This difference in localization might give an
explanation for the ineffectiveness of BCG as a MHC class I / CD8 triggered vaccine.
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
Cveta Tomova PhD Post-doc
Matthijn Vos PhD Post-doc
Abdallah Abdallah 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
Collaborators
37
CeLL BIoLoGy II
Jose Jesus Fernandez: Centro Nacional de
Biotechnologia-Conejo Superior de
Investigaciones Cientificas, Madrid, Spain.
Bram Koster: Department of Molecular Cell
Biology, Leiden University Medical Center,
Leiden, the Netherlands
Henny Zandbergen: Department of Physic,
Technical University Delft, the Netherlands
Helmut Gnaegi: Diatome Ltd, Biel,
Switzerland
Figure 5

38
CeLL BIoLoGy II
Publications
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. 2009
Gregorieff A, Stange DE, Kujala P,
Begthel H, van den Born M, Korving J,
Peters PJ, Clevers H. The ets-domain
transcription factor Spdef promotes
maturation of goblet and paneth cells in the
intestinal epithelium. Gastroenterology.
2009;137:1333-45
Pierson J, Sani M, Tomova C, Godsave S,
Peters PJ. Toward visualization of
nanomachines in their native cellular
environment. Histochem Cell Biol.
2009;132:253-62
Herz J, Pardo J, Kashkar H, Schramm M,
Kuzmenkina E, Bos E, Wiegmann K,
Wallich R, Peters PJ, Herzig S,
Schmelzer E, Krönke M, Simon MM,
Utermöhlen O. Acid sphingomyelinase is a
key regulator of cytotoxic granule secretion
by primary T lymphocytes. Nature
Immunol. 2009;10:761-8
Sato T, Vries RG, Snippert HJ,
van de Wetering M, Barker N, Stange DE,
van Es JH, Abo A, Kujala P, Peters PJ,
Clevers H. Single Lgr5 stem cells build
crypt-villus structures in vitro without a
mesenchymal niche. Nature.
2009;459:262-5
Van der Flier LG, van Gijn ME, Hatzis P,
Kujala P, Haegebarth A, Stange DE,
Begthel H, van den Born M, Guryev V,
Oving I, van Es JH, Barker N, Peters PJ,
van de Wetering M, Clevers H.
Transcription factor achaete scute-like 2
controls intestinal stem cell fate. Cell.
2009;136:903-12
Zhang L, Lee SY, Beznoussenko GV,
Peters PJ, Yang JS, Gilbert HY, Brass AL,
Elledge SJ, Isaacs SN, Moss B, Mironov A,
Hsu VW. A role for the host coatomer and
KDEL receptor in early vaccinia biogenesis.
Proc Natl Acad Sci U S A. 2009;106:163-8
Looking further into the difference in localization can bring us closer to a better
vaccine against tuberculosis but perhaps also against virus induced tumors such as
HPV. The aim of our current work is to determine which regions of difference (RDs)
between the M. tuberculosis and BCG genomes are important for translocation. In
collaboration with the lab of Roland Brosch from Pasteur in Paris, we started with
looking at BCG with a knock-in of the extended RD1 region (BCG:RD1), as this
region is now known to encode the ESX-1 secretion system recently coined as a novel
type VII secretion system (Abdallah AM et al., Type VII secretion. Nat Rev Microbiol.
2007;5:883-91). We found that this bacterium can be seen in the cytosol of the cell
after 7 days of infection and conclude that the ESX-1 system (in a BCG background) is
sufficient for translocation. In addition we are investigating ESX-5, another type VII
secretion system from M. marinum (Bitter, VUmc Amsterdam). One of our next aims
is to characterize the structure of ESX-1 secretion system by cryo electron tomography.
The proposed 3D-EM studies complement and expand upon on-going efforts to
provide novel insights into the spatial organization of molecular machines within
cells as outlined in the paragraph above. Currently we are also investigating by
cryo-immunogold EM several different recombinant BCG based vaccine candidates.
These are hypothesized to translocate from the phagosome to the cytosol using pore
forming proteins from other bacteria, such as Listeriolysin from Listeria
monocytogenes (Kaufmann, Berlin) and Perfringolysin O from Clostridium
perfringens (Fulkerson, Aeras, Rockville USA).
Prions Prion diseases are caused by accumulation of an abnormally folded isoform
(PrPS c ) of the cellular prion protein (PrP C ). We are performing cryo-electron
tomography on prion preparations, in order to solve the 3D structure of prions.
In collaboration with Jesus Requena (University of Santiago de Compostela, Spain)
we have examined purified samples of the proteinase K-resistant core of hamster
PrP Sc , vitrified in the recently obtained FEI vitrobot. The preparations were found to
contain 2 nm fibres, sometimes twisted together with other fibres. The data were
consistent with a model in which individual fibres are comprised of ‘strings’ of
monomers. We are currently reproducing these data.
Single intestinal stem cells The intestinal epithelium is the most rapidly selfrenewing
tissue in adult mammals. In collaboration with the lab of Hans Clevers
(Hubrecht Institute, Utrecht) we have recently demonstrated the presence of
approximately six cycling Lgr5 +ve stem cells at the bottom of a small intestinal crypt
(Barker N, et al., Nature. 2007;449:1003-7). We have now established long-term
culture conditions under which single crypts undergo multiple crypt fission events,
whilst simultaneously generating villus-like epithelial domains in which all
differentiated cell types are present. Single sorted Lgr5 +ve stem cells can also initiate
these crypt-villus organoids. We conclude that intestinal crypt-villus units are selforganizing
structures, which can be built from a single stem cell in the absence of a
non-epithelial cellular niche. We recently demonstrated the existence of a long-lived
pool of cycling stem cells defined by Lgr5 expression and intermingled with postmitotic
Paneth cells at crypt bottoms. We have now determined a gene signature for
these Lgr5 stem cells. One of the genes within this stem cell signature is the Wnt
target Achaete scute-like 2 (Ascl2). Transgenic expression of the Ascl2 transcription
factor throughout the intestinal epithelium induces crypt hyperplasia and ectopic
crypts on villi. Induced deletion of the Ascl2 gene in adult small intestine leads to
disappearance of the Lgr5 stem cells within days. The combined results from these
gain- and loss-of-function experiments imply that Ascl2 controls intestinal stem cell
fate.

DIVISION OF EXPERIMENTAL THERAPY
MOLECULAR PATHOLOGY AND MOLECULAR EPIDEMIOLOGY
OF BREAST AND COLORECTAL CANCER
Genome-wide genomic profiling in hereditary breast cancer (PI Petra Nederlof)
Breast tumors from BRCA1 and BRCA2 mutation carriers show specific chromosomal
changes, and these genomic profiles 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-fixed paraffin embedded (FFPE) tumor material. We showed
that tumors (N=48) from BRCA1/2 negative breast and ovarian cancer (HBOC)
families only two tumors showed a BRCA1-like profile, inactivation of BRCA1 was
confirmed in one case (promoter methylation). In collaboration with Ans van den
Ouweland (Rotterdam), we could show that deletions in exon1-2 of BRCA1 resulted in
a BRCA1-like profile. In collaboration with Dr. Peter Devilee (Leiden) we have
analyzed tumors from BRCA1/2 negative HBC families (BRCAx). The tumors show
heterogeneous profiles but within families the differences were smaller. This group
will be extended in order to identify BRCAx subgroups that may allow linkage
analyses within subgroups, in order to find additional breast cancer genes. We further
evaluated the p53 mutations in BRCA1 and sporadic cases, and showed a high
incidence of truncating mutations in BRCA1 tumors. Furthermore, sporadic basallike
breast tumors resemble BRCA1 tumors in their aCGH profile, IHC profile and
p53 mutations, and frequently show inactivation of BRCA1 by methylation.
Genetic determinants of breast cancer risk and breast cancer outcome We
validated a number of breast cancer risk susceptibility loci (common SNPs) as part of
our ‘Breast Cancer Outcome Study Of Mutation carriers’ (BOSOM) cohort (breast
cancer patients aged C) and MDM2
SNP309 (-410T>G) play a role in breast cancer survival, specifically within the group
of patients with p53-mutated tumors (by p53 IHC). We confirmed this finding in a
cohort of patients for which tumors were p53 genotyped. These findings are in line
with the available biological evidence, and in the future, may attain clinical
significance for models of breast cancer prognosis or treatment
Validation of the 70-gene prognosis-signature in breast cancer subpopulations
Previous studies of the 70-gene prognosis-signature demonstrated that women with
cancerous lymph node involvement and a ‘low-risk’ genomic signature had a
considerably better prognosis than their ‘high-risk’-signature counterparts.
We undertook a validation project to test the prognostic value of the 70-gene
signature in women with 1-3 involved lymph nodes as well as for postmenopausal
women. The majority of breast cancer patients are older, postmenopausal women
who are increasingly being offered adjuvant chemotherapy despite the more
favorable biological characteristics of their tumors, such as a lower proliferation rate
and a high endocrine sensitivity. This validation study provides evidence that the 70gene
prognosis-signature (MammaPrint), is also a predictor of disease outcome in
postmenopausal breast cancer patients and in women with 1-3 involved lymph nodes.
The detection and prediction of circulating tumor cells in breast cancer
patients We previously developed a multi-marker mRNA QPCR-based detection
platform for the semi-quantitation of tumor cell load in the peripheral blood of breast
39
EXPERIMENTAL THERAPY
Division head, group leader Laura van ‘t Veer
Laura Van ’t Veer PhD Group leader
Petra Nederlof PhD PI and Academic staff
Frans Hogervorst PhD Academic staff
Flora Van Leeuwen PhD Academic staff
Sabine Linn MD PhD Academic staff
Senno Verhoef MD PhD Academic staff
Annegien Broeks PhD Academic staff
Marjanka Schmidt PhD Senior Post-doc
Youji He MD PhD Post-doc
Tim Molloy PhD Post-doc
Esther Lips PhD Post-doc
Lorenza Mittempergher PhD Post-doc
Mihaela Didraga PhD Post-doc
Marleen Kok MD PhD student
Stella Mook MD PhD student
Sjoerd Bruin MD Clinical fellow
Astrid Bosma Technical staff
Linde Braaf Technical staff
Renske Fles Technical staff
Richard Van Hien Technical staff
Sten Cornelissen Technical staff
Izabela Mikolajewska Technical staff
Kim Brandwijk Technical staff
Lennart Mulder Technical staff
Selected publications
Ahmed S, BCAC Working group,
Schmidt MK, Broeks A, van Hien RR,
Cornelissen S, Easton DF. Newly
discovered breast cancer susceptibility loci
on 3p24 and 17q23.2. Nat Genet.
2009;41:585-90
Broeks A, Braaf L, Wessels LFA,
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
profile. IJROBP. 2009 (in press)
Joosse SA, Van Beers EH, Tielen IH,
Horlings H, Peterse JL, Hoogerbrugge N,
Ligtenberg MJ, Wessels LF, Axwijk P,
Verhoef S, Hogervorst FB, Nederlof PM.
Prediction of BRCA1-association in
hereditary non-BRCA1/2 breast carcinomas
with array-CGH. Breast Cancer Res Treat.
2009;116:479-89

40
EXPERIMENTAL THERAPY
Publications (continued)
Kok M, Holm-Wigerup C, Hauptmann M,
Michalides R, Stål O, Linn S, Landberg G.
Estrogen Receptor-{alpha} Phosphorylation
at Serine-118 and Tamoxifen Response in
Breast Cancer. J Natl Cancer Inst. 2009
Kok M, Koornstra RH, Margarido TC,
Fles R, Armstrong NJ, Linn SC,
Van 't Veer LJ, Weigelt B. Mammospherederived
gene set predicts outcome in patients
with ER-positive breast cancer. J Pathol.
2009;218:316-26
Mook S, Schmidt MK, Weigelt B, Kreike B,
Eekhout I, Van de Vijver MJ, Glas AM,
Floore A, Rutgers EJT, 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. 2009
(in press)
Mook S, Bonnefoi H, Pruneri G,
Larsimont D, Jaskiewicz J, Sabadell MD,
Macgrogan G, Van't Veer LJ, Cardoso F,
Rutgers EJ. Daily clinical practice of fresh
tumour tissue freezing and gene expression
profiling; logistics pilot study preceding the
MINDACT trial. Eur J Cancer.
2009;45:1201-8
Ruijs MW, Broeks A, Menko FH,
Ausems MG, Wagner A, Oldenburg R,
Meijers-Heijboer H, Van 't Veer LJ,
Verhoef S. The contribution of CHEK2 to
the TP53-negative Li-Fraumeni phenotype.
Hered Cancer Clin Pract. 2009;1:4
Schmidt MK, Tommiska J, Broeks A,
Van Leeuwen FE, Van 't Veer LJ,
Pharoah PDP, Easton DF, Shah M,
Humphreys M, Dork 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
Research, 2009 (in press)
He Y, Van ’t Veer LJ,
Mikolajewska-Hanclich I,
Van Velthuysen M, Zeestraten ECM,
Nagtegaal ID, Van de Velde CJH,
Marijnen CAM. PIK3CA mutations predict
local recurrences in rectal cancer patients.
Clinical Cancer Research, 2009 (in press)
cancer patients, which showed both high specificity and sensitivity. Recently we
validated this assay in collaboration with the Radiumhospitalet in Oslo, Norway
(B Naume), assaying a retrospective series of 776 early-stage breast cancer patients
from which blood was collected at time of diagnosis and stored frozen (median
follow-up for relapse free survival 6.2 years and 7.6 for breast cancer specific survival).
The assay proved to be a powerful outcome predictor, with patients in which circulating
tumor cells were detected having a significantly poorer relapse-free and overall
survival. This assay represents a powerful tool that could be used for prognostication
in the clinic, and is currently being adapted and validated in other tumor types and
also as a predictor of treatment response in patients.
Specific DNA aberrations associated with colorectal cancer metastasis The
overall five year survival of colorectal cancer (CRC) is 57% and up to 50% of all
patients will develop metastasis. We are using array-CGH to investigate genome-wide
chromosomal aberrations in primary and liver metastasized colorectal tumors. We
found that primary colorectal tumors that developed liver metastasis have a unique
chromosomal signature. We are now working on a validation study to validate the
liver metastasis classifier in combination with mutation analysis. Secondary research
will be done in operated colorectal liver metastasis for understanding the clinical
behavior after surgery. The ability to predict liver metastasis and understanding of
their clinical behavior by genomic aberrations is important for understanding the
biology of the tumor and may lead to more individualized disease management.
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 profile for the risk of LR in rectal cancer patients, allowing for selection of
patients for pre-radiotherapy (RT). 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 short-term pre-RT
followed by TME or TME alone. 215 samples with > 50% tumor content and high
RNA quality were subjected to Illumine bead-array and gene expression analysis is
in processing. 2. By using parallel extracted DNA, we have indicated that PIK3CA
mutations can be used as a biomarker in identifying rectal cancer patients with an
increased risk for LR. Comparison to irradiated patients within the TME trial
revealed a beneficial effect of pre-RT on PIK3CA mutant patients in preventing LR.
Our findings suggest that prospective evaluation of PIK3CA mutation status could
reduce overtreatment by preoperative radiotherapy for the low-risk patients who
might otherwise only experience the side-effects.
Quantitative Expression Profiling of RNA from FFPE and from Fresh Frozen
Tissues using DASL Illumina platform Tissues samples collected during surgeries
and biopsies are often fixed in formalin, followed by embedding in paraffin. Many of
these samples represent clinical outcomes with the potential to provide critical
insight into expression profiles associated with complex disease as the breast cancer.
Unfortunately, FFPE archival methods often lead to partial RNA degradation, limiting
the amount of information that can be derived from such samples. The project aims
to evaluate if the DASL-based expression profiling assay, specifically designed as a
gene expression profiling system to generate reproducible data from 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 paraffin 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)).
In light of the promising results obtained with the 502 cancer-related genes DASL
Illumina platform we decided to enlarge the cohort of breast samples pairs (20 paired
breast cancer samples from 2008) and analyze them with the whole genome DASL
platform.

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 response
at all in others. This response is highly dependent on the hormone receptor status of
tumors, where HER2 positive and Triple Negative (TN; ER, PR and HER2 negative)
patients show a response of around 40-50% and HER2- ER+ patients showing a
response in only 5-10% of cases. Therefore there is a strong need for tests that predict
chemotherapy response.
The first idea for such a test was to assess homologous recombination deficiency
(HRD) in tumors from breast cancer patients. These tumors, which include BRCA1
associated breast cancers, are not able to reliably repair DNA double strand breaks
(DSBs), and consequently are hypersensitive to alkylating agents. We have
investigated several markers in HER2 negative breast cancer biopsies that may
correlate with HRD. Forty-three TN and 91 ER+ breast tumors scheduled to be
treated with neoadjuvant chemotherapy were studied. aCGH BRCA1-like and
BRCA2-like patterns were assessed (Figure 1). BRCA1 gene expression and promoter
methylation were determined, and the amplification of the BRCA2 inhibiting gene
EMSY was assessed. From these analyses we concluded that abnormalities associated
with BRCA1 inactivation are found in over half of the TN breast cancers and may
identify tumors sensitive to chemotherapy that causes DNA DSBs. In ER+/HER2-
tumors, a BRCA2-like aCGH profile may be predictive for chemotherapy response.
These findings are now being validated in independent sample series.
Figure 1: aCGH-profile from sporadic, BRCA1-like and BRCA2-like breast carcinoma.
A second idea for such a test is based upon the results of gene expression microarray
analysis. We performed up till now 190 microarrays from tumors of patients treated
with neoadjuvant chemotherapy. The dataset contains now information about 48,000
different transcripts for 97 ER+ samples, 42 TN samples and 51 HER2+ samples. We
plan to perform unsupervised clustering and statistical analysis of microarrays to find
genes differently expressed between responders and non-responders to neoadjuvant
chemotherapy. From these data we try to find markers predictive for neoadjuvant
chemotherapy response.
Development of a human in mouse model of breast carcinoma to optimize
alkylating or targeted treatment In recent years, significant progress has been
made in identifying the various types of breast cancer by histogenetic criteria.
The best known classification concerning invasive ductal carcinoma not otherwise
specified (IDC NOS) comprises ER-positive luminal A and B tumors, HER2-positive
tumors, and basal-like tumors (Sorlie et al., PNAS 2001; 98:10869). Such classifications
have far reaching consequences for the prognosis and response to therapy in breast
cancer. The basal-like, for which no adequate targeted therapy is available yet, and the
HER2-positive tumors have a worse prognosis and occur at a younger age. Moreover,
these tumors tend to develop resistance to conventional and targeted therapy very
rapidly. Therefore, more efficient therapy is warranted to improve patient survival.
In the development of new therapies, the availability of preclinical in vivo models is of
41
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

42
EXPERIMENTAL THERAPY
great importance. Ideally, these in vivo models would recapitulate the heterogeneity in
histogenetic characteristics, expression profiles, signaling pathways, and response to
therapy found in human breast cancers. Xenograft models, in which human tumor
tissue is engrafted directly into recipient mice, could be very suitable for this purpose
(Marangoni et al., Clin. Cancer Res. 2007;13:3989).
We have set up a panel of xenografts in which human breast carcinomas can be studied
in a dynamic and reproducible manner. For this, fresh breast cancer fragments are
obtained from the Department of Pathology and implanted into immunocompromised
mice. Tumor outgrowth occurs 3 to 10 months after implantation. After outgrowth,
tumors are serially transplanted followed by analysis of cytogenetic features, gene
expression profile and histology from both the original tumor and the xenografts.
The breast cancer xenograft models can be used to analyze response to (combination
of) new targeted therapy, and acquisition of drug resistance. We will use results from
histology, gene expression profiling and cytogenetic profiling to assign tumors to
different groups based on possible homologous recombination deficiency. We will
then use the breast cancer xenografts to analyze the response to alkylating agents and
PARP inhibitors.
We have started therapeutic intervention studies on six xenograft models so far. Of
these, one model was derived from a Brca2-mutated tumor, one model was assessed
to be Brca2-like based on array-CGH analysis (Joosse et al., Breast Cancer Res Treat
2009;116:479) and four models were assigned to a Brca1-like group. Preliminary
results show a very good response to cisplatin treatment in the Brca1-like group
(Figure 2), but not in Brca2-like tumors.
Figure 2: aCGH profile and response to cisplatin of xenograft tumor T127.
A: Comparison of aCGH pattern of primary human tumor and xenograft tumor showing highly similar
BRCA1-like aCGH patterns.
B: After implanted tumors reached a size of 200mm3 (100%), tumor size was monitored in animals
without treatment (controls, black lines) or treated with cisplatin (6mg/kg, days 0 and 14). Treatment on
1 tumor was started again after relapse to 100%. Response monitoring in the animals will continue to
study if tumors eventually become resistant to cisplatin treatments.
By conducting therapeutic intervention studies in our breast xenograft models we try
to correlate responses to tumor characteristics in different tumor types to find
predictive factors for different therapeutic strategies. This to optimize tailored
systemic treatment of breast cancer patients.

MECHANISMS AND PREDICTION OF TUMOR RESPONSE
TO RADIATION
We are pursuing two lines of research. The first involves how the cell handles DNA
base damage and single strand breaks, an aspect of radiation damage that has
indicated some novel targets for radiotherapy for cancer. The second focuses on
prediction of local control after radiotherapy using genome wide screening methods
on human tumor biopsies, which has also indicated some potential targets for
improving cancer treatments involving radiotherapy.
Mechanisms and modulation of radiosensitivity
Mechanisms of sensitization by PARP inhibitors
We have shown that expression of a polbeta dominant negative (polbDN)
radiosensitized mammalian cells, of potential clinical relevance, since a significant
proportion of human cancers have polbeta mutations resembling this polbDN. We
also showed that inhibition of base excision and single strand break repair pathways
are involved in this radiosensitization. Since PARP inhibitors also affect these
pathways and are showing great clinical promise, we further pursued the mechanisms
of sensitization by these compounds. We found that PARP inhibition by Olaparib
sensitized cells to killing by alkylating agents (MMS) and radiation but was largely
independent of XRCC1, indicating inhibition of alternative BER sub-pathways not
requiring XRCC1. Alkaline comet assays confirmed inhibition of an alternative BER/
SSBR pathway. In polbeta deficient cells, sensitization by Olaparib to MMS increased
synergistically, indicating inhibition of a backup repair pathway acting on polbetadependent
BER intermediates. Expression of the polbDN (polymerase dead but dRPlyase
proficient) restored sensitization to wildtype levels, demonstrating that PARP
inhibition affected 5’dRP removal. Radiosensitization by Olaparib in polbeta-deficient
cells and was increased by the expression of the polbDN. We conclude that Olaparib
sensitization to MMS and IR resulted mainly from inhibition of polb-independent
alternative repair pathways.
Testing the hypothesis that BER-deficient cells are sensitive to HR inhibition
We hypothesized that synthetic lethality would arise if cells with mutations in the BER
pathway, frequent in human tumors, are treated with homologous recombination
(HR) inhibitors, since HR forms a backup repair pathway for unrepaired single
strand breaks after conversion to double strand breaks. We are currently testing
this hypothesis. We previously showed that the model compound caffeine
(radiosensitization by which has been shown to be HR-dependent) radiosensitized
polbDN-expressing cells more than controls. Since caffeine also inhibits ATM and
ATR, we have further tested an ATM inhibitor. As with caffeine, we saw greater
radiosensitization by the ATM inhibitor in polbDN expressing cells. We are now
testing caffeine and other compounds in additional BER deficient models, including
in XRCC1 knockout cells.
Fanconi Anemia mutations in sporadic head and neck cancer
We have begun a project to study deficiencies in the Fanconi Anemia (FANC)
pathway in head and neck tumors (HNSCC), since FANC patients have an increased
incidence of HNSCC. We are screening for mutations in FANC genes initially with
conventional DNA sequencing, and recently with high throughput deep sequencing.
DNA has been extracted from a first cohort of 30 tumors, of which all FANCA and
FANCG exons have been sequenced (conventional) in four patients. We are presently
testing sensitivity and specificity of clonal sequencing and DNA capture strategies on
a panel of 33 HNSCC cell lines. Results from this project are of potential clinical
relevance since FANC deficiencies are likely to indicate an increased sensitivity to
cisplatin and related drugs, often used in combination with radiotherapy.
Prediction of outcome The purpose of these studies is to understand and predict
causes of failure after radiotherapy, allowing selection of patients for alternative more
effective therapies. We studied a series of early stage larynx tumors from patients
treated with radiotherapy alone. Each patient with a local recurrence was matched
for subsite and T-stage with one or two cured patients, resulting in 19 recurrences
Adrian Begg PhD Group leader
Conchita Vens PhD Research Associate
Sari Neijenhuis MSc PhD student
Monique De Jong MD PhD student
Caroline Verhagen MD PhD student
Ingrid Hofland Technical staff
Manon Verwijs Technical staff
Publications
43
EXPERIMENTAL THERAPY
Group leader Adrian Begg
Begg AC. Predicting response to
radiotherapy: Evolutions and revolutions.
Int J Radiat Biol. 2009 85:825-36
Begg AC. Radiobiology: state of the present
art. A conference report. Int J Radiat Biol
2009 (in press)
Eschrich SA, Pramana J, Zhang H,
Zhao H, Boulware D, Lee JH, Bloom G,
Rocha-Lima C, Kelley S, Calvin DP,
Yeatman TJ, Begg AC, Torres-Roca JF.
A gene expression model of intrinsic tumor
radiosensitivity: prediction of response and
prognosis after chemoradiation. Int J
Radiat Oncol Biol Phys. 2009;75:489-96
Neijenhuis S, Verwijs-Janssen M,
Kasten-Pisula U, Rumping G,
Borgmann K, Dikomey E, Begg AC,
Vens C. Mechanism of cell killing after
ionizing radiation by a dominant negative
DNA polymerase beta. DNA Repair
(Amst). 2009;8:336-46
Van den Broek GB, Wildeman M,
Rasch CR, Armstrong N, Schuuring E,
Begg AC, Looijenga LH, Scheper R,
van der Wal JE, Menkema L, Van Diest PJ,
Balm AJ, Van Velthuysen MF,
Van den Brekel MW. Molecular markers
predict outcome in squamous cell carcinoma
of the head and neck after concomitant
cisplatin-based chemoradiation. Int J
Cancer. 2009;124:2643-50

44
EXPERIMENTAL THERAPY
Publications (continued)
Wildeman MA, Gibcus JH, Hauptmann M,
Begg AC, Van Velthuysen ML, Hoebers FJ,
Mastik MF, Schuuring E, Van der Wal JE,
Van den Brekel MW. Radiotherapy in
laryngeal carcinoma: can a panel of 13
markers predict response? Laryngoscope.
2009;119:316-22
and 33 cures. Pretreatment biopsies were taken and expression profiles measured
(Illumina platform). As reported last year, the putative stem cell marker CD44
emerged as the most significant predictor of local control, employing both data-driven
and hypothesis-driven bioinformatics approaches. To validate these results, we have
now studied a separate matched series of 76 larynx tumors of similar stages and
treatment, 29 with a local recurrence. A tissue microarray (TMA) made from
pretreatment biopsies were stained with and anti-CD44 antibody and each core
scored for fraction of positive cells and staining intensity. A score combining these
two parameters correlated significantly with mRNA expression from the microarray
analyses in a subgroup of the patients where material was available for both methods.
In addition, the immunohistochemistry score correlated with local control in the whole
group, providing a validation of the microarray data in an independent clinical series.
In addition, we measured expression profiles on a series of 33 head and neck
carcinoma cell lines with a range of radiosensitivities both before and up to 6h after
4Gy in vitro (collaboration with R Grenman, Turku, Finland). This resulted in a
‘classifier’ of 288 genes showing a significant correlation with intrinsic radiosensitivity.
Of interest was the presence of epithelial-mesenchymal transiation (EMT) genes. To
study the role of EMT genes more directly, we tested a cell line in which HIF1alpha
containing a deletion in the oxygen dependent degradation domain (HIF1-deltaODD)
was expressed (provided by K.J.Wu, Taiwan). Constitutive expression led to a
mesenchymal phenotype compared to vector controls, and expression profiling
showed corresponding changes in EMT genes. Cells expressing the HIF1-deltaODD
were found to be the more radioresistant, consistent with data on our HNSCC cell
line panel. We are pursuing the role of EMT in determining radiosensitivity,
especially given the known links between EMT and CD44 expression.

RADIATION-INDUCED VASCULAR DAMAGE
Radiotherapy has been shown to be an independent risk factor for cardiovascular
and cerebrovascular disease in long-term survivors of cancer. This manifests as
atherosclerosis in large vessels and telangiectasia 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 telangiectasia (in collaboration with Peter ten Dijke, Leiden)
Radiation-induced telangiectasia strongly resembles the vascular phenotype in the
human disorder hereditary hemorrhagic telangiectasia (HHT). HHT patients display
mutations in genes encoding the TGF-b receptors ALK1 or endoglin, resulting in
reduced receptor levels on the endothelium. ALK1 and endoglin, together with the
receptor ALK5, regulate endothelial cell proliferation and migration. It was thought
that decreased ALK1/endoglin expression disturbed the ALK1/ALK5 signaling
balance, thereby eventually leading to vessel dilation. However, it is now known that
ALK5 is also downregulated in endothelial cells from HHT patients, probably to
compensate for reduced ALK1/endoglin levels, and that telangiectasia only form
when an additional vascular insult triggers erroneous repair.
To study the influence of irradiation on ALK1/ALK5 signaling and telangiectasia
formation, we irradiated human microvascular endothelial cells (HMVEC) in vitro
and kidneys of wild type (Eng +/+ ) and endoglin deficient (Eng +/– ) mice. Irradiation of
HMVEC with 5 Gy increased ALK5 and decreased ALK1 signaling, and was associated
with decreased cell migration and tube formation. At 20 weeks after kidney irradiation
of Eng +/+ mice, endoglin expression was increased but the ALK1 pathway was not
affected. In contrast, ALK5 signaling was enhanced, leading to strong expression of
profibrotic genes CTGF, PAI-1 and collagen type III. This was associated with increased
fibrosis and vascular telangiectasia in the irradiated kidneys. Expression of profibrotic
genes was significantly less in irradiated kidneys of Eng +/– mice than in Eng +/+ mice.
Moreover, Eng +/– mice developed significantly less fibrosis and irradiation-induced
telangiectasia. The latter was somewhat surprising, as endoglin is thought to be
crucial for vessel repair and neovessel formation. However, it is consistent with other
reports that, both reductions and overexpression of endoglin can result in defective
blood vessels, suggesting that a fine regulation of endoglin expression levels is
required to maintain proper vascular morphology.
Endoglin is also expressed in bone marrow derived mononuclear cells. These
circulating cells can contribute to vascular repair by infiltrating perivascular sites
where they secrete angiogenic factors. It has previously been shown that mononuclear
cells from HHT patients have a decreased potential to contribute to vascular repair,
probably because of defective homing and/or secretion of angiogenic growth factors.
Our studies showed that infiltration of CD45 + mononuclear and granulocytic cells
was increased at 20 weeks after irradiation of Eng +/+ mouse kidneys but infiltration
was reduced in Eng +/– mice. We are currently investigating the composition of the
cellular infiltrate, and the respective cytokines and growth factors produced, to gain
insight into how these cells might influence vascular repair and ALK1/ALK5
signalling after irradiation.
Radiation induced atherosclerosis in large vessels (in collaboration with Mat
Daemen, Cardiovascular Research Institute, Maastricht) We established a mouse model
for studying radiation-induced atherosclerosis in the carotid arteries of ApoE-/- mice,
which have increased cholesterol levels and develop atherosclerosis spontaneously
with age, unlike most mice. We have previously shown that single doses (8-14 Gy)
or clinically relevant fractionated irradiation (20 x 2 Gy in 4 weeks) to the neck of
ApoE-/- mice accelerated the development of atherosclerotic plaque. Irradiation also
predisposed to the formation of a vulnerable, inflammatory, thrombotic plaque
phenotype with a thin fibrous cap.
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
45
EXPERIMENTAL THERAPY
Group leader Fiona Stewart
Scharpfenecker M, Floot B, Russell NS,
Ten Dijke P, Stewart FA. Endoglin
haploinsufficiency reduces radiationinduced
fibrosis and telangiectasia
formation in mouse kidneys. Radiother
Oncol. 2009;92:482-91
Scharpfenecker M, Kruse JJCM, Sprong D,
Russell NS, ten Dijke P, Stewart FA.
Ionizing radiation shifts the PAI1/ID1
balance and activates Notch signaling in
endothelial cells. Int J Radiat Oncol Biol
Phys. 2009;73:506-13
Kruse JJCM, Floot BGJ, Te Poele JAM,
Russell NS, Stewart FA. Radiation-induced
activation of TGF-beta signaling pathways
in relation to vascular damage in mouse
kidneys. Radiat Res. 2009;73:506-13
Russell NS, Hoving S, Heeneman S.
Hage JJ, Woerdeman LAE, de Bree R,
Lohuis PJFM, Smeele L, Cleutjens J,
Valenkamp A, Dorresteijn LDA, Dalesio O,
Daemen MJ, Stewart FA. Novel insights
into pathological changes in muscular
arteries of radiotherapy changes. Radiother
Ocol. 2009;92:477-83
Stewart FA, Dörr W. Milestones in normal
tissue radiation biology over the past 50
years: From clonogenic cell survival to
cytokine networks and back to stem cell
recovery. Int J Radiat Biol. 2009;85:574-86

46
EXPERIMENTAL THERAPY
Publications (continued)
De Vreeze RSA, De Jong D, Haas RL,
Stewart F, Van Coevorden F. Effectiveness
of radiotherapy in myxoid sarcomas is
associated with a dense vascular pattern.
Int J Radiat Oncol Biol Phys
2008;72:1480-7
Nyst HJ, Tan IB, Stewart FA, Balm AJM.
Is photodynamic therapy a good alternative
to surgery and radiotherapy in the
treatment of head and neck cancer?
Photodiagnosis Photodyn Ther. 2009;6:3-11
Based on these results, we set up intervention studies, to investigate the potential of
anti-platelet, anti-inflammatory and anti-cholesterol drugs to inhibit development of
radiation-induced atherosclerosis. Drugs were given in the chow from 1 week before
irradiation until termination of the experiment. High dose aspirin (ASA) and
clopidogrel effectively blocked platelet aggregation, while the low dose ASA, nitricoxide
releasing aspirin (NCX 4016) and atorvastatin had no significant effect on
platelet aggregation. ASA inhibited endothelial cell expression of VCAM-1 and
thrombomodulin at 4 weeks after irradiation; eNOS and ICAM-1 levels were
unchanged. After 30 weeks of follow-up, NCX 4016 significantly reduced the total
number of lesions and the number of inflammatory, macrophage-rich lesions in
unirradiated mice, but these effects were not maintained in irradiated mice. ASA did
not reduce lesion number or size, but did lead to formation of collagen-rich ‘stable’
lesions in irradiated mice. Neither clopidogrel nor atorvastatin modified the
development or progression of atherosclerotic lesions in irradiated or unirradiated
vessels. These studies showed that the effects of radiation-induced atherosclerosis
could not be circumvented by standard anti-inflammatory and anti-coagulant therapies,
suggesting more complex underlying mechanistic pathways compared to age-related
atherosclerosis.
Radiation induced cardiac damage (in collaboration with Mat Daemen,
Cardiovascular Research Institute, Maastricht) Both coronary artery disease
(atherosclerosis) and microvascular perfusion defects probably contribute to the risk
of heart failure after irradiation. To investigate these processes in more detail we have
set up procedures for local irradiation of mouse hearts, with sequential examination
of cardiac function before sacrifice and evaluation of changes in morphology and
gene and protein expression.
SPECT/CT images ( 99 Tc-HSA) of mice demonstrated a significant decrease in cardiac
blood volume at 20 weeks after 8 or 16 Gy (23-29%), with a non-significant 9%
decrease in blood volume after 2 Gy. Ultrasound imaging also demonstrated a
significant decrease in end diastolic and systolic volume (32%, 52%) and cardiac
output (19%) at 20 weeks after 16 Gy. Morphological examination showed that
epicardial thickness doubled 20 weeks after 8-16 Gy; this was associated with
macrophage invasion in the epicard. Microvascular density in the myocard was
moderately decreased (13%, ns) at 20 weeks after 16 Gy but increased by 16-20% after
2-8 Gy. No atherosclerotic changes were seen in coronary arteries of irradiated or
control wild type (C57Bl6) mice. Ongoing studies are evaluating functional cardiac
perfusion (gated SPECT/CT after injection of 99m Tc-myoview and staining of heart
tissue for FITC-lectin perfused vessels), as well as morphological and gene
expression changes, at 20-60 weeks after irradiation. Cardiac function, morphology
and vascular patency in irradiated wild type mice will be compared with age matched
atherosclerosis prone ApoE-/- mice, and Eng +/– mice.
Combination of Axitinib with standard chemotherapy (cisplatin and
pemetrexed) in malignant mesothelioma Malignant Pleural Mesothelioma (MPM)
have a high microvessel density and express increased levels of vascular endothelial
growth factor (VEGF), receptors VEGFR1-3, and other angiogenic factors like Platelet-
Derived Growth Factor (PDGF). Recent studies have reported a negative correlation
between the number of vessels in specimens of MPM, high VEGF levels, 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 toxicity of the addition of Axitinib to standard chemotherapy for
patients with previously untreated MPM. One of the major limitations in the
evaluation of ‘targeted agents’ is the lack of non-invasive methods to determine the
effect of a new treatment in an early stage. In an investigational arm of this trial, we
are therefore studying the effects of Axitinib on tumor vascularization. Thoracoscopic
biopsies (obtained before treatment and after three treatment courses) are being
examined for microvessel density, endothelial cell proliferation, expression and
activation of VEGFR2 and PDGFR- b. Moreover, we will measure VEGF levels in
patient serum and test the ‘angiogenic potential’ of patient serum in in vitro
angiogenesis assays. Results from these analyses will be correlated to clinical
chemistry data and non-invasive patient data obtained from CT or X-ray.

PHARMACODYNAMICS OF ANTICANCER DRUGS
DPYD single nucleotide polymorphisms in patients with metastatic colorectal
cancer and toxicity of capecitabine Capecitabine, an oral prodrug of 5-fluorouracil
(5-FU), is indicated amongst others for the first-line treatment of metastatic colorectal
cancer. The enzyme dihydropyrimidine dehydrogenase (DPD), encoded by its gene
DPYD, metabolizes 5-FU for about 85% to its inactive form dihydro-5-fluorouracil.
Approximately 2-3% of the population has a DPD-deficiency; as a consequence these
patients are at increased risk for developing severe toxicity when treated with
standard doses of capecitabine or 5-FU. We investigated the role of polymorphisms in
DPYD on the toxicity of capecitabine.
Methods: Out of 569 patients with metastatic colorectal cancer treated with
capecitabine, oxaliplatin, bevacizumab with or without cetuximab, we selected 50
patients presenting with severe capecitabine-related toxicity. In these cases the entire
DPYD coding region was sequenced, and allele frequencies of observed polymorphisms
were compared to those in 100 control patients, which were randomly selected from
these 569 patients. Subsequently, SNPs that appeared to be of special interest from
this nested case-cohort analysis were assessed in all 569 individuals. These SNPs
were associated with toxicity, survival and dose modifications of capecitabine.
Figure 3: Probability of grade 3-4
diarrhea by genotype
Results: 29 SNPs in DPYD were identified by sequencing the 50 case and 100 cohort
patients. A multivariate analysis including treatment arm (± cetuximab) and gender
revealed that DPYD*2A, IVS9-51T>G and 496A>G were significantly associated with
capecitabine-related toxicity. Furthermore, 2 exonic and 3 intronic SNPs were only
detected in the case population, whereas these were not detected in the cohort patients.
8 of these 29 SNPs were assessed in all 569 individuals. 5 SNPs (DPYD*2A, 2846A>T,
1236A>G, DPYD*6 and 496A>G) were significantly associated with diarrhea grade
3-4 (Figure 3). Patients heterozygous mutant for DPYD*2A or 2846A>T were more
frequently hospitalized due to toxicity and required significant dose reductions of
capecitabine of approximately 50% and 25% on average, respectively (Figure 4).
Figure 4: Dose modifications of capecitabine
by genotype. Cumulative dose of capecitabine
expressed as percentage of the planned dose
according to the protocol for wild type (black
bars) and mutant patients for DPYD*2A or
2846A>T.
SNPs in DPYD were not associated with progression-free or overall survival Conclusions:
Several SNPs in DPYD are predictive for toxicity of capecitabine, especially DPYD*2A
and 2846A>T. A priori dose reductions could be considered for these patients.
Jan Schellens MD PhD Group leader
Jos Beijnen PhD Academic staff
Irma Meijerman PhD External staff
Serena Marchetti MD PhD Academic staff
Maarten Deenen MSc PhD student
Lot Devriese MD MSc PhD student
Geert Frederix MSc PhD student
Suzanne Leijen MD PhD student
Roos Oostendorp MSc PhD student
Artur Burylo Technical staff
Dick Pluim Technical staff
Publications
47
EXPERIMENTAL THERAPY
Group leader Jan Schellens
Oostendorp RL, Beijnen JH,
Schellens JHM. The biological and clinical
role of drug transporters at the intestinal
barrier. Cancer Treat Rev 2009;35:137-47
Oostendorp RL, Buckle T, Beijnen JH,
Van Tellingen O, Schellens JHM. The effect
of P-gp (Mdr1a/1b), BCRP (Bcrp1) and
P-gp/BCRP inhibitors on the in vivo
absorption, distribution, metabolism and
excretion of imatinib. Invest New Drugs
2009;27:31-40
Oostendorp RL, Van de Steeg E,
Van der Kruijssen CM, Beijnen JH,
Kenworthy KE, Schinkel AH, Schellens JH.
OATP1B1 mediates transport of gimatecan
and BNP1350 and can be inhibited by
several classical ABCB1 and/or ABCG2
inhibitors. Drug Metab Dispos
2009;37:917-23
Oostendorp RL, Witteveen PO,
Schwartz B, Vainchtein LD, Schot M,
Nol A, Rosing H, Beijnen JH, Voest EE,
Schellens JH. Dose-finding and
pharmacokinetic study of orally
administered indibulin (D-24851) to
patients with advanced solid tumors.
Invest New Drugs 2009; (in press)

48
EXPERIMENTAL THERAPY
Publications (continued)
Oostendorp RL, Huitema A, Rosing H,
Jansen RS, Ter Heine R, Keessen M,
Beijnen JH, Schellens JH.
Coadministration of ritonavir strongly
enhances the apparent oral bioavailability
of docetaxel in patients with solid tumors.
Clin Cancer Res 2009;15:4228-33
Pluim D, Beijnen JH, Schellens JH,
Van Tellingen O. Simultaneous
determination of AZD1152 (prodrug) and
AZD1152-hydroxyquinazoline pyrazol
anilide by reversed phase liquid
chromatography. J Chromatogr B Analyt
Technol Biomed Life Sci 2009;877:3549-55
Schellens JH, Beijnen JH. Novel clinical
trial designs for innovative therapies. Clin
Pharmacol Ther 2009;85:212-16
Schellens JH. Phase 0 (zero) clinical trials:
More than zero benefit? Eur J Cancer
2009;45:728-29
Circulating tumor cell detection in advanced solid cancer: validation of
QPCR-based detection In the development of metastases, tumor cells intravasate
into the bloodstream giving rise to circulating tumor cells (CTCs). We are performing
a pilot study to explore the potential of CTC detection as a pharmacodynamic and
prognostic marker in various types of solid cancer. In carcinoma patients we are
performing positive selection with anti-EpCam(CD326) (melanoma: anti-MSCP) and
depletion with anti-CD45. Ten types of solid cancer are being investigated: colorectal,
esophageal, gastric, pancreatic, breast, ovarian, prostate, NSCLC, ACUP and
melanoma. After mRNA isolation and cDNA synthesis, selected potential marker
genes are being assessed in QPCR. (This project is carried out in collaboration with
Astrid Bosma, Tim Molloy and Laura van ’t Veer.)
Phase 1 dose escalation study of MEK inhibitor RO4987655, administered
orally as monotherapy in patients with advanced tumors The RAS/RAF/MEK/
ERK signaling pathway plays a central role in cell growth by transferring extracellular
signals from ligand bound cell surface tyrosine kinase receptors (i.e. EGFR
and HER2) to the nucleus via a cascade of specific phosphorylation events. This
pathway is dysregulated in many human cancers and can lead to components being
constantly turned on. RO4987655 is a potent, highly selective ATP non-competetive
MEK inhibitor.
Pre-clinical data have shown specific inhibition of ERK phosphorylation and strong
anti-tumor activity against various tumors. In addition, RO4987655 increased antitumor
activity significantly in combination with different anticancer agents.
Preliminary results in patients as single agent show that RO4987655 is well tolerated.
Skin rash is frequently reported as an adverse effect related to the study drug.
A substantial amount of patients show stable disease.
Phase 1 dose escalation study of Wee1 inhibitor MK1775 in both monotherapy
and in combination with either gemcitabine, cisplatin or carboplatin in adult
patients with advanced solid tumors Tumor cells rely on cell cycle checkpoints for
repair of DNA damage induced by toxic agents. Wee1 is a tyrosine kinase involved in
regulation of cell cycle checkpoints, particularly the G2/M checkpoint. MK-1775 is a
highly selective, small molecule, and an inhibitor of Wee1 kinase activity; it
potentiates the activity of a cytotoxic agent and sensitizes tumor cells to cytotoxic
agents. The protein p53 regulates the cell cycle checkpoints different from those
regulated by Wee1, including the G1 checkpoint. Therefore, the efficacy of MK-1775 is
expected to be greater in p53 deficient tumors.
Pre-clinical data have shown that MK-1775 in vitro inhibits Wee1 activity and induces
G2 checkpoint escape in cell based assays. Furthermore, MK-1775 showed synergistic
effects on cell death induction in p53 deficient cell lines combined with DNA
damaging agents (gemcitabine, cisplatin and carboplatin). In vivo MK-1775 was well
tolerated and showed enhancement of anti-tumor efficacy by gemcitabine,
carboplatin and cisplatin in a nude xenograft tumor model.
Preliminary results in patients show that MK-1775 with chemotherapy is well
tolerated and a substantial amount of patients show stable disease.
Cost-effectiveness of adjuvant breast cancer therapies New and existing
interventions in cancer care have brought significant improvement, however such
improvement can come at a substantial cost. In order to efficiently use limited health
care resources, beyond safety and efficacy, it is necessary to evaluate the relative costs
and benefits of (new) medical technologies. Therefore cost-effectiveness analyses
(CEA)s are performed to allocate resources as efficiently as possible.
Within recent (2000-2009) published CEA’s for the adjuvant treatment of breast
cancer we have seen a large variation in outcomes and methodological quality. To
overcome these variations we develop a new pharmacoeconomic guideline for
oncolytic products specifically, in which essential characteristics of the disease are
taken into consideration. This guideline will increase the usefulness and reliability of
CEA’s in oncology, and therefore enhance it’s value in decision making.

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 obtained on these genes will allow
us to design in the future 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 and novel components of DNA damage signaling
components.
In addition, we lately 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 cancerpreventing
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 some functional miRNA
targets (identified by our genetic screens) are highly conserved throughout evolution.
We postulated that these regions recruit RNA binding proteins (RBPs) that regulate
miRNA function. We performed genetic screens and identified RBPs that can inhibit
or potentiate the accessibility of miRNAs to their target mRNAs. We suggest that the
genetic interaction between miRNAs and RBPs determines developmental processes
and cellular proliferation. We are working now to establish these interactions and
their role in cancer development and progression.
Functional genetic approaches identify cancerous miRNAs MicroRNAs
(miRNAs) are potent post-transcriptional regulators of protein coding genes.
Patterns of mis-expression of miRNAs in cancer suggest key functions of miRNAs in
tumorigenesis. However, current bioinformatics tools do not fully support the
identification and characterization of the mode of action of such miRNAs.
To perform genetic screens for novel functions of miRNAs we developed a library of
vectors expressing the majority of cloned human miRNAs and created corresponding
DNA barcode arrays. In a screen for miRNAs that cooperate with oncogenes in
cellular transformation we identified miR-372 and miR-373, each permitting
proliferation and tumorigenesis of primary human cells that harbor both oncogenic
RAS and active wild type p53 (figure 1). We provide evidence that these miRNAs are
novel oncogenes participating in the development of human testicular germ cell
tumors. By numbing the p53 pathway they allow tumorigenic growth in the presence
of wild type p53. Intriguingly, in a genetic screen for miRNA genes that promote
cellular migration we identified the same miRNA family. We characterized the role of
miR-373 in the induction of tumor metastasis of breast cancer (figure 2).
Second, we have used a novel functional genetic approach and identified miR-221
and miR-222 as potent regulators of p27Kip1, a cell cycle inhibitor and tumor
suppressor. Interestingly, high miR-221 level appears in signatures of poor prognosis
cancers. Using miRNA-inhibitors we demonstrated that certain cancer cell lines
require high activity of miR-221 for the maintenance of low p27Kip1 levels and
continuous proliferation. We show that this interaction plays a role in human
glioblastoma development.
49
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
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
Remco Nagel MSc PhD student
Henning Schaefer MD
Mariëtte Schrier PhD Technical staff
Joachim Oude Vrielink BSc Technical staff
Figure 1: A. A flow-chart of the genetic
screen. Cells transduced with the miR-Lib
were grown for two to three weeks in the
presence or absence of RAS V12 .
Subsequently, the population of inserts in
each condition was recovered and compared
using a barcode miR-array. B. Three
independent miR-Array experiments were
performed. The position of the reproducibly
upregulated miR-Vecs is indicated for each
experiment.

50
GENE REGULATION
Publications
Van Haaften G and Agami R.
Tumorigenicity of the miR-17-92 cluster
distilled. Genes and development (in press)
Drost J and Agami R. Transformation
locked in a loop. Cell 2009;139:654-656
Bartolome RA, Ferreiro S,
Miquilena-Colina ME, Martinez-Prats L,
Soto-Montenegro ML, Garcia-Bernal D,
Vaquero JJ, Agami R, Delgado R,
Desco M, et al. The chemokine receptor
CXCR4 and the metalloproteinase
MT1-MMP are mutually required during
melanoma metastasis to lungs.
Am J Pathol 2009;174:602-612
Huse JT, Brennan C, Hambardzumyan D,
Wee B, Pena J, Rouhanifard SH,
Sohn-Lee C, le Sage C, Agami R, Tuschl T,
et al. The PTEN-regulating microRNA
miR-26a is amplified in high-grade glioma
and facilitates gliomagenesis in vivo.
Genes Dev 2009;23:1327-1337
Nagel R, Clijsters L, Agami R. The
miRNA-192/194 cluster regulates the
Period gene family and the circadian clock.
FEBS J 2009;276:5447-5455
Schaap-Oziemlak A, 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 (USSCs).
Stem Cells Dev. 2009
Scheel AH, Beyer U, Agami R and
Dobbelstein M. Immunofluorescence-based
screening identifies germ cell associated
microRNA 302 as an antagonist to p63
expression. Cell Cycle 2009;8:1426-1432
Figure 2: MCF7 cells stably expressing luciferase and miR-373 were transplanted into SCID mice via tail
vein injection. Bone metastasis in skull and pulmonary metastasis were observed in these mice.
Interplay between RNA-binding proteins and miRNAs on mRNAs regulate
gene expression MicroRNAs (miRNAs) are inhibitors of gene expression capable
of controlling processes in normal development and cancer. In mammals, miRNAs
use a seed sequence of 6-8 nucleotides to associate with 3’ UnTranslated Regions
(3’UTRs) of mRNAs and inhibit their expression. Intriguingly, occasionally not only
the miRNA-targeting site but also sequences in its vicinity are highly conserved
throughout evolution. We therefore hypothesized that conserved regions in mRNAs
may serve as docking platforms for modulators of miRNA activity. Here we
demonstrate that the expression of dead end 1 (DND1), an evolutionary conserved
RNA-binding protein, counteracts the function of several miRNAs in human cells
and in primordial germ cells of zebrafish by binding mRNAs and prohibiting
miRNAs from associating with their target sites (figure 3). These effects of DND1 are
mediated through uridine-rich regions present in the miRNA-targeted mRNAs.
Thus, our data unravel a novel role of DND1 in protecting certain mRNAs from
miRNA-mediated repression. We are now performing large scale analysis to broaden
our early observations and our molecular understanding as to what extent this
phenomena affect cellular growth and cancer
Figure 3: A schematic model depicting the mechanism of DND1 action. The miRNA-RISC loaded with
miRNAs targeting a 3’UTR inhibits its translation. By binding to U-rich regions in the 3’UTR of Nanos,
DND1 block miRNAs from binding to and inhibiting translation, thereby prohibiting miRNA function.

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 specific 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 fly 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 whole-genome
approaches to study the structure and composition of chromatin and the
mechanisms of gene regulation.
One of our major workhorses is our DamID technology, which allows 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 efficiently 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
unprecedented detail.
Reorganization of genome – nuclear lamina interactions during differentiation
The nuclear lamina (NL) has long been thought to be an anchoring site of chromatin
and to participate in the regulation of gene expression, but genomic sequences that
interact with the NL in vivo have remained unknown. Using DamID, we previously
constructed a ~1kb resolution map of the interaction sites of the entire human
genome with the NL in fibroblasts (Guelen et al, Nature 2008). This map showed
that human genome-NL interactions occur via more than 1,300 sharply defined large
domains of 0.1-10Mb in size. We have now analyzed the dynamics of this
organization during cellular differentiation. We generated high-resolution maps of
genome – nuclear lamina interactions in mouse embryonic stem cells and derivative
neural precursor cells and astrocytes. This revealed that a basal chromosome
architecture present in embryonic stem cells is cumulatively altered at hundreds of
sites during lineage commitment and subsequent terminal differentiation. These
alterations involve both single transcription units and clusters of genes. Loss of
lamina contacts often coincides with activation of gene expression. However, some
genes that detach from the lamina remain initially silent and become activated in a
next differentiation step, suggesting that release from the lamina can unlock genes
for activation at a later stage. These results reveal an intricate choreography of
chromosome reorganization during differentiation.
Determinants of Histone H1 binding Linker histones are involved in the
formation of higher-order chromatin structure and the regulation of specific genes,
yet it remains unclear what their principal binding determinants are. We generated a
genome-wide high-resolution binding map for linker histone H1 in Drosophila cells,
using DamID. H1 binds at similar levels across much of the genome, both in classic
euchromatin and heterochromatin. Strikingly, there are pronounced dips of low H1
occupancy around transcription start sites for active genes and at many distant cisregulatory
sites. H1 dips are not due to lack of nucleosomes; rather, all regions with
low binding of H1 show enrichment of the histone variant H3.3. Knockdown of H3.3
causes H1 levels to increase at these sites, with a concomitant increase in nucleosome
repeat length. These changes are independent of transcriptional changes. Our results
show that the H3.3 protein counteracts association of H1, providing a mechanism to
keep diverse genomic sites in an open chromatin conformation.
Group leader Bas van Steensel
51
GENE REGULATION
Bas van Steensel PhD Group leader
Guillaume Filion PhD Post-doc
Jop Kind PhD Post-doc
Katy Kolodziej PhD Post-doc
Alexey Pindyurin PhD Post-doc
Ludo Pagie PhD Bioinformatician
Joke van Bemmel MSc PhD student
Dominika Bijos MSc PhD student
Ulrich Braunschweig MSc PhD student
Wouter Meuleman MSc PhD student
Daniel Peric-Hupkes MSc PhD student
Wendy Talhout BAS Technical staff
Arantxa Rosado BSc Technical staff
Figure 4: Model of the folding of
chromosomes inside the cell nucleus.
Large chromosomal domains (LADs)
contact the nuclear lamina. Genes located
in LADs are typically repressed. LADs are
often demarcated by specific sequences that
may act as borders of LADs.

52
GENE REGULATION
Publications
Vogel MJ, Pagie L, Talhout W, Nieuwland
M, Kerkhoven RM, van Steensel B. Highresolution
mapping of heterochromatin
redistribution in a Drosophila positioneffect
variegation model. Epigenetics
Chromatin 2009;2:1
de Wit E, van Steensel B. Chromatin
domains in higher eukaryotes: insights
from genome-wide mapping studies.
Chromosoma 2009;118:25-36
Braunschweig U, Hogan GJ, Pagie L,
van Steensel B. Histone H1 binding is
inhibited by histone variant H3.3.
EMBO J. 2009; 28:3635-45
Filion G, van Steensel B. H3K9me2
domains are abundant in chromatin of
ES cells (Correspondence). Nature Genet.
(in press)
Van Steensel B, Braunschweig U,
Filion G, Chen M, van Bemmel J, Ideker T.
Bayesian network analysis of targeting
interactions in chromatin. Genome Res.
(in press)
Heterochromatin organization in Drosophila Chromosome rearrangements
can disrupt chromatin domain organization and thereby lead to aberrant regulation
of genes. A classic example (known for more than 70 years) are white-mottled
X-chromosomal inversions in Drosophila. In these inversions, the white eye color
gene becomes partially silenced due to its relocation next to pericentric
heterochromatin. This silencing has been interpreted as spreading of repressive
heterochromatin across the rearrangement breakpoint. However, the extent of this
spreading and the precise pattern of heterochromatin redistribution have remained
unclear. By high-resolution DamID mapping we found that HP1 invades
euchromatin across the inversion breakpoints over ~175kb and ~30kb, causing
de novo association of HP1 with 20 genes. However, HP1 binding levels in these
regions show substantial local variation, and white is the most strongly bound gene.
Remarkably, white is also the only gene that is detectably repressed by heterochromatin.
Thus, heterochromatin can invade a normally euchromatic region, yet the strength
of HP1 binding and the effects on gene expression are highly dependent on local
context. These results reveal how a chromosomal rearrangement can affect
chromatin domain organization and alter the expression of genes in the affected
genomic regions.
A systematic approach to 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 43 chromatin components in Drosophila
cells, based on the DamID profiles of these proteins. Experimental and computational
validation confirm 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 refined 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, high-resolution DamID
maps, which are expected to reveal many new molecular interactions and functions
of the hitherto uncharacterized proteins. At present we have generated informative
binding maps of more than 10 novel proteins, and many others are being tested.
This project will broaden our view of chromatin by identifying dozens of novel
components and predicting their functions and molecular interactions.
Figure 5: Bayesian Network model
of the targeting interactions between
43 chromatin components, based on
genome-wide binding maps. Each
arrow indicates a predicted targeting
interaction; the width of the arrowhead
reflects the confidence level of the
prediction.

EPIGENETIC REGULATION OF GENE EXPRESSION
In eukaryotic cells the DNA is packaged into chromatin by histone proteins. Posttranslational
modifications of the histone proteins and methylation of DNA can affect
chromatin structure and function. Indeed, chromatin modifications are involved in
regulation of gene expression and DNA damage response. Changes in chromatin
modification 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 modifiers 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.
Figure 6. Telomeric silencing in budding yeast;
dark and light sectors in a yeast colony indicate
epigenetic inheritance of active and silent chromatin.
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, influences
heterochromatin structure and the DNA damage response, and has been implicated
in oncogenic transformation in mammals. Our recent studies indicate that the role of
Dot1 in gene silencing and DNA damage response is modulated by other pathways.
We are currently investigating how Dot1 interacts with other factors to promote gene
silencing and to mount a cellular response upon induction of DNA damage. A major
goal of our research is to understand how H3-Lys79 methylation affects chromatin
structure and function. Through a combination of yeast genetics and biochemistry
we found that Dot1, in contrast to other known protein methyltransferases, 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 specific functions in chromatin.
In contrast, the different methyl forms of histone H3K79 showed functional overlap,
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, our results show that H3K79 methylation acts as an antibinding
signal. Since human and yeast Dot1 are structurally very similar, we expect
that similar rules apply to human cells.
53
GENE REGULATION
Group leader Fred van Leeuwen
Fred van Leeuwen PhD Group leader
Iris Stulemeijer PhD Post-doc
Floor Frederiks MSc PhD student
Kitty Verzijlbergen MSc PhD student
Marit Terweij MSc PhD student
Tibor van Welsem Technical staff

54
GENE REGULATION
Publications
Verzijlbergen KF, Faber AW, Stulemeijer IJ,
van Leeuwen F. Multiple histone
modifications in euchromatin promote
heterochromatin formation by redundant
mechanisms in Saccharomyces cerevisiae.
BMC Mol Biol 2009;10:76
Frederiks F, Heynen GJ, van Deventer SJ,
Janssen H, van Leeuwen F. Two Dot1
isoforms in Saccharomyces cerevisiae as a
result of leaky scanning by the ribosome.
Nucleic Acids Res 2009;37:7047-58
Martino F, Kueng S, Robinson P,
Tsai-Pflugfelder M, van Leeuwen F,
Ziegler M et al. Reconstitution of Yeast
Silent Chromatin: Multiple Contact Sites
and O-AADPR Binding Load SIR
Complexes onto Nucleosomes In Vitro.
Mol Cell 2009;33:323-34
Sampath V, Yuan P, Wang IX, Prugar E,
van Leeuwen F, Sternglanz R. Mutational
analysis of the Sir3 BAH domain reveals
multiple points of interaction with
nucleosomes. Mol Cell Biol 2009;29:2532-45
Verzijlbergen KF, Menendez-Benito VM,
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. 2010;107:64-68
Figure 7. A novel 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.
Chromatin dynamics One of the main goals of our group is to understand how
chromatin modifications can have long-term effects on gene expression. Posttranslational
modifications 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 (unmodified 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. Since yeast has only two copies of each
histone gene, it provides a unique system to manipulate histones and study their
inheritance. We developed a novel assay in yeast to determine protein turnover
in vivo. This universally applicable assay called RITE involves differential labeling of
existing and newly synthesized proteins by a genetic epitope-tag switch system.
Using this assay we found that histones throughout the genome are subject to
extensive replication-independent exchange, suggesting that histones and their posttranslational
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.

DYNAMIC INTERACTIONS AT THE NUCLEAR ENVELOPE
The nuclear envelope arguably is the most important border within the eukaryotic
cell. Borders in general are stable, but can reflect the dynamics of an entire
organization. This makes them an attractive platform from which to study a complex
system – such as the eukaryotic cell. The current focus of the lab is nuclear transport,
chromatin dynamics at the nuclear envelope and connections between these two
processes.
Chromatin interactions with nuclear pore complex components Apart from
forming a physical barrier between the nucleus and the cytoplasm, allowing selective
molecular exchange through nuclear pore complexes (NPCs, figure 8), the nuclear
envelope plays an important role in chromatin organization. In particular the nuclear
lamina, which is a thin layer of microfilaments coating the inner nuclear membrane,
had been implicated in the spacial organization of the eukaryotic genome. Less is
known about the nature of chromatin interacting with the NPC.
In yeast, nuclear pore complexes also interact with active genes, attracting or retaining
them at the nuclear periphery. In higher eukaryotes, some NPC components
(nucleoporins) are also found in the nucleoplasm, with so far unknown function.
We have functionally characterized nucleoporin-chromatin interactions specifically at
the NPC or within the nucleoplasm in Drosophila. We analyzed genomic interactions
of full-length nucleoporins Nup98, Nup50 and Nup62 and nucleoplasmic and NPCtethered
forms of Nup98. We found that nucleoporins predominantly interacted with
transcriptionally active genes inside the nucleoplasm. A smaller set of non-active
genes interacted with the NPC. Genes strongly interacting with nucleoplasmic
Nup98 were downregulated upon Nup98 depletion and activated on nucleoplasmic
Nup98 overexpression. Thus, nucleoporins stimulate gene expression away from the
NPC by interacting with these genes inside the nucleoplasm.
Nuclear pore complex components and cancer Genes that interact with and
respond to nucleoplasmic pools of Nup98 or Nup50 are highly enriched in
developmental genes, suggesting an important function of the nucleoplasmic pool of
nucleoporins on fly development. In adition, genes that interact with and respond to
nucleoplasmic Nup98 are enriched in genes that are directly linked to the cell cycle.
These include for example Cyclin B, Bub1 and Mad2. Interestingly, several of the
nucleporin-regulated cell cycle genes have also been implicated in human cancer.
For example, overexpression of Mad2 leads to tumors in transgenic mice and the
human homologues of several of the group are included in ‘death-from-cancer’ gene
signatures: high expression of this signature set of genes correlates with an
unfavourable outcome in several types of cancer.
Nup98 plays a causative yet incompletely understood role in human leukemia.
A large number of different chromosome translocations in mainly acute myeloid
leukemia (AML) result in chimaeric proteins containing the FG repeat part of Nup98
and a wide set of proteins, including homeobox transcription factors such as HoxA9.
Common to all oncogenic Nup98 fusion proteins is that their localization is inside
the nucleoplasm, not at the NPC.
Future research will focus on the role of nucleoplasmic nucleoporins expressed as a
consequence of leukemia-associated chromosome translocations, and whether they
contribute to oncogenesis by promoting expression of cell cycle genes.
Group leader Maarten Fornerod
Maarten Fornerod PhD Group leader
Nikos Xylourgidis PhD Post-doc
Bernike Kalverda MSc PhD student
Michael Röling MSc PhD student
Publications
55
GENE REGULATION
Kalverda B, Pickersgill H, Shloma VV,
Fornerod M. Nucleoporins directly
stimulate expression of cell cycle and
developmental genes inside the nucleoplasm.
Cell (in press)
Xylourgidis N, Fornerod M. Acting out of
character: Regulatory Roles of Nuclear
Pore Complex Proteins. Dev. Cell
2009;17:617-625
Gontan C, Güttler T, Engelen E,
Demmers J, Fornerod M, Grosveld FG,
Tibboel D, Görlich D, Poot RA, Rottier RJ.
Exportin 4 mediates a novel nuclear import
pathway for Sox family transcription
factors. J. Cell Biol. 2009;185:27-34
Figure 8. Nuclear pore complexes in
Xenopus laevis oocyte nuclear envelopes
visualised from the nuclear side by scanning
electron microscopy and showing a
characteristic basket structure. Image
courtesy of Prof. Dr. Terry Allen, Paterson
Cancer Research Institute, Manchester, UK.

56
IMMUNOLOGY
Division head, group leader Jannie Borst
Jannie Borst PhD Group leader
Jonathan Coquet PhD Post-doc
Ulf Geumann PhD Post-doc
Yanling Xiao MD 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
Inge Verbrugge MSc PhD student
Evert De Vries Technical staff
Gerda Van der Horst Technical staff
Publications
Keller AM, Xiao Y, Peperzak V, Naik SH,
Borst J. Costimulatory ligand CD70 allows
induction of CD8+ T-cell immunity by
immature dendritic cells in a vaccination
setting. Blood. 2009;113:5167-75
Manocha M, Rietdijk S, Laouar A, Bhan A,
Borst J, Terhorst C, Manjunath N. Blocking
CD27-CD70 costimulatory pathway
suppresses experimental colitis. J Immunol.
2009;183:270-6
Middendorp S, Xiao Y, Song J-Y, Peperzak V,
Krijger P, Jacobs H, Borst J. Mice deficient for
CD137 ligand are predisposed to develop
germinal center-derived B cell lymphoma.
Blood. 2009;114:2280-9
Peperzak V, Xiao Y, Veraar EAM, Borst J.
CD27 sustains survival of CTL in virusinfected
non-lymphoid tissue by inducing
autocrine IL-2 production. J Clin Invest.
2010;120:168-78
Ribot JC, deBarros A, Pang DJ, Neves JF,
Peperzak V, Roberts SJ, Girardi M, Borst J,
Hayday AC, Pennington DJ, and
Silva-Santos B. CD27 is a thymic
determinant of the balance between
interferon gamma- and interleukin
17-producing gamma/delta T cell subsets.
Nat Immunol. 2009;10:427-36
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.
To determine by which molecular mechanisms CD27 directs the T cell response, we
have used genome-wide expression profiling. In this way, we have identified IL-2 as a
key CD27-directed gene product in primed CD8 T cells. Retrovirus-mediated
reconstitution of influenza virus-specific CD8 T cells showed 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 another mechanism. CD27 upregulates Bcl-x L, but
neutralization of the mitochondrial apoptosis pathway did not restore the survival
defect of CD27-deficient CD8 T cells in vivo. We have identified a serine/threonine
kinase as a direct target of CD27 in recently primed cells that acts as an additional
important downstream component in the pro-survival effect of CD27 during clonal
expansion.
We have also found a chemokine that is under control of CD27 signaling in CD8 T
cells. CD8 donor T cells expressing this chemokine have a profound trans-effect on
naive CD8 T cells in recipient mice and strongly increment their recruitment into a
responder pool. There is evidence that presentation of chemokines on high endothelial
venules promotes the recruitment of naive T cells from circulation into the priming
lymph node tissue. This potential mechanism is currently under investigation.
We demonstrated that CD4 T cells require CD27 to support their own survival, but
also to enable them to imprint memory characteristics into CD8 T cells. In primed
CD4 T cells, CD27 directs the expression of MS4A4B, a tetraspan-like molecule of
unknown function that is diagnostic for T helper-1 cells. Strikingly, the same molecule
was identified as a unique marker for CD8 memory T cells that had received CD27proficient
CD4 T cell help. Our findings suggest that MS4A4B is instrumental in
programming memory CD8 T cells for secondary expansion. Diagnostics with novel
antibodies, biochemical approaches and genetic interventions are underway to
determine its functional contribution.
Recent findings suggest that the functional capabilities of T cells are to a large extent
imprinted in the priming phase, during T cell-dendritic cell (DC) contact.
Development and use of transgenic mice that constitutively express CD70 on immature
conventional DC highlighted the potential of the CD27-CD70 costimulatory pathway.
Transgenic CD70 expression on DC converted immunological tolerance to induction
of full CD8 T cell responsiveness to tumors and viral infection. The transgenic DC
also induced an effective anti-tumor immune response upon adoptive transfer, which
mimics a clinical situation of cancer immunotherapy. We have generated CD70deficient
mice, which will be used as recipients to address the contribution of CD70
on different cell types to the development of the T cell response.
Using CD70- and CD27-deficient mice, we have established that CD27-CD70
interactions support the thymic development of natural regulatory T cells (nTreg).
In addition, it was found that CD27 expression discriminates between functional

categories of gamma delta T cells. Gene expression profiling has suggested a
mechanism by which CD27 directs this functional dissociation.
CD27 is closely related to 4-1BB (CD137) and both molecules sustain lymphocyte
survival. We discovered that mice lacking the 4-1BB costimulatory pathway are
predisposed to develop germinal center B cell lymphoma with characteristics of
human follicular lymphoma. Gene array provided evidence that 4-1BB ligand acts as
a tumor suppressor during the germinal center B cell reaction.
Apoptosis signaling and cancer therapy Death receptors, such as the TRAIL
receptors and Fas/CD95 are TNF receptor family members that share the ability to
induce apoptosis. We aim to further unravel pro-apoptotic signaling by death
receptors and to exploit this knowledge for cancer therapy.
In collaboration with Marcel Verheij (Division of Radiotherapy) we carry out a project
aimed to exploit death ligands to enhance the efficacy of radiotherapy. As proof of
principle, we have used human lymphoid tumor cells. In these cells, radiation can
sensitize for TRAIL receptor- and Fas/CD95 induced apoptosis when the
mitochondrial pathway is blocked and p53 is lacking. We found a significant
combined effect of both treatment regimens on cell death induction, as read out by
apoptosis and clonogenic survival. Not only ionizing radiation, but also other stress
stimuli used in cancer treatment could sensitize the cells to apoptosis induction by
death receptors via the mitochondrion-independent pathway. We found that, in the
cells employed, sensitization occurred by downregulation of c-FLIP molecules that
block death receptor-induced caspase activation at an upstream level. The possibility
to execute tumor cells via a p53 independent extrinsic pathway for caspase activation
is very attractive for combined modality treatment.
After death receptor stimulation, the Bcl-2 family member Bid is cleaved by
Caspase-8 and its carboxy-terminal fragment translocates to mitochondria where it
induces release of pro-apoptotic mediators. It is stipulated in the field that certain cell
types rely on the mitochondrial pathway for death induction (Type II), whereas other
cell types do not (Type I). However, we have established that also Type I cells need the
mitochondrial pathway for execution at the clonogenic level. The pathway makes an
essential contribution to arrest of clonogenicity by overruling the action of Inhibitor
of Apoptosis proteins that block the function of Caspase-9 and effector Caspases after
initial cleavage and are often overexpressed in cancer cells.
We have discovered that Bid function is regulated by a novel form of serine/
threonine/cysteine ubiquitination. This serves to target Bid’s amino-terminal
fragment for degradation, thus liberating its BH3 domain for apoptosis-induction.
Although Bid was seen as a unique mediator of death receptor-induced apoptosis, we
have found that it can also be essential for apoptosis-induction by DNA damaging
stimuli, particularly in case functional p53 is lacking. Use of a large array of Bid
mutants indicated that its mode of regulation is distinct in this scenario, since it is
not cleaved at the conventional sites by upstream caspases or other proteolytic
enzymes.
In a functional genetic screen for mediators of TRAIL-induced apoptosis in MCF-7
breast cancer cells, we have identified a molecule that controls cell surface expression
of TRAIL receptor-1, but not TRAIL receptor-2. Our data indicate that the two
receptors are subject to differential control with regards to their turnover from the
plasma membrane. This is important, since receptor-selective targeted agents
(antibodies and recombinant TRAIL forms) are currently in phase I and II clinical
trials for cancer treatment. It appears that tumor cells can readily escape from
apoptosis-induction by TRAIL receptor-1 by downregulating plasma membrane
expression of the receptor. In collaboration with the group of Jacques Neefjes
(Division of Cell Biology), we have identified a novel mechanism of TRAIL receptor
endocytosis that impinges on a distinct family of ubiquitin ligases.
Publications (continued)
57
IMMUNOLOGY
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
(in press)
Verbrugge I, Wissink EHJ, Rooswinkel RW,
Jongsma J, Beltraminelli N, Dupuis M,
Borst J, Verheij M. Combining
radiotherapy with APO010 in cancer
treatment. Clin Cancer Res. 2009;15:2031-8
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 Regen Med. 2010
(in press)
Figure 1: Germinal center B cell lymphoma
development in CD137 ligand-deficient mice.
Histochemistry with Peanut agglutinin to
define germinal center B cells on spleen
sections of mice diagnosed with progressive
stages of follicular lymphoma (FL)
development in CD137-ligand deficient
mice: I) healthy aged wild-type control, II)
pre-neoplastic lesions, III) early-stage FL,
IV) late-stage FL.
Original magnification: 5x. Specific
staining is shown in brown and
hematoxylin counterstaining in blue.
The diagnosis FL is based on defined
criteria (see Middendorp S et al. Blood
2009;114:2280-9).

58
IMMUNOLOGY
Group leader Ton Schumacher
Ton Schumacher PhD Group leader
Gavin Bendle PhD Post-doc
Andrew Kaiser PhD Post-doc
Shalin Naik PhD Post-doc
Remko Schotte PhD Post-doc
Jenny Shu PhD Post-doc
Carmen Gerlach MSc PhD student
Jeroen Van Heijst MSc PhD student
Carsten Linnemann MSc PhD student
Laura Bies Technical staff
Nienke van Rooij Technical staff
Erwin Swart Technical staff
Mireille Toebes Technical staff
Jos Urbanus Technical staff
DISSECTING VIRUS AND TUMOR-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-specific T cell immunity, and 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 Peptide-MHC tetramers and other
types of pMHC multimers have become an essential tool for the analysis of T cell
immunity. However, the inefficiency of the classical strategy for pMHC generation
has precluded the development of large libraries of MHC multimers for highthroughput
screening, or of a collection of clinical grade pMHC multimer reagents
for cellular therapy. In collaboration with Huib Ovaa (Division of Cell Biology) we
have in the past years addressed this issue, by designing MHC class I molecules
occupied with UV-sensitive ‘conditional’ peptide ligands. This technology that can be
utilized to generate collections of thousands of pMHC reagents has now been
established for 6 different human MHC class I alleles.
While this UV-induced ligand exchange technology has successfully been utilized to
identify T cell antigens in animal model systems by us and others, limitations on
patient sample size preclude an equally comprehensive analysis of T cell immunity
for most human diseases. To tackle this issue, we have developed a combinatorial
coding strategy that allows the parallel detection of a large number of (at least 25)
different T cell populations within a single sample. This technology is based on the
concept that a given T cell specificity is not encoded by a single identifier (in this case
fluorescent proteins or quantum dots), but is encoded by a defined combination of
two or more identifiers. Following establishment of proof of concept (Hadrup et al.,
2009), this technology is currently utilized in a number of epitope identification and
immunomonitoring efforts. To provide two specific examples: First, in collaboration
with Sine Hadrup (now at University of Copenhagen), we have assembled a
collection of 216 melanoma-associated epitopes for the monitoring of both disease-
and therapy-induced melanoma-reactive T cell responses. Second, we have utilized
combinatorial coding to screen for T cell responses against human prostate
carcinoma-associated antigens in an HLA-transgenic mouse model. Early data appear
promising and if confirmed this project should yield prostate carcinoma-reactive
TCRs for future use in our T cell engineering program (vide infra).
Dissection of T cell immunity through genetic tagging The ability to visualize
antigen-specific 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 such lineage tracking we have invested in the development of technologies
with which individual T cells can be tagged with genetic barcodes. A first 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 micro-array analysis can then be used to trace the
progeny of individual cells.
Using a strategy for the retroviral barcode labeling of naïve T lymphocytes we have
analyzed to what extent 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. Furthermore, the observed multiple fates of single CD8+ T cells
can be demonstrated for TCRs of different affinities. In a second line of research, we
have utilized retroviral barcoding to determine to what extent the magnitude of T cell
responses is either determined by the recruitment of antigen-specific T cells from the
naive T cell pool, or by the magnitude of their subsequent clonal burst (van Heijst,
2009). These data demonstrate that for both monoclonal and oligoclonal antigenspecific
T cell populations, regulation of the magnitude of T cell responses occurs

primarily at the level of T cell expansion (figure 2). Consequently, efforts to maximize
vaccine-induced T cell responses should focus on optimizing the magnitude of T cell
clonal bursts. In ongoing work we also aim to establish in vivo strategies for the
genetic tagging of immune cells and other cell types. A transgenic model has been
established to this purpose and is currently the subject of analysis.
Figure 2: Regulation of T cell responses by clonal burst size. Genetic tagging was used to examine how the
magnitude of CD8 T cell responses is regulated. Solid line indicates expected result if T cell response
magnitude is solely determined by alterations in naive T cell recruitment. Dashed line indicates expected
result if T cell response magnitude is solely determined by alterations in clonal burst size. Note that for all
biological variables, regulation of T cell response size occurs primarily through alterations in clonal burst size.
TCR gene therapy (collaboration with Haanen lab) In the past years our group
has developed the retroviral introduction of antigen-specific T cell receptors into
peripheral T cells as a means to induce tumor-specific T cell immunity. In this
strategy, autologous or donor-derived T cell populations are equipped with a TCR of
defined reactivity in short-term ex vivo procedures, and re-infusion of the redirected
cells is used to supply T cell reactivity against defined antigens.
An important recent development in this line of research has been the observation of
acute Graft-versus-Host-Disease (GVHD) in mouse models for TCR gene transfer.
This pathology occurs under conditions in which the in vivo function of TCR
modified T cells is strongly promoted and further experiments have provided formal
proof that this pathology is caused by the action of gene-modified T cells (figure 3).
Furthermore, we have demonstrated that this pathology can be explained by the
generation of so-called mixed dimers that are formed by the joint assembly of
endogenous and exogenous TCR chains. In line with this, the use of engineering
approaches that limit the formation of mixed TCR dimers leads to a substantial
reduction in the occurrence of TCR gene transfer-induced GvHD.
Figure 3: Depletion of TCR-modified T cells reduces TCR gene transfer-induced GvHD. Mice received
T cells modified with either an unmodified OT-I TCR alpha chain or an OT-I TCR alpha chain that
carries an NH2 Myc tag. Note that anti-Myc antibody administration prevents pathology in recipients of
Myc-OT-I T cells but not of untagged OT-I T cells.
With respect to the testing of TCR gene-engineered T cells in clinical trials, we have
now produced a clinical grade batch retrovirus encoding a melanoma-reactive TCR
for the gene modification of T cells of patients with metastatic melanoma.
Importantly, in view of the safety issues seen in mouse models, this TCR has been
modified such that the formation of mixed dimers is to a large extent suppressed.
In a joint effort with the Haanen lab and NKI-AVL pharmacy (Bastiaan Nuijen), we have
over the past year developed the pharmaceutical process that will be utilized to generate
gene-modified T cells for this trial, and recruitment is expected to start in 2010.
Publications
59
IMMUNOLOGY
Bendle G, Haanen JB, Schumacher TNM.
Preclinical development of TCR gene
therapy. Curr Opin Immunol.
2009;21:209-14
Celie PHN, Toebes M, Rodenko B,
Ovaa H, Perrakis A, Schumacher TNM.
UV-induced ligand exchange in MHC class
I protein crystals. J Am Chem Soc.
2009;131:12298-304
Hadrup SR, Bakker AH, Shu CJ,
Andersen RS, van Veluw J, Thor Straten P,
Blank C, Haanen JB, Heemskerk MH,
Schumacher TN. Parallel detection of
antigen-specific T-cell responses by
multidimensional encoding of peptide-Major
Histocompatibility Complexes. Nat
Methods. 2009;6:520-6
Rodenko B, Toebes M, Celie P, Perrakis A,
Schumacher TNM, Ovaa H. MHC class I
complexes loaded by a periodate trigger. J
Am Chem Soc. 2009;131:12305-13
Schumacher TN, Restifo NP. Adoptive T
cell therapy of Cancer. Curr Opin Immunol.
2009;21:187-9
Van den Berg JH, Oosterhuis K,
Hennink WE, Storm G, van der Aa LJ,
Engbersen JFJ, Haanen JBAG, Beijnen JH,
Schumacher TN, Nuijen B. Shielding of
charge is essential for antigen expression
and immunogenicity of nanoparticleformulated
dermal DNA vaccines. J Contr
Rel. 2009 (in press)
Van Heijst JWJ, Gerlach C, Swart E, Sie D,
Kerkhoven RM, Arens R, Schepers K,
Schumacher TNM. Recruitment of
antigen-specific CD8+ T cells in response to
infection is markedly efficient. Science.
2009;325:1265-9
Wright GP, Notley CA, Xue S-A,
Bendle GM, Holler A, Schumacher TN,
Ehrenstein MR, Stauss HJ. Adoptive
therapy with redirected primary regulatory
T cells results in antigen-specific suppression
of arthritis. Proc Natl Acad Sci USA.
2009;106:19078-83

60
IMMUNOLOGY
Group leader John Haanen
John Haanen MD PhD Group leader
Florry Vyth-Dreese PhD Senior staff scientist
Joost van den Berg PhD Post-doc
Bianca Heemskerk Post-doc
Annelies Jorritsma PhD Post-doc
Susanne Quaak PhD Post-doc
Esther Tjin PhD Post-doc
Silvia Ariotti MSc PhD student
Koen Oosterhuis MSc PhD student
Trees Dellemijn Technical staff
Raquel Gomez MSc Technical staff
Willeke van de Kasteele Technical staff
Martin van der Maas Technical staff
Publications
Bendle GM, Haanen JB, Schumacher TN.
Preclinical development of T cell receptor gene
therapy. Curr Opin Immunol. 2009;21:209-14
Griffioen AW, Vyth-Dreese FA. Angiostasis
as a way to improve immunotherapy.
Thromb Haemost. 2009;101:1025-31
Hadrup SR, Bakker AH, Shu CJ, Andersen RS,
van Veluw J, Hombrink P, Castermans E,
Thor Straten P, Blank C, Haanen JB,
Heemskerk MH, Schumacher TN.
Parallel detection of antigen-specific T-cell
responses by multidimensional encoding of
MHC multimers. Nat Methods. 2009;6:520-6
Quaak SG, Nuijen B, Haanen JB, Beijnen JH.
Development and validation of an anionexchange
LC-UV method for the quantification
and purity determination of the DNA plasmid
pDERMATT. J Pharm Biomed Anal.
2009;49:282-8
Quaak SG, van den Berg JH, Oosterhuis K,
Beijnen JH, Haanen JB, Nuijen B. DNA tattoo
vaccination: effect on plasmid purity and
transfection efficiency of different topoisoforms.
J Control Release. 2009;139:153-9
Van den Berg JH, Nujien B, Beijnen JH,
Vincent A, van Tinteren H, Kluge J,
Woerdeman LA, Hennink WE, Storm G,
Schumacher TN, Haanen JB. Optimization of
intradermal vaccination by DNA tattooing in
human skin. Hum Gene Ther. 2009;20:181-9
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 to clinical application. This work is
performed in close collaboration with Ton Schumacher’s and Christian Blank’s
group. The focus is on treatment of patients with solid tumors, especially melanoma
and renal cell carcinoma. A second line of research is focusing on
immunomonitoring. This work is performed under supervision of Florry Vyth-
Dreese. Its primary aim is to evaluate specific and cytokine-based immunotherapies,
using advanced technologies to characterize immune responses in peripheral blood
and at the tumor site. These studies are conducted in collaboration with Ton
Schumacher and Christian Blank (Division of Immunology), Axel Bex (Division of
Surgical Oncology), Michel van den Heuvel (Division of Medical Oncology), Willem
Nooijen (Division of Diagnostic Oncology), Arjan Griffioen (Free University,
Amsterdam) and E Jonash (University of Texas, Houston, Texas, USA).
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 (tattoo), robust T cell responses, of a magnitude sufficient to reject established
subcutaneous tumors, are generated within 14 days. These results were confirmed in
a non-human primate model (in collaboration with BPRC, Rijswijk). We showed that
DNA tattooing results in 10-100-fold increase in vaccine-specific T cells compared to
intramuscular vaccination.
Together with Bastiaan Nuijen (Pharmacy, Slotervaart Hospital) and Wim Hennink
and Gert Storm (Biopharmacy, Utrecht University) 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 (figure 4). 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, four stage IV melanoma
patients have been enrolled in a first phase I clinical trial with DNA tattoo vaccination.
Figure 4: Tattooing procedure of the human skin (A) and typical expression of luciferase (B), visualized
with a light sensitive camera, 18 hours after tattooing. Each area of 50mm2 was tattooed with a different
tattoo setting. Note the marked variation in luciferase signal obtained with different vaccination conditions.
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 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
immunotherapy. Indeed, therapeutic vaccinations targeting these viral antigens have
shown promise in women suffering from cervical cancer.
In the coming years, we will perform a phase I/II study in patients with HPV
16-positive squamous cell cancer of the penis or cervix using the outlined DNA tattoo
vaccination strategy. In preclinical studies we have developed highly immunogenic
and safe HPV 16 E6 and E7 containing DNA vaccines and GMP production for a first
clinical trial has been started.

Adoptive immunotherapy program TIL therapy Adoptive therapy with Tumorinfiltrating
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 IL-2.
Our goals are: to show that this treatment can be given safely at the NKI-AVL, to show
in an randomized controlled phase II trial that this treatment improves progressionfree
survival compared to standard chemotherapy and to perform a comprehensive
analysis of the T cell specificities of the melanoma-reactive TIL prior and after
adoptive transfer. We expect to start treatment of the first patients in spring 2010.
T-cell receptor gene therapy In close collaboration with the group of Ton Schumacher,
we have selected a highly avid T cell receptor (TCR) specific 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.
It has been equipped with extra safeguards to prevent mispairing with endogenous
TCR chains after transduction of peripheral T cells to prevent potential side effects.
Clinical grade culturing and transduction of peripheral T cells with the 1D3-MP-71
retrovirus is now being validated in our GMP facility in order to start a phase I
clinical study in melanoma patients in 2010.
Immunomonitoring of DNA tattoo vaccination trial Recently, we analysed
samples from the first 3 melanoma patients treated in the DNA tattoo vaccination
trial. Biopsies were taken from the vaccination site and studied by immunohistochemistry
showing the presence of CD8 T lymphocytes and activated DCs or put in
culture in the presence of high dose IL-2, resulting in outgrowth of MART-1-specific
CD8 T lymphocytes in 10-500 fold higher numbers compared to healthy donor skin
samples (figure 5). These result show that DNA tattoo vaccination can induce local
CD8 T cell responses. We are awaiting further analyses.
Immune infiltrates in renal cell carcinoma 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 Interferon alpha show enhanced immune
cell infiltration 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)-specific T cells in a human ex vivo in situ skin
explant model of Graft versus Host reactivity (in collaboration with the groups of Els
Goulmy (Leiden University Medical Center) and Eric Spierings (Utrecht University).
Recently, we developed and validated a method, using so-called MHC-dextramers, to
directly enumerate specific T lymphocytes in cryopreserved tissues. Currently, we are
implementing this technique for the study of HY- and HA-1 directed T lymphocytes
in Graft versus Host Disease and could detect HY-directed T lymphocytes in patient
skin lesions.
Human ex vivo in situ skin model for vitiligo and melanoma In collaboration
with Rosalie Luiten (SNIP and Dermatology, Academical Medical Center, University
of Amsterdam) and Cees Melief (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 specific T cell clones
or bulk T cells obtained from vitiligo lesions co-cultured with normal skin tissues, the
induction of vitiligo could be mimicked ex vivo resulting in induction of severe
lesions in this skin model (Van der Boorn, 2009).
Publications (continued)
61
IMMUNOLOGY
Van den Berg JH, Nuijen B,
Schumacher TN, Haanen JB, Storm G,
Beijnen JH, Hennink WE. Synthetic
vehicles for DNA vaccination. J Drug
Target. 2009
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. 2009
Van den Berg JH, Quaak SG, Beijnen JH,
Hennink WE, Storm G, Schumacher TN,
Haanen JB, Nuijen B. Lipopolysaccharide
contamination in intradermal DNA
vaccination: Toxic impurity or adjuvant?
Int J Pharm. 2009
Van den Boorn JG, Konijnenberg D,
Dellemijn TAM, Van der Veen JPW,
Bos JD, Melief CJM, Vyth-Dreese FA,
Luiten RM. Autoimmune Destruction of
Skin Melanocytes by Perilesional T Cells
from Vitiligo Patients. J Invest Dermatol.
2009;129:2220-32
Figure 5: Analysis of the injection site of a
DNA Mart vaccinated melanoma patient
(a) Immunohistochemical analysis of
CD8 T lymphocytes
(b) Flow cytometric analysis of specific
Mart-wild type directed T lymphocytes
detectable after 2 weeks culture

62
IMMUNOLOGY
Group leader Heinz Jacobs
Heinz Jacobs PhD Group leader
Marinus Heideman MSc PhD student
Marc Hogenbirk MSc PhD student
Peter Krijger MSc PhD student
Niek Wit MSc PhD student
Paul van den Berk Technical staff
PROGRAMMED MUTAGENESIS AND LYMPHOMAGENESIS
B cells have the unique capacity to mutate their immunoglobulin genes by
programmed mutagenesis. Our research aims to: i) Unravel the molecular
mechanism of programmed mutagenesis, ii) Determine the targeting specificity of
the mutation process and its contribution to lymphoma development, and iii)
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. We aimed to establish a genome-wide AID binding profile and
identify molecular parameters controlling the targeting of AID. Therefore, we applied
the DamID technique for AID in a hypermutation-competent Burkitt lymphoma cell
line. This unbiased approach revealed that AID is targeted 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 provide a detailed, genome-wide binding profile of AID. This will reveal
abbarant AID target sites (ATS), and help to identify 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.
Ubiquitination of PCNA controls DNA damage responses throughout the cell
cycle Damage-inducible mono-ubiquitination at lysine residue 164 of proliferating
cell nuclear antigen (PCNA-Ub) stimulates translesion synthesis (TLS). We aimed to
determine the role of PCNA-Ub in controlling DNA damage responses throughout
the cell cycle. We show that primary pre-B cells from PCNAK164R-mutant mice are
highly sensitive to methyl methanesulphonate (MMS), cisplatin (CisPt) and UV-C.
Interestingly, the PCNA K164R mutation impedes but does not abolish the progression
of replication forks on UV-C-induced lesions in vivo, suggesting that fork progression
at damaged templates can occur independently of ubiquitination of PCNA.
Furthermore, UV-C treatment of PCNA K164R cells delays cell cycle progression also
outside S phase, suggesting a role for PCNA-Ub-dependent damage tolerance during
DNA replication and DNA repair synthesis. UV-C treatment of wild type and
PCNA K164R cells reveals a major, PCNA-Ub-dependent and a minor PCNA-Ubindependent
subset of TLS-polymerase h (Polh)-foci forming cells, indicating two
distinct modes of Polh recruitment. Our data support a model in which TLS by Polη
requires PCNA-Ub to survive UV-C-induced DNA lesions and generate high affinity
Ig variants during SHM at template A/T.
Dependence of nucleotide substitutions on Ung2, Msh2 and PCNA-Ub during
SHM During SHM, B cells introduce mutations into their Ig genes to generate high
affinity antibodies. Current models suggest a separation in the generation of G/C
transversions by the Ung2 dependent pathway and the generation of A/T mutations
by the Msh2/PCNA-Ub dependent pathway. Our data indicate that PCNA-Ub is
required for A/T mutagenesis downstream of both Msh2 and Ung2. Furthermore, we
provide evidence that both pathways are non-competitive to initiate mutagenesis and
even collaborate to generate half of all G/C transversions. These findings necessitate
an update of present SHM models.

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-specificity. Using specific, non-functional IgH knock-in and transgenic mice,
we showed in collaboration with J Lutz and H-M Jäck from the University of
Erlangen, 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.
Publications
63
IMMUNOLOGY
Krijger PHL, Storb U, Jacobs H. Errorprone
and Error-free Resolution of AID
lesions in SHM. (Book chapter) DNA
deamination and the Immune System.
Editors: Fugmann S, Diaz M, Papavasiliou
Publisher: Imperial College Press. 2010
(in press)
Krijger PHL, Langerak P,
van den Berk PCM, Jacobs H. Dependence
of nucleotide substitutions on Ung2,
Msh2, and PCNA-Ub during somatic
hypermutation. J Exp Med.
200;206:2603-11
Middendorp S, Xiao Y, Song JY,
Peperzak V, Krijger PH, Jacobs H, Borst J.
Mice deficient for CD137 ligand are
predisposed to develop germinal centerderived
B-cell lymphoma. Blood.
2009;114:2280-9
Langerak P, Krijger PHL, Heideman MR,
van den Berk PC, Jacobs H. Somatic
hypermutation of immunoglobulin genes;
Lessons from PCNAK164R mutant mice.
Philos Trans R Soc Lond B Biol Sci.
2009;364:621-9
Van Maldegem F, Scheeren FA,
Aarti Jibodh R, Bende RJ, Jacobs H,
van Noesel CJM. AID splice variants lack
deaminase-activity. Blood. 2009;113:1862-4

64
IMMUNOLOGY
Group leader Christian Blank
Christian Blank MD PhD Group leader
Andrew Kaiser PhD Post-doc
Lisa Borkner MSc PhD student
Anna Hooijkaas MSc PhD student
Jules Gadiot Technical staff
Publications
Bex A, van der Veldt AA, Blank C,
van den Eertwegh AJ, Boven E, Horenblas S,
Haanen J. Neo-adjuvant sunitinib for
surgically complex advanced renal cell cancer
of doubtful resectability: initial experience with
downsizing to reconsider cytoreductive surgery.
World J Urol. 2009;27:533-9
Hadrup SR, Bakker AH, Shu CJ, Andersen RS,
van Veluw J, Hombrink P, Castermans E,
Thor Straten P, Blank C, Haanen JB,
Heemskerk MH, Schumacher TN.
Parallel detection of antigen-specific T-cell
responses by multidimensional encoding of
MHC multimers. Nat Methods.
2009;6:520-6
Schuster K, Gadiot J, Andreesen R,
Mackensen A, Gajewski TF, Blank C.
Homeostatic proliferation of naïve CD8 + T
cells depends on CD62L/L-selectin-mediated
homing to peripheral LN. Eur J Immunol.
2009;39:2981-90
Strizzi L, Postovit LM, Margaryan NV,
Lipavsky A, Gadiot J, Blank C, Seftor RE,
Seftor EA, Hendrix MJ. Nodal as a
biomarker for melanoma progression and
a new therapeutic target for clinical
intervention. Expert Rev Dermatol.
2009;4:67-78
COMBATING TUMOR IMMUNE ESCAPE
Tumor immune escape, homeostatic proliferation and Nodal The aim of our
research is to identify mechanisms that tumors use to escape from anti-tumor
immune responses. One of these immunotherapy approaches is adoptive transfer of
tumor-reactive T cells. A prerequisite for effective tumor growth control is sufficient
expansion and survival of tumor-reactive T cells without exhaustion in vivo. This may
best be achieved by transferring T cells into lymphopenic hosts. Upon the transfer of
these peripheral T cells into lymphopenic recipients, homeostatic proliferation (HP)
is observed. We will characterize these HP T cells further aiming at identifying
markers that we can use to select these T cells for future adoptive T cell therapy.
Another possibility to overcome tumor immune escape is to target tumor antigens
that are specific and essential for tumor stem cells by transduction of autologous
T cells with genes encoding T cell receptor specific for these antigens. One of such
molecules is the embryologic molecule Nodal, which is expressed in melanomas.
We are analyzing this molecule as a target for immunotherapy.
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 co-inhibitory
molecules on the T cell surface. 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 against tumor cells. We could show
that the susceptibility of T cells for PD-1 mediated signals inversely depends on the
tumor antigen-density on tumor cells suggesting that a decreased tumor antigen
expression combined with higher PD-L1 expression may play a role in tumor
immune escape.
To study whether PD-L1 expression is correlated with melanoma progression, we
examined primary tumor and metastases samples from melanoma patients treated at
the NKI-AVL for PD-L1 and MHC expression, T cell infiltration, and tumor associated
antigen (TAA) expression. Clinical approaches to interfere with PD-L1/PD-1
interaction would either be blockade with monoclonal antibodies (currently tested in
phase I and II clinical trials in humans) or suppression of PD-1 T cell surface
expression via siRNA. Our in vitro data show that siRNAs designed to target PD-1
were equally effective as anti-PD-1/PD-L1 mAb.
Homeostatic proliferating T cells for the treatment of cancer Transfer of naïve
peripheral T cells into lymphopenic recipients results in their slow, cytokine-driven
proliferation. During this HP, T cells acquire effector functions, 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. To allow for HP of infused T cells, patients must first be rendered
lymphopenic by chemotherapy alone or combined with total body irradiation.
We dissect the characteristics of these HP T cells with the aim to induce HP in vitro
and avoid the intensive pretreatment of the patient. Characterization of HP T cells
using microarray analysis point to a specific HP phenotype. Currently, the role of
these newly identified molecules in the onset of HP is being tested by means of
transgenic overexpression or siRNA-mediated silencing.
Targeting Nodal in human melanoma Adoptive transfer of T cells genetically
modified to express a TCR specific for tumor-associated antigen (TAA can successfully
target tumors. However, many of these targets are also expressed in normal tissues
resulting in autoimmune side effects. Therefore, there is a need for targets that are
exclusively expressed on the melanoma cell. Recently Nodal was shown to maintain
stem cell like characteristics in melanoma and to be expressed in more aggressive
stages of melanomas. We therefore analyzed tumor samples from stage III/IV
melanoma patients treated at the NKI-AVL, comparing Nodal expression in the
primary tumors to that in metastases of the same patients. We found a higher
percentage of Nodal positive tumor cells within in-transit and lymph node metastases
than in primary tumors.

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.
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. These complexes recognize base.base mismatches and
small loops of unpaired bases, respectively. Subsequent steps involve recruitment of
another heterodimeric protein complex, MLH1/PMS2, activation of exonucleolytic
activity to remove the error-containing DNA strand and resynthesis of a new strand.
MMR can also recognize mismatches that arise by replication of damaged bases. In
particular, O 6 -methylguanine lesions in DNA are processed by the MMR system into
toxic lesions causing cell death. Thus, DNA MMR acts anti-mutagenic and mediates
the toxicity of methylating agents.
Oligonucleotide-directed gene modification Mismatches can also artificially arise
in cells, e.g., during genetic modification of embryonic stem cells (ESC) by classical
gene targeting or by use of single-stranded oligo-deoxyribonucleotides (ssODN).
ssODN are identical to the chromosomal target sequence except for one or a few
centrally located nucleotides that comprise the desired modification. The initial step
in ssODN-mediated gene modification is base paring between the ssODN and its
chromosomal complement. We have found that the resulting heteroduplex is
recognized by the MMR system, imposing a strong impediment to subtle ssONmediated
gene modification in mouse ESC. As readout for ‘oligo targeting’ we measure
re-activation of a chromosomally-located neomycin (neo) reporter gene that carries a
mutation in the start codon. ssODN containing 1-4 altered nucleotides could generate a
novel ATG start codon, resulting in G418 resistance, albeit at extremely low frequency.
We found that MMR deficiency increased the efficacy of oligo targeting 200-500-fold,
reaching frequencies up to 10 -5 -10 -4 (Dekker et al., NAR 2003). We have demonstrated
that ESC can be rendered permissive for oligo targeting by transiently suppressing MSH2
protein levels by RNA interference (Aarts et al., Nucleic Acids Res 2006). A detailed
protocol of our procedure is described in Aarts et al., Methods Mol Biol 2009;530:79-99.
In an attempt to increase the efficacy of oligo targeting, we have tested ssODN of
variable length and found 35-40 nucleotides to perform best. Remarkably,
phosphorothioate linkages in ssODN (to protect them from exonucleolytic
degradation) reduced the efficacy of oligo targeting. The addition of locked nucleic
acids slightly improved the performance of short ssODN, however, at the optimum
length of 35-40 nucleotides, there was no benefit of chemical modification. Sense
ssODN performed better than anti-sense. However, reversion of the neo reporter
reduced the efficacy of sense ssODN. Together with our previous findings discarding
homologous recombination and transcription as a critical mediators, this suggests
that oligo targeting in ESC occurs in the context of DNA replication. Indeed, when
the speed of DNA replication was decelerated by low doses of hydroxyurea (HU), the
frequency of oligo targeting raised 1.8-fold. This level of improvement does not
warrant inclusion of HU in our protocol, but strongly corroborates the role of DNA
replication in ssODN-directed gene modification.
Unclassified variants of MMR genes Oligo targeting is now a routine procedure in
our laboratory to introduce subtle modifications into the ESC genome. We use this
technology to recreate suspected gene variants of MMR genes in ESC, which allows
us to assess their capacity to sustain MMR functions, in particular mutation avoidance
and sensitivity to methylating agents. MMR gene variants affecting only a single
hein te Riele phD Group leader
Rob Dekker Post-doc
henri van de Vrugt Post-doc
camiel Wielders phD Post-doc
Marieke Aarts Msc PhD student
sietske Bakker 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
65
MOLecULAR BiOLOGY
Division head, group leader Hein Te Riele
Aarts M, Dekker M, Dekker R, de Vries S,
van der Wal A, Wielders E, Te Riele H.
Gene modification in embryonic stem cells
by single-stranded DNA oligonucleotides.
Methods Mol Biol 2009;530:79-99
Aarts M, Te Riele H. Parameters of
oligonucleotide-mediated gene modification
in mouse ES cells. J Cell Mol Med 2009
Bakker ST, van de Vrugt HJ,
Rooimans MA, Oostra AB, Steltenpool J,
Delzenne-Goette E, van der Wal A,
van der Valk M, Joenje H, Te Riele H,
de Winter JP. Fancm-deficient mice reveal
unique features of Fanconi anemia
complementation group M. Hum Mol
Genet 2009;18:3484-95
Ferrás C, Oude Vrielink JA, Verspuy JW,
Te Riele H, Tsaalbi-Shtylik A, de Wind N.
Abrogation of Microsatellite-instable
Tumors Using a Highly Selective Suicide
Gene/Prodrug Combination. Mol Ther
2009;17:1373-80
Wielders E, Dekker M, Te Riele H.
Generation of double-knockout
embryonic stem cells. Methods Mol Biol
2009;530:205-18
Te Riele H. Recreating stem cells: a novel
entrance to the fountain of youth - Preview.
Cell Stem Cell 2009;4:279-80

66
MOLecULAR BiOLOGY
Figure 1: Mitogen starvation of TKO MEFs
(lacking Rb, p107 and p130) causes DNA
damage as evidenced by the neutral comet
assay. Bcl2 was expressed in TKO and wildtype
MEFs to suppress apoptosis. MEFs were
cultured in the presence (10% FCS) or
absence (w/o 10% FCS) of foetal calf
serum. (A) Representative comets of nuclei
of TKO-Bcl2 and WT-Bcl2 MEFs stained
with Propidium Iodide. Cells exposed to 0.5
or 20 Gy of g–irradiation served as controls.
(B) Box plot representation showing
interquartile ranges of tail moments in
~50 nuclei of each condition shown in
A. Horizontal bars denote the median,
+ indicates the mean value and points
indicate outliers.
codon are widespread in the human population and in (suspected) Lynch syndrome
patients. As the consequences of single amino acid substitutions are often difficult to
predict, they are termed ‘Variants of uncertain significance’ (VUS) or ‘Unclassified
Variants’ (UV). We have thus far analyzed six VUS and found one to show complete
and another partial MMR deficiency. As we use mouse ES cells, we were able to
introduce the latter variant into the germ line of mice. We found that this variant
predisposed to cancer to the same extend 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 developmental abnormalities and
a 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 significance of the FA genome maintenance pathway in suppression of
cancer, we have generated Fancm-deficient mice by deleting exon 2. FANCM is part
of the FA core complex that is essential for mono-ubiquitination of FANCD2.
FANCM deficiency caused hypogonadism in mice and hypersensitivity to crosslinking
agents in mouse embryonic fibroblasts (MEFs), thus phenocopying other FA
mouse models. However, Fancm ∆2/∆2 mice also showed features atypical for FA mice,
including under-representation of females and decreased overall and tumor-free
survival. The increased cancer incidence may be correlated to the role of FANCM in
the suppression of spontaneous sister chromatid exchanges as we observed in MEFs.
In addition, FANCM appeared to have a stimulatory rather than essential role in
FANCD2 monoubiquitination. The FA-M mouse model presented here suggests that
FANCM functions both inside and outside the FA core complex to maintain genome
stability and to suppress tumorigenesis. The Fancm knockout allele has been crossed
into cancer prone Apc +/- mice to study how FANCM deficiency promotes 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 identify residual cell cycle control mechanisms and 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 fraction that survives arrests in the G 2 phase of the cell cycle. G 2 arrest
was effectuated through inhibitory interactions of the cyclin-dependent-kinase
inhibitors p21CIP1 and p27 KIP1 with Cyclins A and B1. We found that G 2-arrested
TKO cells had accumulated DNA double-strand breaks (figure 1), which only
gradually disappeared upon mitogen re-stimulation and cells entering mitosis
showed excessive chromosomal aberrations. None of these effects were seen in TKO
cells that were cultured in the presence of mitogens. These results suggest that
combined lack of pocket proteins and mitogenic signaling leads to genomic instability.
Anchorage independence We have also shown that full pocket protein ablation was
not sufficient 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 screen for
genetic events that permit anchorage-independent growth of RAS V12 -expressing
pocket-protein-defective MEFs, we identified the immortalizing oncogene TBX2 that
was shown to interfere with the p53 pathway through downregulation of p19ARF.
We are currently performing loss-of-function screens to identify genes that impair
oncogenic transformation of TKO MEFs.

DNA BAse J
b-glucosyl-hydroxymethyluracil (base J), which we discovered in African trypanosomes
in 1993 (Gommers-Ampt et al., Cell 1993;75:1129-36), is a base present in
kinetoplastid flagellates and in Euglena. It replaces 1% of thymine in DNA and is
predominantly located in repetitive sequences, such as telomeric repeats. We have
characterized a J-binding protein (JBP1) that binds with high specificity to
J-containing duplex DNA (Cross et al. EMBO J 1999;18:6573-81). We have shown that
JBP1 is a thymidine hydroxylase that catalyses the first step of J biosynthesis, the
conversion of T in DNA into hydroxymethyluracil. JBP1 appears to belong 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 under normal growth conditions, 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 find 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 marking transcriptional stop
sites. Using immuno-precipitation of J-DNA and deep sequencing, we have also
found the 1% of non-telomeric DNA in Leishmania in specific chromosome-internal
positions, partly at transcriptional stops (figure 2). We have also found an interesting
interaction between J and histone H3/H3V (H3 variant). We think that the tools are
now on board to solve the elusive function of base J. We are also continuing attempts
to set up assays for hydroxylase activity and for the putative glucosyl transferase
catalyzing the second step in J biosynthesis. With Anastassis Perrakis (Division of
Biochemistry) we are trying to determine the structure of JBP1-J-DNA complexes by
crystallography; with Paul Wentworth (Scripps, California), we are looking for
inhibitors of the binding of JBP1 to DNA and of its hydroxylase function.
Figure 2: Chromosome internal base J is highly enriched at convergent transcription termination sites.
The sequence of the Leishmania tarentolae genome was assembled by F. Raymond and M. Ouellette
(Laval University, Quebec, Canada). Base J containing DNA fragments were precipitated using an anti
J antiserum or the J-binding protein 1, sequenced and mapped on the genome by R. Kerkhoven,
M. Nieuwland and A. Velds (NKI Microarray facility). The distribution of J on chromosome 5 is shown.
The large arrows represent the polycistronic transcription units which were mapped in the close homolog
L. major. The height of the peaks correspond to the number of hits (the J-level) at a specific genomic region.
MULtiDRUG ResistANce OF cANceR ceLLs
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 specificity and sensitivity to
inhibitors in transfected cells. To study the physiological function in metabolism and
defense of the body against drugs and xenotoxins of these transporters, we have
inactivated genes for several drug transporters by targeted gene disruption in mice.
Initially we looked at P-glycoproteins (ABCB1 family); most recently we have studied
the Multidrug Resistance Protein (ABCC) family members MRP2, 3, 4, 5 and 6.
MRp3 (ABcc3) is an organic anion transporter contributing to the cellular export of
endogenous or exogenous (toxic) compounds, conjugated to glutathione, sulphate or
glucuronate. Using KO mice, we have shown that murine MRP3 is important for
basolateral export of glucuronated compounds from the gut epithelium into blood.
Group leader Piet Borst
piet Borst MD phD Group leader
henri Van Luenen phD Academic staff
sven Rottenberg DVM phD Dipl ecVp
Senior post-doc
pankaj tripathi phD Post-doc
Koen Van de Wetering DVM phD Post-doc
Nikola Banishki Msc PhD student
petra Krumpochova Msc PhD student
Guotai Xu Msc PhD student
serge Zander DVM Msc PhD student
Maaike Gonggrijp DVM Research associate
Marcel De haas Technician
Liesbeth Van Deemter Technician
sabrina Jan Technician
Ariena Kersbergen Technician
sunny sapthu Technician
Wendy sol Technician
publications
67
MOLecULAR BiOLOGY
Beedholm-Ebsen R, Van de Wetering K,
Hardlei T, Nexo E, Borst P, Moestrup SK.
Identification of multidrug resistance
protein 1 (MRP1/ABCC1) as a molecular
gate for cellular export of cobalamin. Blood
2009 (in press)
Jaspers JE, Rottenberg S, Jonkers J.
Therapeutic options for triple-negative
breast cancers with defective homologous
recombination. Biochim Biophys Acta 2009
(in press)
Lagas JS, van der Kruijssen CM,
Van de Wetering K, Beijnen JH,
Schinkel AH. Transport of diclofenac by
BCRP (ABCG2) and stimulation of. Drug
Metab Dispos 2009;37:129-36
Pajic M, Iyer JK, Kersbergen A,
Van der Burg E, Nygren AO, Jonkers J,
Borst P, Rottenberg S. Moderate increase
in Mdr1a/1b expression causes in vivo
resistance to doxorubicin in a mouse model
for hereditary breast cancer. Cancer Res
2009;69:6396-404

68
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, NJ: Humana Press Inc., 2009
(in press)
Sabatini R, Cliffe L, Vainio S, Borst P.
Enzymatic formation of the hypermodified
DNA base J (b-D-glucopyranosyloxymethyluracil).
In: Grosjean H (ed). DNA
and RNA modification enzymes:
comparative structure, mechanism,
function and evolution. Austin: Landes
Bioscience, 2009:144-156
Vainio S, Genest PA, ter Riet B,
van Luenen H, Borst P. Evidence that
J-binding protein 2 is a thymidine
hydroxylase catalyzing the first step in the
biosynthesis of DNA base J. Mol Biochem
Parasitol 2009;164:157-61
Van de Wetering K, Burkon A, Feddema W,
Bot A, de Jonge H, Somoza V, Borst P.
Intestinal breast cancer resistance protein
(BCRP)/Bcrp1 and multidrug resistance
protein 3 (MRP3)/Mrp3 are involved in the
pharmacokinetics of resveratrol. Mol
Pharmacol 2009;75:876-85
Van de Wetering K, Feddema W, Helms JB,
Brouwers JF, Borst P. Targeted
metabolomics identifies glucuronides of
dietary phytoestrogens as a major class of
MRP3 substrates in vivo. Gastroenterol
2009;137:1725-35
Van Leeuwen FW, Buckle T, Kersbergen A,
Rottenberg S, Gilhuijs KG. Noninvasive
functional imaging of P-glycoproteinmediated
doxorubicin resistance in a mouse
model of hereditary breast cancer to predict
response, and assign P-gp inhibitor
sensitivity. Eur J Nucl Med Mol Imaging
2009;36:406-12
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-deficient mouse mammary
tumors to the topoisomerase I inhibitor
topotecan. Cancer Res 2009 (in press)
We have initiated a systematic search for compounds conjugated to glucuronide or
sulphate that are transported by MRPs by comparing the derivatives in plasma/urine
of WT and KO mice using Mass Spectrometry. We have identified several
glucuronidated and sulphated phyto-estrogens, derived from food, as novel MRP3
substrates by this approach and anticipate that it may also be helpful to find substrates
of other MRPs and BCRP (ABCG2).
In collaboration with Sören Moestrup (University of Arhus, Denmark) we have shown
that the ABC transporter MRP1 (ABCC1) is able to transport vitamin B12 (cobalamin)
out of cells. Using our Mrp1 KO mice, we have shown that Mrp1 has a physiological
role in cobalamine homeostasis.
DRUG ResistANce iN ‘spONtANeOUs’ MOUse tUMORs
In collaboration with Jos Jonkers (Divion of Molecular Biology), we are studying
resistance mechanisms in ‘spontaneous’ tumors arising in mice, conditionally
defective in p53 and Brca1. These mice contain floxed alleles of these two genes and a
Cre recombinase gene driven by a Keratin14 promoter, active only in epithelial cells,
resulting in breast (and skin) cancer. 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 and we have shown with specific inhibitors that remarkably low
levels of Abcb1 upregulation (5-fold the levels in sensitive tumors) suffice 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) in
collaboration with KuDOS/AstraZeneca. We have crossed disrupted alleles for the
Abcb1 and Abcg2 genes into our mouse model to further test the importance of these
transporters in drug resistance and to uncover other forms of resistance not mediated
by these transporters. Upregulation of Abcg2 is important for topotecan resistance in
this model, but even in Abcg2 -/- mice tumors eventually become resistant, often by
downregulation of the topotecan target topoisomerase I (figure 3).
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 identified 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 specific 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 O 2 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 and what needs to be done to kill them.
Figure 3: Mechanisms of topotecan resistance in Brca1,p53deficient
mouse mammary tumors (Zander et al. in press).
(A) Overall survival (%) over time (days) after repeated
topotecan MTD treatment (4mg/kg i.p. on days 0-4 and
14-18) of wild-type animals carrying orthotopically
transplanted Abcg2-proficient or -deficient Brca1∆/∆ , p53∆/∆ mammary tumors. (B) Western Blot analysis of Top1 levels
in topotecan-resistant tumors. Lysates of 9 resistant
Abcg2∆/∆ , Brca1∆/∆ , p53∆/∆ tumors (TB1-4 and TB6-10) were probed with an anti-Top1 antibody and
compared with matched topotecan-sensitive controls (Top1 MW=100 kDa). As loading control Lamin A/C
(Santa Cruz; Lamin A MW=69 kDa, Lamin C MW=62 KDa) was used. Resistant/control ratios of
relative Top1 band intensities are indicated for each individual tumor.

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. We have developed models
for BRCA1- and BRCA2- associated hereditary breast cancer and E-cadherin-mutated
invasive lobular breast cancer. These models are used to (1) investigate genotypephenotype
relations in mammary tumorigenesis; (2) perform therapeutic
intervention studies; (3) identify genetic changes underlying breast tumorigenesis;
(4) study the role of innate and adaptive immunity in breast cancer development.
Mouse models for BRcA-associated breast cancer We have previously generated
mouse mutants with tissue-specific loss of Brca1/2 and p53 to establish models for
BRCA1- and BRCA2-associated breast cancer. The Brca1 –/– ;p53 –/– mammary tumors
closely mimic BRCA1-deficient, basal-like breast cancers in women: they are highly
proliferative, poorly differentiated, hormone receptor- and HER2-negative
adenocarcinomas with a high degree of genomic instability. Interestingly, we have
found that mammary tumor formation in our BRCA1 model is still estrogendependent.
We are currently investigating whether this estrogen dependence is due
to autocrine or paracrine mechanisms.
In collaboration with Hein te Riele, we have used oligo targeting to generate Brca1
mouse mutants mimicking two common founder mutations, BRCA1-185delAG and
BRCA1-5382insC. We have also obtained from Jo Morris (King’s College London) a
mouse mutant mimicking the BRCA1-C61G variant. We have crossed these 3 mutants
into our BRCA1 model to study the impact of these defined Brca1 mutations on
mammary tumor development, therapy response and resistance. Brca1 185delAG/– ;p53 –/– ,
Brca1 5382insC/– ;p53 –/– and Brca1 C61G/– ;p53 –/– mammary tumors developed with similar
latency as Brca1 –/– ;p53 –/– tumors and are currently treated with cisplatin. Whereas
Brca1 –/– ;p53 –/– tumors never develop resistance to cisplatin due to irreversible
deletion of Brca1 exons 5-13, the Brca1 C61G/– ;p53 –/– tumors readily become resistant to
this drug while retaining the Brca1-C61G mutation. We are currently studying the
cause of cisplatin resistance in these tumors.
tumor intervention studies in the BRcA mammary tumor models The central
role of BRCA1 and BRCA2 in DNA double-strand break (DSB) repair by homologous
recombination (HR) implies that BRCA-deficient tumors are especially sensitive to
therapeutics that directly or indirectly induce DSBs. In collaboration with Sven
Rottenberg, Piet Borst and KuDOS-AstraZeneca, we have used our BRCA1/2 models
to test the clinical PARP inhibitor olaparib (AZD2281), which may be selectively toxic
to HR-deficient cells because it suppresses base excision repair. Olaparib induced
regression but not eradication of Brca1 -/- ;p53 -/- or Brca2 -/- ;p53 -/- mammary tumors
without signs of toxicity. However, long-term treatment with olaparib resulted in
acquired drug resistance due to upregulation of the Mdr1 genes encoding
P-glycoprotein (Pgp) drug efflux transporters. To study Pgp-independent mechanisms
of olaparib resistance, Sven has crossed the BRCA1 mammary tumor model onto an
Mdr1 -/- background and transplanted the resulting Brca1 -/- ;p53 -/- ;Mdr1 -/- tumors into
wild-type recipients. Treatment of these mice with olaparib resulted in delayed
induction of resistance. We are currently analyzing these tumors to identify novel
mechanisms of olaparib resistance.
Functional screens and complementation assays in BRcA1-deficient es cells
Although BRCA1 suppresses tumorigenesis in breast and ovarian epithelium, BRCA1
loss in most cells induces growth arrest or apoptosis. To identify factors that rescue
cells from BRCA1 defects, we have performed clonal survival screens in R26cre-
ER T2 ;Brca1 sko/∆ ES cells using PiggyBac (PB)-based insertional mutagenesis. We
found that inactivating PB insertions in 53BP1 rescued ES cells from BRCA1
deficiency. We are currently investigating the underlying mechanism and whether
53BP1 loss plays a role in human BRCA1-associated breast cancer.
We have also used our R26cre-ER T2 ;Brca1 sko/∆ ES cells to perform functional
complementation assays to test BRCA1 variants of unknown clinical significance
(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).
Group leader Jos Jonkers
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
Metamia ciampricotti PhD student
Rinske Drost Msc PhD student
Bastiaan evers 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
chris Doornebal Msc Visiting Scientist
selected publications
69
MOLecULAR BiOLOGY
Holstege H, Joosse SA, van Oostrom CT,
Nederlof PM, de Vries A, Jonkers J. High
incidence of protein-truncating TP53
mutations in BRCA1-related breast cancer.
Cancer Res 2009;69:3625-33
Jonkers J, Moolenaar WH. Mammary
tumorigenesis through LPA receptor
signaling. Cancer Cell 2009;15:457-9
de Visser K, Jonkers J. Towards
understanding the role of cancer-associated
inflammation in chemoresistance. Curr
Pharm Des 2009;15:1844-53

70
MOLecULAR BiOLOGY
selected publications (continued)
Pajic M, Iyer JK, Kersbergen A,
van der Burg E, Nygren AO, Jonkers J,
Borst P, Rottenberg S. A moderate
increase in Mdr1a/1b expression causes in
vivo resistance to doxorubicin in a mouse
model for hereditary breast cancer. Cancer
Res 2009;69:6396-404
Puppe J, Drost R, Liu X, Joosse SA,
Evers B, Cornelissen-Steijger P,
Nederlof P, Yu Q, Jonkers J*,
van Lohuizen M*, Pietersen AM*.
BRCA1-deficient mammary tumor cells are
dependent on EZH2 expression and
sensitive to PRC2-inhibitor DZNep.
Breast Cancer Res 2009;11:R63 (* joint
corresponding authors)
Jaspers J, Rottenberg S*, Jonkers J*.
Therapeutic options for triple-negative
breast cancers with defective homologous
recombination. Biochim Biophys Acta
2009;1796:266-80 (* joint corresponding
authors)
Rottenberg R, Pajic M, Jonkers J. Studying
drug resistance using genetically engineered
mouse models for breast cancer. Methods
Mol Biol 2009 (in press)
Drost R, Jonkers J. Preclinical mouse
models for BRCA1-associated breast cancer.
Br J Cancer 2009;101:1651-7
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 2009
(in press)
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 identifies compounds
with specific toxicity against BRCA2deficient
tumors. Clin Cancer Res 2009
(in press)
Klijn C, Bot J, Adams DJ, Reinders M,
Wessels LF*, Jonkers J*. Identification of
networks of co-occurring, tumor-related
DNA copy number changes using a
genome-wide scoring approach. PLoS
Comput Biol 2009 (in press) (* joint
corresponding authors).
Introduction of wild-type BRCA1 – but not BRCA1-185delAG or BRCA1-5382insC – rescued
the growth defect of switched R26cre-ER T2 ;Brca1 sko/∆ cells. We have generated 45
defined BRCA1 VUS mutations, which are currently being tested in our functional
complementation assay.
Mouse models for iLc Loss of E-cadherin is associated with invasive lobular
carcinoma (ILC), which accounts for 10-15% of all breast cancers. We have generated
a mouse ILC model based on epithelium-specific inactivation of E-cadherin and p53.
Compared to p53 -/- mammary carcinomas, Ecad -/- ;p53 -/- mammary tumors show a
significantly reduced latency, a morphological switch from ductal to lobular carcinoma,
and a phenotypic change from non-invasive to highly invasive and metastatic tumors.
Moreover, Ecad -/- ;p53 -/- mammary tumor cell lines – but not p53 -/- cell lines – are
resistant to detachment-induced apoptosis, aka anoikis.
We have performed kinome-wide siRNA screens to identify factors that modulate
anoikis resistance of Ecad -/- ;p53 -/- tumor cells, which may serve as a surrogate assay
for survival of circulating tumor cells. We have identified 5 kinases that are essential
for survival of Ecad -/- ;p53 -/- cells under non-adherent culture conditions. We are currently
validating these kinases in vitro using growth assays and soft agar assays. In vivo
validation studies will be performed using (inducible) expression of shRNAs against
these kinases in Ecad -/- ;p53 -/- mouse mammary tumors.
We are also using the E-cadherin model for tumor intervention studies. For this purpose
we have derived luciferase-marked mammary tumor cell lines. Together with the
spontaneous models, these reagents permit preclinical testing of (combinations of)
anticancer drugs at different levels: (1) in vitro, on the panels of tumor cell lines; (2) in
vivo, on tumor outgrowths from orthotopically grafted cell lines or tumor fragments
(allowing parallel treatments on genetically identical tumors); (3) in vivo, on the
spontaneous tumor model (allowing intervention at early stages of tumor development).
impact of the inflammatory tumor-microenvironment on breast cancer
development and therapy response 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
influence of the inflammatory tumor-microenvironment on response and resistance
of tumors to chemotherapy is addressed. For these studies, the E-cadherin mammary
tumor model is employed, which faithfully recapitulates human invasive and
metastatic lobular carcinoma. 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-inflammatory mediators. By genetic
elimination and pharmacological inhibition of specific subsets of the adaptive and
innate immune system, we are currently investigating their functional significance in
a tumor-stage specific manner. We have identified 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.
acGh 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. To identify regions with significantly recurrent DNA
copy number alterations (CNAs), we have developed a novel algorithm (KC-SMART) for
multi-experiment analysis of aCGH data and comparative KC-SMART to identify
significant differences in recurrent CNAs between sample groups. We have used both
algorithms in cross-species comparisons to identify conserved BRCA1- or BRCA2specific
CNAs in mouse mammary tumors and human BRCA-associated breast cancers.
Furthermore, we have developed a novel approach to find co-occurring or mutual
exclusive CNAs. Using this method, we identified several mutual exclusive amplifi cations
in mouse mammary tumors that might represent redundant oncogenic pathways.

MOLecULAR DissectiON OF BReAst cANceR BY
DiFFeReNtiAL DRUG seNsitiVitY
In the clinic, we generally use anticancer drugs selected by clinical trials to perform
best for the whole group of breast cancer patients, whereas little is known about the
molecular mechanisms underlying differential drug sensitivity. The focus of our
research line is to unravel these 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-defined cohorts of breast cancer patients
into resistant and sensitive to a particular drug. In addition, we have a close
collaboration with the group of Jos Jonkers, who uses 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 classifiers on
adjuvant systemic treatment advice in breast cancer.
the 70-gene prognosis signature in node-negative breast cancer in daily clinical
practice We evaluated the additional value of the 70-gene prognosis signature in
701 patients from three previously described series. Median follow-up time of the
patients who had not received adjuvant systemic treatment (AST) was 9 years
(N=186). Clinical risk was assessed on the basis of Adjuvant! Online, St Gallen
guidelines and Nottingham Prognostic Index. The signature had additional value in
patients with estrogen receptor (ER)-positive tumors and a low or discordant clinical
risk according to the three clinical risk indexes. The signature was not useful for
estrogen receptor (ER)-positive tumors with concordant high clinical risk. The 10-year
overall survival estimate for good signature tumors with these characteristics was

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MOLecULAR BiOLOGY
publications (continued)
Linn SC, Wesseling J. Molecular tests for
breast-cancer diagnosis? Lancet Oncol
2009;10:314-5
Ploem MC, Retel VP, Linn SC,
van Boven HH, Schmidt MK, de Jong JP,
Gevers JK, van Harten WH. Tumour
tissue: who is in control? Lancet Oncol
2009
Schilder CM, Seynaeve 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
breast cancer patients: results from the
neuropsychological side study of the
Tamoxifen and Exemestane Adjuvant
Multicenter (TEAM) trial. J Clin Oncol
2009 (in press)
Sonke GS, Linn SC. Isolated tumor cells in
breast cancer. N Engl J Med.
2009;361:1995-6
Retel VP, Bueno-de-Mesquita JM,
Hummel MJ, van de Vijver MJ, Douma KF,
Karsenberg K, van Dam FS,
van Krimpen C, Bellot FE, Roumen RM,
Linn SC, van Harten WH. Constructive
Technology Assessment (CTA) as a tool in
coverage with evidence development: the
case of the 70-gene prognosis signature for
breast cancer diagnostics. Int J Technol
Assess Health Care 2009;25:73-83
Teunissen SF, Rosing H, Koornstra RH,
Linn SC, Schellens JH, Schinkel AH,
Beijnen JH. Development and validation of
a quantitative assay for the analysis of
tamoxifen with its four main metabolites
and the flavonoids daidzein, genistein and
glycitein in human serum using liquid
chromatography coupled with tandem mass
spectrometry. J Chromatogr B Analyt
Technol Biomed Life Sci 2009;877:2519-29
Roepman P, Horlings HM, Krijgsman O,
Kok M, Bueno-de-Mesquita JM, Bender R,
Linn SC, Glas AM, van de Vijver MJ.
Microarray-based determination of estrogen
receptor, progesterone receptor, and HER2
receptor status in breast cancer. Clin Cancer
Res 2009;15:7003-11
Figure 5: Association of ERaS305-P, PAK1 and pPKA with outcome after adjuvant tamoxifen treatment.
Kaplan-Meier analysis according to the algorithm based on PAK1 nuclear and pPKA-associated
ERaS305-P expression in 231 patients. Recurrence-free survival (RFS) of patients who had been randomly
assigned to tamoxifen or no adjuvant systemic treatment. Tumors with no PAK1 and no pPKA-associated
ERaS305-P expression (A), and tumors with either PAK1 and/or pPKA-associated ERaS305-P expression
(B) were analyzed separately. (C) RFS according to the algorithm among patients who did not receive any
adjuvant treatment (controls).
cytochrome p450 enzyme system, tamoxifen and breast cancer outcome
Tamoxifen is metabolized by several cytochrome P450 (CYP450) enzymes into
compounds with altered affinity for the estrogen receptor, among which the very
active metabolite endoxifen. It has been suggested that polymorphisms of 2D6 and
2C19 are predictive of outcome after adjuvant tamoxifen therapy in ER-positive
patients with early breast cancer. In collaboration with Van Schaik and Berns
(Erasmus Medical Center, Rotterdam) we have investigated their role in relation to
outcome after tamoxifen treatment in the metastatic setting. We found a longer time
to treatment failure (TTF) for CYP2C19*2 carriers, encoding lower enzymatic activity,
compared to non-carriers (HR (95% CI): 0.72 (0.57-0.90), p=0.004) (N~500).
Neither the ultra-active CYP2C19*17 nor the inactive CYP2D6*4 allele were associated
with TTF. Similar results were obtained in multivariable analysis. As these results are
counterintuitive, further investigations are ongoing to understand these results.
In collaboration with Jos Beijnen (Division of Medical Oncology) and Hans Bonfrer
(Division of Diagnostic Oncology) we have investigated correlations of these
polymorphisms with steady-state serum concentrations of tamoxifen and its
metabolites in 143 patients who have been treated with adjuvant tamoxifen in the
nineties. A weak correlation between the CYP2D6*4 allele and lower endoxifen levels
was demonstrated, however no relation between breast cancer outcome and
metabolite concentrations or CYP450 polymorphisms was found.
Molecular mechanisms underlying sensitivity for high dose alkylating agents
Homologous recombination deficient (HRD) cancer cells are hypersensitive to agents
inducing double strand DNA breaks (DSB), such as bifunctional alkylators and
poly(ADP-ribose)polymerase(PARP)-inhibitors. BRCA1-mutated breast cancers, which
are considered HRD, can be detected with a Comparative Genomic Hybridization
classifier. We hypothesized that the BRCA1-classifier could also detect HRD in
sporadic tumors (BRCAness) and therefore would predict sensitivity to DSB-inducing
agents. We trained this BRCA1-classifier in a series of metastatic breast cancer
patients who had received high-dose alkylating chemotherapy with autologous stem
cell rescue. We chose the cut-off for prediction of progression-free survival with the
best positive predictive value and called the optimized classifier ‘BRCA1-like’ classifier.
We tested the performance of our BRCA1-like classifier in an independent patient
series, by using a randomized clinical trial that compared 5 x fluorouracil-epirubicincyclophosphamide
(FEC) with 4 x FEC followed by 1x carboplatin-thiotepacyclophosphamide
(CTC) for high risk, primary operable breast cancer [Rodenhuis et
al., New Engl J Med, 2003]. We first evaluated the performance of the BRCA1-like
classifier in a subgroup of ER low (

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 influence 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 active 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)
(Figure 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 specificity 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.
p-gp, Bcrp1, and Mrp2 (compound) knockout mice We generated and characterized
Mrp2/Bcrp1 combination knockout mice, which were viable and fertile, and had a
normal life span. We then used these mice to investigate the possibly overlapping
roles of Bcrp1 and Mrp2 in the elimination of the anti-cancer drug methotrexate
(MTX) and its toxic metabolite 7-hydroxymethotrexate (7OH-MTX) after i.v.
administration of 50 mg/kg MTX. Compared to wild-type, the plasma areas under
the curve (AUCs) for MTX were 1.6-fold and 2.0-fold higher in Bcrp1 and Mrp2
knockout mice, respectively, and 3.3-fold increased in Mrp2/Bcrp1 knockout mice.
Biliary excretion of MTX was 23-fold reduced in Mrp2/Bcrp1 knockout mice, and
MTX levels in the small intestine were dramatically decreased. Plasma levels of 7OH-
MTX were not significantly altered in Bcrp1 knockout mice, but the AUCs were 6.2fold
and even 12.4-fold increased in Mrp2 and Mrp2/Bcrp1 knockout mice,
respectively. This indicates that Mrp2 compensates for Bcrp1 deficiency, but that
Bcrp1 can only partly compensate for Mrp2 absence. Furthermore, in Mrp2/Bcrp1
knockout mice, 21-fold decreased biliary 7OH-MTX excretion and substantial 7OH-
MTX accumulation in liver and kidney were seen. We additionally found that in
absence of Mrp2, Bcrp1 mediated substantial urinary excretion of MTX and 7OH-
MTX. Collectively, the data indicate that Mrp2 and Bcrp1 together are major
determinants of MTX and 7OH-MTX pharmacokinetics. Variation in MRP2 and/or
BCRP activity due to polymorphisms or co-administered inhibitors may therefore
substantially affect the therapeutic efficacy and toxicity in patients treated with MTX.
The brain penetration of many drugs is limited by P-gp and BCRP, which, for
anticancer drugs, might limit their therapeutic activity against brain tumors and
brain metastases. It has previously been found that the anticancer drug imatinib, a
BCR-ABL tyrosine kinase inhibitor, is a substrate of P-gp and BCRP, and that its
brain penetration is restricted by both transporters. For dasatinib, an inhibitor of
SRC/BCR-ABL kinases, in vivo interactions with P-gp and BCRP are not fully
established yet. We therefore used Mdr1a/1b, Bcrp1 and Mdr1a/1b/Bcrp1 combination
knockout mice to establish the roles of P-gp and BCRP in the pharmacokinetics and
brain penetration of dasatinib. We found that the oral uptake of dasatinib is limited
Alfred schinkel phD Group leader
Birk poller phD Post-doc
Robert Van Waterschoot phD Post-doc
Dilek iusuf Msc PhD student
Jurjen Lagas Msc PhD student
evita Van de steeg Msc PhD student
Marijn Vlaming Msc PhD student
selvi Durmus Msc PhD student
Anita Van esch Technical staff
els Wagenaar Technical staff
publications
73
MOLecULAR BiOLOGY
Group leader Alfred Schinkel
Lagas JS, van der Kruijssen CM,
van de Wetering K, Beijnen JH,
Schinkel AH. Transport of diclofenac by
breast cancer resistance protein (ABCG2)
and stimulation of multidrug resistance
protein 2 (ABCC2)-mediated drug transport
by diclofenac and benzbromarone. Drug
Metab Dispos 2009;37:129-36
Lagas JS, van Waterschoot RA,
van Tilburg VA, Hillebrand MJ,
Lankheet N, Rosing H, Beijnen JH,
Schinkel AH. Brain accumulation of
dasatinib is restricted by P-glycoprotein
(ABCB1) and breast cancer resistance
protein (ABCG2) and can be enhanced by
elacridar treatment. Clin Cancer Res
2009;15:2344-51
Figure 6: Putative structure of a prototypic
ABC drug transporter.

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MOLecULAR BiOLOGY
publications (continued)
Lagas JS, Vlaming ML, Schinkel AH.
Pharmacokinetic assessment of multiple
ATP-binding cassette transporters: the
power of combination knockout mice.
Mol Interv 2009;9:136-45
Van de Steeg E, van der Kruijssen CM,
Wagenaar E, Burggraaff JE, Mesman E,
Kenworthy KE, Schinkel AH. Methotrexate
pharmacokinetics in transgenic mice with
liver-specific expression of human organic
anion-transporting polypeptide 1B1
(SLCO1B1). Drug Metab Dispos
2009;37:277-81
Van Herwaarden AE, van Waterschoot RA,
Schinkel AH. How important is intestinal
cytochrome P450 3A metabolism? Trends
Pharmacol Sci 2009;30:223-7
Van Waterschoot RA, Rooswinkel RW,
Sparidans RW, van Herwaarden AE,
Beijnen JH, Schinkel AH. Inhibition and
stimulation of intestinal and hepatic
CYP3A activity: studies in humanized
CYP3A4 transgenic mice using triazolam.
Drug Metab Dispos 2009;37:2305-13
Van Waterschoot RA, Rooswinkel RW,
Wagenaar E, van der Kruijssen CM,
van Herwaarden AE, Schinkel AH.
Intestinal cytochrome P450 3A plays an
important role in the regulation of
detoxifying systems in the liver. FASEB J
2009;23:224-31
Vlaming ML, Lagas JS, Schinkel AH.
Physiological and pharmacological roles of
ABCG2 (BCRP): recent findings in Abcg2
knockout mice. Adv Drug Deliv Rev
2009;61:14-25
Vlaming ML, Pala Z, van Esch A,
Wagenaar E, de Waart DR,
van de Wetering K, van der Kruijssen CM,
Oude Elferink RP, van Tellingen O,
Schinkel AH. Functionally overlapping
roles of Abcg2 (Bcrp1) and Abcc2 (Mrp2)
in the elimination of methotrexate and its
main toxic metabolite
7-hydroxymethotrexate in vivo. Clin
Cancer Res 2009;15:3084-93
Zimmermann C, van Waterschoot RA,
Harmsen S, Maier A, Gutmann H,
Schinkel AH. PXR-mediated induction of
human CYP3A4 and mouse Cyp3a11 by the
glucocorticoid budesonide. Eur J Pharm Sci
2009;36:565-71
by P-gp. Furthermore, relative brain accumulation, 6 hr after administration, was not
affected by Bcrp deficiency, but absence of P-gp resulted in a 3.6-fold increase after
oral and 4.8-fold higher accumulation after i.p. administration. Mdr1a/1b/Bcrp1
knockout mice had the most pronounced increase in relative brain accumulation,
which was 13.2-fold higher after oral and 22.7-fold increased after i.p. administration.
Moreover, coadministration to wild-type mice of dasatinib with the dual P-gp and
BCRP inhibitor elacridar resulted in a similar dasatinib brain accumulation as
observed for Mdr1a/1b/Bcrp1 knockout mice (figure 7). Collectively, our data indicate
that brain accumulation of dasatinib is primarily restricted by P-gp, but Bcrp1 can partly
take over this protective function at the blood-brain-barrier. Consequently, when both
transporters are absent, brain uptake of dasatinib is highly increased. These findings
might be clinically relevant for patients with central nervous system Philadelphia
chromosome-positive leukemia, as coadministration of an inhibitor of P-gp and BCRP
with dasatinib might result in better therapeutic responses in these patients.
cyp3a and p-gp/cyp3a 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 influence 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, we previously generated Cyp3a knockout mice, and
showed a pronounced effect of Cyp3a deficiency on the oral bioavailability, i.v.
clearance and toxicity of the anticancer drug and CYP3A substrate docetaxel.
Docetaxel is one of the most widely used anti-cancer drugs. A major problem with
docetaxel treatment, however, is the considerable inter-patient variability in docetaxel
exposure. Another disadvantage of the drug is that it has a very low oral bioavailability
and can therefore only be administered intravenously. The drug-metabolizing
enzyme CYP3A and the drug transporter P-glycoprotein (P-gp; MDR1) are considered
to be major determinants of docetaxel pharmacokinetics. It had been hypothesized
that CYP3A and P-gp work synergistically in limiting the systemic exposure to many
orally ingested drugs. However, it has been difficult to examine this interplay in vivo.
We therefore generated mice lacking all CYP3A and P-gp genes. Although missing
two primary detoxification systems, Cyp3a/Mdr1a/1b knockout mice are viable, fertile
and without spontaneous abnormalities. When orally challenged with docetaxel, a
disproportionate (>70-fold) increase in systemic exposure was observed compared to
the increases in single Cyp3a knockout (12-fold) or Mdr1a/1b knockout mice (3-fold).
Unexpectedly, although CYP3A and P-gp collaborated extremely efficiently in
lowering docetaxel exposure, their individual efficacy was not dependent on activity of
the other protein. Upon reflection, this absence of functional synergism makes
biological sense as synergism would conflict with a robust detoxification defense.
Importantly, the disproportionate increase in docetaxel exposure in Cyp3a/Mdr1a/1b
knockout mice resulted in dramatically altered and lethal toxicity, with severe
intestinal lesions as a major cause of death. Simultaneous inhibition of CYP3A/P-gp
might thus be a highly effective strategy to improve oral drug bioavailability, but with
serious risks when applied to drugs with narrow therapeutic windows.
Figure 7: Plasma (A) and brain (B) concentrations and brain-to-plasma ratios (C) of dasatinib (5 mg/
kg) for wild-type (WT) and Mdr1a/1b/Bcrp1 knockout mice (KO) 60 min after i.v. administration.
Elacridar (10 mg/kg) or vehicle were administered i.v. 15 min before dasatinib. *, P < 0.05; **,
P < 0.01; ***, P < 0.001 compared with WT mice treated with vehicle.

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 performing state of the
art analyses of a wide array of data types, including laboratory and animal experiments,
clinical trials, and epidemiologic studies. The members of the group also conduct
research in bioinformatics and statistics, for example on stratifying tumors into
groups with distinct and homogeneous outcome and therapy response; on the
function of genes and pathways involved in tumorigenesis and understanding
molecular regulatory mechanisms. A number of exemplary projects are presented
below in more detail.
extracting oncogenes and oncogenic pathways from insertional mutagenesis
screens To find oncogenic lesions which are collaborating events in tumorigenesis,
we developed an approach to detect the significantly frequent co-occurrence of
independent insertions within one tumor. We have extended this approach to detect
combinatorial association logic networks (CALs): simple logic circuits which employ
combinations of co-occurring and mutually exclusive insertions 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, finding
known and novel associations between genes involved in Notch signaling.
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 identified co-occurring
recombinations at the T-cell receptor and immunoglobulin loci in T- and B-cell
malignancies, respectively. This demonstrates that we can recover truly co-occurring
genomic alterations. In addition, our analysis revealed networks of co-occurring
genomic losses and gains that are enriched for cancer genes. The detected cooccurrences
are highly enriched for functional relationships. The co-occurring losses
we find are independent of the canonical cancer genes within the network. Our
findings 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.
integration of clinical and gene expression data for breast cancer outcome
prediction Several models exist that can be used to predict disease outcome of breast
cancer patients. Only a few studies have created a single prediction model using both
expression and clinical data. These studies often remain inconclusive regarding an
obtained improvement (if any). We rigorously compared three different integration
strategies (early, intermediate, and late integration) and no integration (only one data
source) using five classifiers of varying complexity. We performed our analysis on a
set of 295 breast cancer samples, for which expression data and an extensive set of
clinical parameters are available.
A nearest mean classifier employing a logical OR operation on clinical and expression
classifier outputs significantly outperforms all other classifiers. Moreover, regardless
of the integration strategy, the nearest mean classifier achieves the best performance.
All five classifiers achieve their best performance when employing an integration
strategy. The late integration strategy performed best for four out of five classifiers,
Lodewyk Wessels phD Group leader
Michael hauptmann phD Academic staff
Marta Lopez phD Academic staff
Wilma heemsbergen phD Academic staff
sander canisius 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 Msc Bioinformatician
Jelle ten hoeve Msc Bioinformatician
publications
75
MOLecULAR BiOLOGY
Group leader Lodewyk Wessels
Klijn C, Bot J, Adams D, Reinders MJT,
Wessels LFA and Jonkers J. Identification
of a network of co-occurring, tumor-related
DNA copy number losses using a genomewide
scoring approach. PLoS
Computational Biology 2009 (in press)
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. 2009
(in press)
Knijnenburg TA, Wessels LFA,
Reinders MJT, Shmulevich I. Fewer
permutations, more accurate P-values.
Bioinformatics 2009;25:i161-8
Knijnenburg TA, Daran JM,
van den Broek MA, Daran-Lapujade PA,
de Winde JH, Pronk JT, Reinders MJT,
Wessels LFA. Combinatorial effects of
environmental parameters on transcriptional
regulation in Saccharomyces cerevisiae:
A quantitative analysis of a compendium of
chemostat-based transcriptome data. BMC
Genomics 2009;10:53

76
MOLecULAR BiOLOGY
publications (continued)
Meuleman W, Engwegen JY, Gast MC,
Wessels LFA, Reinders MJT. Analysis of
mass spectrometry data using sub-spectra.
BMC Bioinformatics 2009;10 Suppl 1:S51
van Vliet MH, Wessels LFA, Reinders MJT.
Knowledge driven decomposition of tumor
expression profiles. BMC Bioinformatics
2009;10 Suppl 1:S20
Rasch CR, Hauptmann M, Schornagel J,
Wijers O, Buter J, Wiggenraad R, Gregor T,
De Boer JP, Hilgers FJ, Ackerstaff AH,
Kroger R, Hoebers FJ, Balm AJ. Intraarterial
versus intravenous chemoradiation
for advanced head and neck cancer: results
of a randomized phase III trial. Cancer
2009 (in press)
Kok M, Holm-Wigerup C, Hauptmann M,
Michalides R, Stål O, Linn S, Landberg G.
Estrogen receptor-alpha phosphorylation at
serine-118 and tamoxifen response in breast
cancer. J Natl Cancer Inst. 2009;101:1725-9
Van den Belt-Dusebout AW, Aleman BM,
Besseling G, De Bruin ML, Hauptmann M,
Van ‘t Veer MB, De Wit R, Ribot JG,
Noordijk EM, Kerst JM, Gietema JA,
Van Leeuwen FE. Roles of radiation dose
and chemotherapy in the etiology of
stomach cancer as a second malignancy. Int
J Radiat Oncol Biol Phys 2009;75:1420-29
Beane Freeman LE, Blair A, Lubin JH,
Stewart PA, Hayes RB, Hoover RN,
Hauptmann M. Mortality from
lymphohematopoietic malignancies among
workers in formaldehyde industries: update
of the NCI cohort. J Natl Cancer Inst
2009;101:751-61
Hauptmann M, Stewart PA, Lubin JH,
Beane Freeman LE, Hornung RW,
Herrick RF, Hoover RN, Fraumeni JF Jr,
Blair A, Hayes RB. Mortality from
lymphohematopoietic malignancies and
brain cancer among embalmers exposed
to formaldehyde in the funeral industry.
J Natl Cancer Inst 2009;101:1696-1708
and early integration once. A nearest mean classifier that is trained on the originally
published clinical variables performs worse than an expression based nearest mean
classifier. However, adding the outputs from clinical prediction models, and a set of
new pathological variables, results in a performance equivalent to that of the
expression based classifier. Thus, there is no longer a significant performance
argument to choose one data source over the other, but rather employ a late
integration strategy based on nearest mean classifiers for optimal results.
Dynamics of genome - nuclear lamina interactions In collaboration with the Van
Steensel group we study genome – nuclear lamina interactions in various cell types.
For this, we use DamID data of the LaminB1 protein, which is one of the components
of the nuclear lamina. We are not only interested in how the genome is organized in
a cell nucleus, but more specifically how it is reorganized during, for example,
differentiation. To this end we employed an in vitro differentiation system in which
cultured mouse embryonic stem cells are differentiated into neural precursor cells,
which in turn are induced to form astrocytes. For all three stages DamID profiles
were collected. We developed a statistical test to discriminate between ‘constitutive’
and more dynamic, or ‘facultative’, genomic regions across these stages. Our data are
currently obtained using high-density genome-wide tiling arrays, for which a strong
dependency between probes adjacent on the genome is observed. The developed test
employs the variance between independent biological replicates and autocorrelation
levels present in the tiling array data to collectively estimate levels of technical and
non-specific biological variance.
statistical evaluation of biomarkers predicting treatment response Biomarkers
predicting treatment response are useful for tailoring treatment to host characteristics
of individual patients in order to maximize treatment benefit and minimize side
effects. Before prospective randomized trials are launched to evaluate a promising
biomarker candidate, the first evaluation in humans often takes place in relatively
small retrospective patient series or trials. Standard analyses use interaction terms in
regression models. However, the impact of the introduction of a predictive biomarker
into clinical practice can also be estimated retrospectively by assigning patients to the
marker-based and non-marker based arm of a hypothetical prospective trial. For
example, this has been done in a retrospective analysis of phosphorylation of the
estrogen receptor and tamoxifen response in a Swedish trial of premenopausal ERpositive
breast cancer, where offering adjuvant tamoxifen treatment to the 52%
patients with phosphorylated tumors (10-year recurrence-free survival of 75%) but not
to the remaining 48% (10-year recurrence free survival of 52%) would result in an
estimated 10-year recurrence-free survival of 64% for patients with phosphorylated
tumors. This value is equal to the estimated 10-year recurrence-free survival if all
patients are treated with adjuvant tamoxifen irrespective of phosphorylation, i.e.,
phosphorylation-guided treatment may save unnecessary treatment for half of the
patients while maintaining approximately the same 10-year recurrence-free survival.
Other examples include homologous recombination deficiency to predict response to
high dose chemotherapy for breast cancer and EGFR ligands and insulin-like growth
factors to predict response to EGFR-inhibitor treatment for lung cancer.
Late treatment effects among cancer survivors: evaluation and prediction of
risks After the introduction of modern radiotherapy and chemotherapy, many
cancers have become curable malignancies, e.g., Hodgkin lymphoma, breast 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 order to evaluate the causes of those effects and to predict their risk of occurrence,
two large studies have been designed. Within a large Dutch cohort of Hodgkin
lymphoma survivors, risk prediction models for second malignancies and
cardiovascular disease are constructed to be used in the setting of a survivors’ clinic
or when making initial treatment decisions. In a multinational cohort of survivors of
testicular cancer and Hodgkin lymphoma, the occurrence of stomach cancer as a
second malignancy is evaluated with a detailed assessment of radiation doses and
their uncertainties.

Division oF moleculaR
caRcinoGenesis
Functional Genomics
My group uses functional genomics technologies to identify mechanisms of resistance
to cancer drugs and to find novel cancer-relevant genes. We use primarily highthroughput
RNA interference-based loss-of-function genetic screens as a tool 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 identification of genes whose
suppression contributes to resistance to retinoic acid (RA)-based therapies. RA is a
vitamin A derivative, which induces differentiation of neuroblastoma cells in vitro
and is used with variable success to treat aggressive forms of this disease.
This variability in clinical response to RA is enigmatic, as no mutations in any of the
core components of the RA signaling cascade have been found in neuroblastoma.
To address this, we performed large-scale RNA interference screens with shRNA
libraries to identify genes whose suppression confers resistance to RA-mediated
differentiation in mouse F9 embryocarcinoma cells and in human neuroblastoma
cells. In the genetic screen using the mouse F9 cells, we identified Zpf423 as a gene
whose suppression confers resistance to RA-induced differentiation and growth
arrest. We found that the protein encoded by human ortholog, ZNF423, binds
directly to the RA receptor in chromatin and that this binding is required for
transcriptional activation of the receptor by RA. ZNF423 knockdown also prevents
differentiation of multiple human neuroblastoma cell lines in vitro. Conversely,
ectopic expression of ZNF423 in ZNF423 deficient human neuroblastoma cells
restores the ability to respond to RA-induced differentiation. Significantly, we found
that ZNF423 is a marker of poor outcome in two cohorts of human neuroblastoma
tumor samples. Our data are consistent with a model in which high levels of ZNF423
expression allow neuroblastoma cells to respond to differentiation-inducing signals,
rending such cells less malignant than cells having low levels of ZNF423, which are
more resistant to such signals.
To find additional genes that are relevant to the ability of neuroblastoma cells to respond
to RA therapy, we performed an additional loss of function genetic screen in human
neuroblastoma. Unexpectedly, we found here that silencing of the tumor suppressor
gene NF1 causes resistance to RA-induced differentiation. Since NF1 is a major
regulator of activity of RAS proteins, this suggests that RAS signaling can modify
responses of neuroblastoma cells to retinoids. In agreement with this, we found that
expression of a KRAS oncogene also confers potent resistance to RA-mediated growth
arrest and differentiation. We found an unexpected link between ZNF423 and NF1 by
showing that loss of NF1 activates RAS-MEK signaling, which in turn represses
ZNF423 expression. Neuroblastomas with low levels of NF1 also have poor outcome,
establishing NF1 as an independent prognostic marker for neuroblastoma disease
progression. Consistent with a role for NF1 in the pathogenesis of neuroblastoma, we
found genomic aberrations of the NF1 gene in primary neuroblastomas. The finding
that NF1-RAS-MEK signaling confers resistance to RA-induced differentiation,
suggests a potential combination therapy to overcome therapy resistance in
neuroblastoma, as inhibition of MEK with small molecule inhibitors should reinstate
sensitivity to RA (figure 1). Indeed, in cell culture, we observed that neuroblastoma
cells lacking NF1 and hence are fully resistant to RA, regain expression of ZNF423
and consequently also responsiveness to RA when RA is used in combination with a
selective MEK inhibitor. These findings highlight the importance of mapping cross
talk between signaling pathways to predict responses to targeted therapies in cancer.
77
moleculaR caRcinoGenesis
Division head, group leader René Bernards
René Bernards PhD Group leader
Katrien Berns PhD Academic staff
annette Dirac PhD Senior Post-doc
mirjam epping PhD Post-doc
michiel van der Heijden mD PhD
Senior Post-doc
michael Hölzel mD PhD Senior Post-doc
sidong Huang PhD Post-doc
Prasanth Kumar PhD Post-doc
ian majewski PhD Post-doc
Rianne oosterkamp mD Clinical fellow
ernst-Jan Geutjes msc PhD student
Guus Heynen msc PhD student
Jasper mullenders msc PhD student
miranda van Dongen Technical staff
marielle Hijmans msc Technical staff
Publications
Eichhorn PJ, Gili M, Scaltriti M, Serra V,
Guzman M, Nijkamp W, Beijersbergen RL,
Valero V, Seoane J, Bernards R, Baselga J.
Phosphatidylinositol 3-kinase
hyperactivation results in lapatinib
resistance that is reversed by the mTOR/
phosphatidylinositol 3-kinase inhibitor
NVP-BEZ235. Cancer Res. 2008;68:9221-30
Eichhorn PJ, Creyghton MP, Bernards R.
Protein phosphatase 2A regulatory subunits
and cancer. Biochem. Biophys. Acta
2009;1795:1-15
Epping MT, Bernards R. Molecular basis of
the anti-cancer effects of histone deacetylase
inhibitors. Int. J Biochem. Cell. Biol.
2009;41:16-20
Fotheringham S, Epping MT, Stimson L,
Kahn O, Wood V, Pezzella F, Bernards R,
La Thangue NB. Genome-wide loss-offunction
screen reveals an important role for
the proteasome in HDAC inhibitor-induced
apoptosis. Cancer Cell 2009;15:57-66

78
moleculaR caRcinoGenesis
Publications (continued)
Huang S, Laoukili J, Epping MT, Koster J,
Holzel M, Westerman BA, Nijkamp W,
Hata A, Asgharzadeh S, Seeger RC,
Versteeg R, Beijersbergen RL, Bernards R.
ZNF423 Is Critically Required for Retinoic
Acid-Induced Differentiation and Is a
Marker of Neuroblastoma Outcome.
Cancer Cell 2009;15:328-40
Mullenders J, Fabius AWM, Madiredjo M,
Bernards R, Beijersbergen RL. A largescale
shRNA barcode screen identifies the
circadian clock component ARNTL as
putative regulator of the p53 tumor
suppressor pathway. PLoS ONE
2009;4:e4798
Mullenders J. Big screens with small
RNAs: Loss of function genetic screens to
identify novel cancer genes. Thesis, 2009.
Utrecht University
Rutgers EJTh, Linn SC, Wesseling J,
van der Hoeven JJM, Klinkenbijl JHG,
van ’t Veer LJ, Bernards R. Nuttige
aan vulling. Medisch Contact 2008;63:
1822-26
Mullenders J, von der Saal W,
van Dongen MMW, Reiff U,
van Willigen R, Beijersbergen RL,
Tiefenthaler G, Klein C, Bernards R.
Candidate biomarkers of response to an
experimental cancer drug identified
through a large-scale RNA interference
genetic screen. Clin. Cancer Res.
2009;15:5811-19
Epping MT, Meijer LAT, Bos JL,
Bernards R. UNC45A confers resistance to
histone deacetylase inhibitors and retinoic
acid. Mol. Can. Res. 2009;7:1861-70
Mullenders J, Bernards R. Loss of function
genetic screens as a tool to improve the
diagnosis and treatment of cancer.
Oncogene 2009
Vredeveld LCW, Rowland BD, Douma S,
Bernards R, Peeper DS. Functional
identification of LRF as an oncogene that
bypasses RASV12-induced senescence via
upregulation of cyclin E. Carcinogenesis
2009
Figure 1: Cross talk between the NF1-RAS-MEK signaling cascade and retinoic acid signaling in
neuroblastoma.
Loss of NF1 activates RAS-MEK signaling, leading to downregulation of the RAR coactivator ZNF423. In
turn, suppression of ZNF423 hampers the transcriptional response of RAR/RXR to RA causing resistance
to RA-mediated growth arrest, differentiation and apoptosis. Restoration of ZNF423 by use of a selective
MEK inhibitor restores responsiveness to RA in NF1low neuroblastoma cells.
We also studied mechanisms of resistance to histone deacetylase inhibitors (HDACi),
which have recently been approved for the treatment of certain leukemias. Using a
genome-wide loss-of-function screen, we identified HR23B, which shuttles
ubiquitinated cargo proteins to the proteasome, as a sensitivity determinant for
HDACi-induced apoptosis. HR23B also governs tumor cell sensitivity to drugs that
act directly on the proteasome. HR23B is found at high levels in cutaneous T cell
lymphoma in situ, a malignancy that responds favorably to HDAC inhibitor-based
therapy. These results suggest that deregulated proteasome activity contributes to the
anticancer activity of HDAC inhibitors. In a complementary study, we searched for
genes whose over-expression conferred resistance to HDACi. Here we identified
Unc45a as a novel resistance marker of HDACi therapy. The vertebrate Unc45 genes
are known for their roles in muscle development and the assembly and cochaperoning
of the muscle motor protein myosin. Since our previous studies had
identified suppression of RA signaling as a major mechanism of resistance to
HDACi, we also studied a possible role of UNC45A in regulation of the response to
RA. Surprisingly, we found that UNC45A also inhibits signaling through the RA
receptor. We found that expression of UNC45A inhibits RA-induced proliferation
arrest of human neuroblastoma cells and inhibits the induction of retinoic acid
receptor target genes. These data establish an unexpected role for UNC45A in
causing resistance to both HDACi drugs and RA. Moreover, these data lend further
support to the notion that HDACi exert their anticancer effect, at least in part,
through an effect on RA signaling.
Finally, we have studied TSPYL5, a gene of unknown function whose over-expression
is associated with poor prognosis in breast cancer. We were interested in this gene, as
it is located at chromosome 8q22, within a small region that is frequently amplified
in breast cancer. Using mass spectrometry, we found that TSPYL5 physically interacts
with ubiquitin-specific protease 7 (USP7)/herpesvirus-associated ubiquitin-specific
protease (HAUSP). USP7 is the DUB for the p53 tumour suppressor and we find that
TSPYL5 opposes the activity of USP7 towards p53, resulting in increased p53
ubiquitination. We found that TSPYL5 reduces p53 protein levels and inhibits
activation of p53 target genes. Moreover, expression of TSPYL5 overrides p53dependent
proliferation arrest and oncogene-induced senescence in multiple assays.
These data identify TSPYL5 as a novel proto-oncogene that critically modulates the
p53-USP7 network.

tHe Rnai stRateGY in taRGet DiscoveRY
The research in my laboratory continues to evolve around the identification 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 specific tumor-associated genetic
alterations.
synthetic lethal interactions For the effective treatment of cancer, there is a great
need for drugs that specifically 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 defined 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 identification of novel cancer drug targets that are only
cytotoxic in the context of a tumor specific alteration and represent ‘genotype specific’
drug targets. We have generated a panel of isogenic cell lines derived from primary
human BJ fibroblasts that contain single or multiple defined genetic alterations that
together are required for tumorigenic transformation of these primary cells. These
genetic alterations include among others, loss-of-p53, 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
specific genetic alterations. We have completed these screens and have identified
several siRNAs whose lethality is dependent on a specific genotype. We have
identified over 15 genes that are synthetic lethal with the activation of RAS. 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 (figure 2). These results indicate that the synthetic lethal phenotype is
associated with RAS pathway activation. We will extend the analysis of the
interactions in a panel of RAS 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 phenotype.
Figure 2: Synthetic lethal interaction with activation of HRAS, NRAS and KRAS
Isogenic BJ cell lines were generated expressing the oncogenic forms of the RAS protein family. Cell lines
were transfected with siRNAs and cell survival was measured 72 hours after transfection. Knock down of
PLK1 is used as a positive control.
siRna screen for the identification of components of the tGFß signaling
network The TGFb pathway orchestrates an extensive transcriptional program that is
important for many processes in the cell. The TGFb pathway has a dual role in
cancer: it is involved in early stage tumor suppression, but also contributes to tumor
progression by promoting invasion. To identify novel genes involved in TGFb
pathway signaling, we have used a TGFb responsive reporter driving the expression
79
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
cor lieftink msc Bioinformatician
Bram Gerritsen msc Bioinformatician
Wouter nijkamp Technical staff
Ben morris Technical staff

80
moleculaR caRcinoGenesis
Publications
Mullenders J, von der Saal W,
van Dongen MM, Reiff U, van Willigen R,
Beijersbergen RL, Tiefenthaler G, Klein C,
Bernards R. Candidate biomarkers of
response to an experimental cancer drug
identified through a large-scale RNA
interference genetic screen. Clin Cancer Res.
2009;15:5811-9
Birmingham A, Selfors LM, Forster T,
Wrobel D, Kennedy CJ, Shanks E,
Santoyo-Lopez J, Dunican DJ, Long A,
Kelleher D, Smith Q, Beijersbergen RL,
Ghazal P, Shamu CE. Statistical methods
for analysis of high-throughput RNA
interference screens. Nat Methods.
2009;6:569-75
Huang S, Laoukili J, Epping MT, Koster J,
Hölzel M, Westerman BA, Nijkamp W,
Hata A, Asgharzadeh S, Seeger RC,
Versteeg R, Beijersbergen RL, Bernards R.
ZNF423 is critically required for retinoic
acid-induced differentiation and is a
marker of neuroblastoma outcome.
Cancer Cell. 2009;15:328-40
Mullenders J, Fabius AW, Madiredjo M,
Bernards R, Beijersbergen RL. A large
scale shRNA barcode screen identifies the
circadian clock component ARNTL as
putative regulator of the p53 tumor
suppressor pathway. PLoS ONE
2009;4:e4798
Otto T, Horn S, Brockmann M, Eilers U,
Schüttrumpf L, Popov N, Kenney AM,
Schulte JH, Beijersbergen R,
Christiansen H, Berwanger B, Eilers M.
Stabilization of N-Myc is a critical function
of Aurora A in human neuroblastoma.
Cancer Cell. 2009;15:67-78
of luciferase. Upon stimulation with TGFb, reporter activity is enhanced in a
SMAD4-dependent manner. We screened a siRNA library targeting 700 kinases and
kinase related genes using this TGFb responsive reporter construct. Several genes
were identified whose knockdown could repress the reporter signal; among these are
the two cellular receptors for TGFb. In addition to these two known components of
the TGFb pathway, several genes were identified that were previously not linked to
the TGFb signaling. Knockdown of one of these genes, the IRAK2 kinase, resulted
not only in an impaired TGFb target genes response, but also in a reduction of the
nuclear localization and phosphorylation of SMAD2 (figure 3). In addition,
suppression of IRAK2 expression led to a partial override of a TGFb induced cell
cycle arrest. These data demonstrate that IRAK2 is a novel and critical component of
TGFb signaling.
Figure 3: SMAD2/3 nuclear localization
PC3 cells were transfected with siRNAs against GFP or TGFBR2 and 48 hours later treated with TGFb
(+) for 1 hr or left untreated (-). Immunofluorescence for SMAD2/3 was performed and cells were counter
stained with DAPI to determine the position of the nuclei. The ratio of nuclear and cytoplasmic SMAD2/3
was quantified with Cellprofiler.

ReGulation oF cell Division in noRmal anD
canceR cells
In my group we aim to understand how cell division works in both normal and
cancer cells, by using an approach of biochemistry, molecular genetics in human
cells, and automated live-cell fluorescence imaging. We focus on three major
biological processes in the cell cycle: i) how cells enter mitosis from G2 phase; ii) how
cells exit mitosis and divide successfully and iii) how cells that exit mitosis re-initiate
DNA replication. Equally important are 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 under conditions that are unfavorable 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 create further insights into the
mechanisms that trigger abrupt cyclin B1-Cdk1 activation in late G2 and the parameters
of an ‘activation threshold’ for mitosis (figure 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 identification a
novel human kinase that directs mitotic entry of G2 cells. By determining the
conditions for mitotic entry in both normal and cancer cells, we aim to understand the
effects of interfering with cyclin B1-Cdk1 activity as an approach for cancer therapy.
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.
The percentage of remaining Cdk1 protein is indicated in the figure.(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 Cdk1-attenuated 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.
81
moleculaR caRcinoGenesis
Group leader Rob Wolthuis
Rob Wolthuis PhD Group Leader
linda clijsters msc PhD student
Wouter van Zon msc PhD student
erik voets msc PhD student
Janneke ogink Technical staff
Bas ter Riet Technical staff

82
moleculaR caRcinoGenesis
Publications
van der Lelij P, van Zon W, Godthelp BC,
van Gosliga D, Oostra AB, Steltenpool J,
de Groot J, Scheper RJ, Wolthuis RMF,
Waisfisz Q, Darroudi F, Joenje H,
de Winter JP. The cellular phenotype of
Roberts syndrome fibroblasts as revealed by
ectopic expression of ESCO2. PLoS ONE
2009;4:e6936
van Leuken R, van Zon W, Clijsters L,
Lim D, Yao XB, Wolthuis RMF, Yaffe MB,
Medema RH, van Vugt MATM. Polo-like
kinase-1 controls Aurora A destruction by
activating APC/C-Cdh1. PLoS ONE
2009;4:e5282
Wolthuis R, van Zon W, Clay-Farrace L,
Yekezare M, Koop L, Ogink J, Medema R
and Pines J. Cdc20 and Cks Direct the
Spindle Checkpoint-Independent
Destruction of Cyclin A. Molecular Cell
2008;30:290-302
van Zon W, Wolthuis RMF. Cyclin A and
Nek2A: APC/C-Cdc20 substrates invisible
to the mitotic spindle checkpoint.
Biochemical Society Transactions 2009
(in press)
Gurden MDJ, Holland AJ, van Zon W,
Tighe A, Vergnolle MA, Malumbres M,
Wolthuis R, Cleveland DW and Taylor SS.
Cdc20 is required for the post-anaphase,
KEN-dependent degradation of Cenp-F.
J Cell Sci 2009 (in press)
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 first 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 (figure 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.
Figure 5: Measuring APC/C activity by time-lapse fluorescence microscopy of Cyclin B1 destruction.
A plasmid encoding for fluorescent 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 fluorescence channel of the same cells. The cell undergoing division rapidly degrades cyclin B1, as
measured by a decrease in fluorescence signal. This assay allows the quantitation of ubiquitin-dependent
protein destruction in live cells undergoing division and was first established in the laboratory of Jon Pines,
Gurdon Institute, Cambridge, UK.
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 modifications in cancer cells may impinge on critical
steps in mitotic entry, mitotic progression and mitotic exit. It is anticipated that
answers to these questions could create exciting opportunities to improve anti-cancer
therapies.

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
modifications. We study the mechanism of transcriptional repression by Polycombgroup
(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 first (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 identified the Bmi1/Ring1b heterodimer as an E3
ubiquitin ligase for monoubiquitination of histone H2A (collaboration with
G Buchwald and Titia Sixma, Division of Biochemistry). Conditional Ring1b loss-offunction
experiments indicate an essential role for maintenance of Pc-G repression
in development and stem cell maintenance. 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 a genome-wide survey of where PRC1 and
PRC2 complexes bind to the Drosophila genome using DAMid profiling on cDNA
and tilling arrays (collaboration with Bas van Steensel, Division of Gene Regulation).
This highlighted binding of both PRC1 and PRC2 to distinct domains of 10-140 Kb
containing ± 400 target genes. These comprise developmental regulators that control
differentiation and are conserved in mammals. In recent in vivo 4C (chromatin
conformation capture on Chip) experiments we demonstrated that these domains
interact in 3D nuclear space.
Connections between Pc-G gene repression, control of stem cell fate and
cancer formation We originally identified 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 Bmi1deficient
primary embryo fibroblasts (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 transplantation model, we
recently revealed the essential role of Bmi1 in mammary epithelial stem cells and
precursors and ductal tree development (figure 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.
Maarten Van Lohuizen PhD Group leader
Elisabetta Citterio PhD Senior Post-doc
Jean Bernard Beaudry PhD Post-doc
Jaap Kool PhD Post-doc
Karim Nacerddine 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
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
Publications
83
MOLECULAR GENETICS
Division head, group leader Maarten Van Lohuizen
Michael LE, Westerman BA, Ermilov AN,
Wang A, Ferris J, Liu J, Blom M,
Ellison DW, van Lohuizen M, Dlugosz AA.
Bmi1 is required for Hedgehog pathwaydriven
medulloblastoma expansion.
Neoplasia. 2008;10:1343-9
Pietersen AM, Horlings HM,
Hauptmann M, Langerod A, Ajouaou A,
Cornelissen-Steijger P, Wessels LF,
Jonkers J, van de Vijver MJ,
van Lohuizen M. EZH2 and BMI1
inversely correlate with prognosis and TP53
mutation in breast cancer. Breast Cancer
Res. 2008;10:R109
Schuettengruber B, Ganapathi M,
Leblanc B, Portoso M, Jaschek R,
Tolhuis B, van Lohuizen M, Tanay A,
Cavalli G. Functional Anatomy of Polycomb
and Trithorax Chromatin Landscapes in
Drosophila Embryos. PLoS Biol. 2009;7:e13
Bruggeman SW, Hulsman D,
van Lohuizen M. Bmi1 deficient neural
stem cells have increased Integrin dependent
adhesion to self-secreted matrix. Biochim
Biophys Acta. 2009;1790:351-60

84
MOLECULAR GENETICS
Publications (continued)
Liu J, Cao L, Chen J, Song S, Lee IH,
Quijano C, Liu H, Keyvanfar K, Chen H,
Cao LY, Ahn BH, Kumar NG, Rovira II,
Xu XL, van Lohuizen M, Motoyama N,
Deng CX. Finkel TBmi1 regulates
mitochondrial function and the DNA
damage response pathway.
Nature. 2009;19404261
Prieur A, Nacerddine K, van Lohuizen M,
Peeper DS. SUMOylation of DRIL1 directs
its transcriptional activity towards leukocyte
lineage-specific genes. PLoS ONE.
2009;4:e5542
Boukarabila H, Saurin AJ, Batsché E,
Mossadegh N, van Lohuizen M, Otte AP,
Pradel J, Muchardt C, Sieweke M,
Duprez E. The PRC1 Polycomb group
complex interacts with PLZF/RARA to
mediate leukemic transformation. Genes
Dev. 2009;23:1195-206
Uren AG, Mikkers H, Kool J,
van der Weyden L, Lund AH, Wilson CH,
Rance R, Jonkers J, van Lohuizen M,
Berns A, Adams DJ. A high-throughput
splinkerette-PCR method for the isolation
and sequencing of retroviral insertion sites.
Nat Protoc. 2009;4:789-98
Puppe J, Drost R, Liu X, Joosse SA, Evers B,
Cornelissen-Steijger P, Nederlof P, Yu Q,
Jonkers J, van Lohuizen M, Pietersen AM.
BRCA1-deficient mammary tumor cells are
dependent on EZH2 expression and sensitive
to Polycomb Repressive Complex 2-inhibitor
3-deazaneplanocin. Breast Cancer Res.
2009;11:R63
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 EGF-receptor signaling rapidly
form agressive gliomas whereas tumor
formation is delayed upon transplantation
of Bmi1-deficient transformed astrocytes
orthotopically transplanted in the forebrain
of recipient mice.
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
deficient mice.
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 deficient 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 (figure 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 findings for human cancer is further underscored by the
amplification 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.
In vivo genetic screens to identify new groups of collaborating oncogenes or
tumor suppressors In close collaboration with Anton Berns (this Division), Jos
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 first completed screens in hemapoietic
tumors induced in wild type, p53 or p19Arf deficient mice. We recently extended
these screens to p15Ink4b, p21 and p27 deficient mice and to Pten-deficient 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-specific 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. Using Cre/Lox mediated switching
taking advantage of somatic gene transfer methods, expression of multiple oncogenes
and tumor suppressor genes can be regulated in a tissue-specific and spatial-temporal
fashion. This permits a more accurate modeling of tumorigenesis as it occurs in
man, and therefore provides the opportunity for establishing relevant genotypephenotype
correlations. These models also provide an excellent experimental tool to
test prevention and intervention strategies especially when combined with sensitive
in vivo imaging techniques. Finally, these models permit us to identify new oncogenes
and tumor suppressor genes involved in tumor progression using a variety of
techniques, such as array CGH, expression profiling and proviral insertional
mutagenesis.
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 cancers
subtypes and mesotheliomas. To achieve (sporadic) activation of oncogenes and
inactivation of tumor suppressor genes we use Adeno-Cre or Lentivirus-mediated
gene transfer to switch the conditional oncogenes and tumor suppressor gene alleles.
Subsequently, tumor initiation and progression is monitored in longitudinal studies
in which noninvasive imaging techniques are used.
Lung tumors We focus on small cell lung cancer (SCLC), non-small cell lung cancer
(NSCLC) and squamous cell carcinoma (SCC). When Rb and p53 are inactivated
specifically in lung, SCLC ensues. The marker profile of these tumors is closely
resembling that of human SCLC. Even similar genomic aberrations are found such
as the amplification of the L-Myc gene. These tumors are often heterogeneous
consisting of different cell types that either grew as spheres in suspension or attached
to substrate. Cloning of the suspended and attached cells from individual tumors
showed a consistently different marker profile between these two cell types. Cells
growing in suspension carries neuroendocrine markers whereas the adherent nonneuroendocrine
cells showed more progenitor-like features. Interestingly, these
phenotypically very diverse cell lines shared some highly distinct genetic aberrations
indicating that they were derived from a common progenitor. K-ras introduction into
neuroendocrine cells changed the phenotype into non-neuroendocrine
characteristics.
We wondered why these different cell types persisted in the tumor. To search for
possible paracrine effects between these cells, in vitro co-culture and in vivo cografting
experiments were conducted. In serum-free medium co-culture strongly
promoted the proliferation of both cell types. In subcutaneous grafts such growth
stimulation was not found. Grafts of each of the cell types gave rise to a local tumor,
however, grafts of the mixture resulted in a high incidence of metastatic spread of the
neuroendocrine cells indicating that the non-neuroendocrine cells in the graft
empowered the neuroendocrine cells to metastasize. We are currently investigating
which signaling events are responsible for this effect.
To gain insight into the cell of origin of SCLC and NSCLC we have designed a series
of cell-type specific Adeno-Cre viruses that enable us to switch oncogenes and tumor
suppressor genes in distinct lung cell types in vivo. Using promoters specific for
Clara cells, Alveolar type II cells, neuroendocrine cells and basal epithelial cells to
drive Cre expression upon Adeno-Cre infection indicated that Cre driven from a
neuroendocrine-specific promoter gave rise to SCLC with high efficiency, whereas
Cre driven from the alveolar-specific promoter SPC showed a delayed onset of SCLC.
Expression from a Clara cell-specific promoter gave mostly hyperplasias in the
bronchial epithelial lining and resulted only rarely to SCLC. We are performing these
experiments with additional promoters to further substantiate these findings.
85
MOLECULAR GENETICS
Group leader Anton Berns
Anton Berns PhD Group leader
Paul Krimpenfort PhD Academic staff
Margriet Snoek PhD Academic staff
Joaquim Calbo PhD Post-doc
Hilda De Vries PhD Post-doc
Johan Jongsma PhD Post-doc
Jaap Kool PhD Post-doc
Andor Kranenburg PhD Post-doc
Min-chul Kwon PhD Post-doc
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
Figure 3: When neuroendocrine and nonneuroendocrine
are cultured together, both
proliferate much faster indicating that they
interact likely through paracrine signaling
mechanisms

86
MOLECULAR GENETICS
Publications
Kazarian M, Calbo J, Proost N, Carpenter C,
Berns A, Laird-Offringa I. Immune response
in lung cancer mouse model mimics human
anti-Hu reactivity. J Neuroimmunol. 2009
Kool J, Berns A. High-throughput
insertional mutagenesis screens in mice to
identify oncogenic networks. Nat Rev
Cancer 2009;9:389-399
Uren A, Berns A. Jump-starting cancer
gene discovery. Nat Biotechnol 2009;27:
251-252
Uren AG, Mikkers H, Kool J,
van der Weyden L, Lund AH, Wilson CH,
Rance R, Jonkers J, van Lohuizen M,
Berns A. A high-throughput splinkerette-
PCR method for the isolation and
sequencing of retroviral insertion sites. Nat
Protoc 2009;4:789-798
van Amerongen R, Nawijn M, Lambooij J,
Proost N, Jonkers J, Berns A. Frat
oncoproteins act at the crossroad of
canonical and noncanonical Wnt-signaling
pathways. Oncogene 2009
Figure 4: Bioluminescence imaging of mice grafted with either neuroendocrine cells (1 and 2),
non-neuroendocrine cells (# 5) or a mixture (# 3 and 4). Note the extensive metastasis to liver in
mice grafted with the cell mixture.
Mesotheliomas Earlier 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 inactivating these genes in combination with activation of mutant
KiRas results in mesotheliomas with a pronounced epitheloid phenotype, closely
resembling the corresponding predominant subtype found in human mesotheliomas.
We have set up a primary tumor grafting model system that allows us to test various
intervention strategies following subcutaneous and orthotopic grafting of primary
tumor cells. These tumors appear highly refractory to most intervention protocols
and show highly variable responses to drug combinations, indicating that additional
alterations that have occurred in these tumors greatly influence the drug response
profile of these tumors. Therefore, a more thorough analysis of activated signaling
pathways in these tumors will be conducted to gain insight in the molecular basis
of their drug response characteristics. Parallel to these experiments we have begun
to establish cultures from mesothelioma specimen of human mesotheliomas.
While propagation in mice will require more work, these cells grow effectively in
vitro. We are planning to generate response profiles from these tumor cell lines to
determine whether there are good matches between the human and mouse
mesothelioma cell lines.
Ink4 proteins We have produced Ink4-less mice. Mice lacking all the four Ink4a-d
genes are viable. We are currently monitoring the spontaneous tumor incidence in
these mice, will determine the effects of additional germline inactivation of p19Arf,
and establish mouse embryo fibroblast cell lines to look for altered characteristics of
the cells in culture. This will teach us to what extent this tumor suppressor family
plays a role in tumor suppression in the various tissues. In addition, this might reveal
unexpected physiological functions of these proteins.
Functional mutagenesis
The cyclin-dependent kinase (CDK) inhibitors p15, p16, p21, and p27 are frequently
deleted, silenced, or downregulated in many malignancies. We have used mice deficient
for one or a combination of two CDK inhibitors to conduct a high-throughput murine
leukemia virus (MuLV) insertional mutagenesis screen. We retrieved approximately
9000 retroviral insertions from 476 lymphomas and found hundreds of loci that are
mutated significantly more frequently than expected by chance. Many of these are
skewed toward a specific genetic context of predisposing germline and somatic
mutations. We also found associations between these loci and gender, age of tumor
onset, and with lymphocyte lineage (B or T cell). Comparison of retroviral insertion sites
with single nucleotide polymorphisms associated with chronic lymphocytic leukemia
reveals significant over- lap between these data sets. Together, these data highlight the
importance of genetic context within large- scale mutation detection studies and show a
novel use for insertional mutagenesis data in prioritization of disease-associated genes
resulting from genome-wide association studies.

RESOLVING CANCER CELL SIGNALING NETWORKS &
IDENTIFYING CANCER DRUG TARGETS
What are the cellular mechanisms protecting us against cancer? How can we effectively
identify novel cancer genes? How can we resolve tumor-suppressing genetic networks?
Can we use our laboratory results to make a difference in the clinic? For example,
how can we identify novel and specific drug targets? In a nutshell, these are the
fundamental as well as clinically relevant questions that we are taking up in the
Peeper laboratory. In doing so, we aim to get more insight into the central mechanisms
disrupting cellular protection against oncogenic processes. And by exploiting unbiased
genomic approaches, we intend to identify novel and selective cancer drug targets.
To reach these goals, we are combining function-based, genome-wide screens with
classical molecular biological approaches. For example, we are studying the genetic
basis for malignant progression of benign moles (nevi) to malignant melanoma. We,
in collaboration with prof. Wolter Mooi (VUmc), were the first to discover a human
lesion, the melanocytic nevus, or moles) displaying many of the classical hallmarks of
Oncogene-Induced Senescence (OIS) in vivo. We have also designed several functional
genomic screens with retroviral and lentiviral cDNA expression and RNA interference
libraries. For example, using OIS as a cancer-relevant cell-based setting, we have
identified novel oncogenes and tumor suppressor genes. Along these lines, we have
recently discovered an unanticipated role for interleukins and the inflammatory
transcriptome in cellular senescence (named Senescence-Messaging Secretome, or
SMS). This process may contribute to preventing colon cancer progression.
Furthermore, we have designed a new in-vitro screen for metastasis genes, based on
suppression of anoikis (detachment-induced cell death). This screen identified a
tyrosine kinase receptor (TrkB) that is overexpressed in metastasizing human tumors.
Further taking advantage of this experimental system, we have recently discovered a
novel and critical mediator of breast cancer metastasis, which also accurately predicts
clinical outcome. Our laboratory intends to also translate information from its in-vitro
experiments to the clinic. For example, in collaboration with pharma industry we are
currently validating TrkB as a novel drug target. Furthermore, we have begun setting
up so-called synthetic lethality screens as a direct and efficient approach to discover
novel cancer drug targets.
We anticipate that these lines of research, together, will not only unravel genetic
networks that upon deregulation contribute to cancer and metastasis. In addition, the
genes that we are identifying may represent novel and specific targets for therapeutic
intervention of cancer.
Function-based genomic screens for metastasis genes Metastasis commonly
underlies the malignancy of cancers, representing the principal cause of cancertreatment
failure. Tumor colonization is 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. In an attempt to identify metastasisassociated
oncogenes we designed an unbiased, functional genome-wide screen
solely based on anoikis suppression. With this approach, we previously identified
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 TrkB, allowing formation of
spheroid aggregates growing in suspension. Mice readily cleared parental cells, but
TrkB-expressing cells formed many lung and heart metastases. Co-expression of
BDNF, the prototypic TrkB ligand, caused highly invasive metastases in virtually
every tissue, with an extremely short latency.
We have made significant progression in identifying downstream signaling targets of
this receptor, as they, in addition to TrkB, may serve as targets for therapeutic
intervention. RNAi knockdown of some of these newly identified targets impairs the
tumorigenic potential of TrkB. We have recently identified several transcription
factors, including Twist and Snail, whose RNAi-mediated silencing significantly
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MOLECULAR GENETICS
Group leader Daniel Peeper
Daniel Peeper PhD Group leader
Gireesh Anirudhan PhD Post-doc
Tristan Gallenne PhD Post-doc
Thomas Geiger 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
Thomas Kuilman Msc PhD student
Marjon Smit MSc PhD student
Liesbeth Vredeveld MSc PhD student
Sirith Douma MSc Technical staff
Arantxa Rosado Technical staff
Figure 5: Model depicting a critical TrkB
effector pathway contributing to EMT,
anoikis suppression and metastasis. TrkB
activation leads to induction of both Twist
and Snail. These transcription factors
repress E-cadherin, thereby inducing EMT
and anoikis suppression, facilitating
metastasis. Twist, acting upstream of Snail,
conceivably has additional targets, including
those that are primarily required for tumor
growth.

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MOLECULAR GENETICS
Publications
Vredeveld LC, Rowland BD, Douma S,
Bernards R, Peeper DS. Functional
identification of LRF as an oncogene that
bypasses RASV12-induced senescence via
upregulation of cyclin E. Carcinogenesis.
2009 (in press)
Geiger TR, Peeper DS. Metastasis
mechanisms. Biochim Biophys Acta.
2009;1796:293-308
Prieur A, Nacerddine K, van Lohuizen M,
Peeper DS. SUMOylation of DRIL1 directs
its transcriptional activity towards leukocyte
lineage-specific genes. PLoS One.
2009;4:e5542
Smit MA, Geiger TR, Song JY, Gitelman I,
Peeper DS. A Twist-Snail axis critical for
TrkB-induced epithelial-mesenchymal
transition-like transformation, anoikis
resistance, and metastasis. Mol Cell Biol.
2009;29:3722-37
Peeper DS. Ras and pRb: the relationship
gets yet more intimate. Cancer Cell.
2009;15:243-5
Kuilman T, Peeper DS. Senescencemessaging
secretome: SMS-ing cellular
stress. Nat Rev Cancer. 2009;9:81-94
Figure 6: Snail depletion prevents
morphologic transformation of epithelial
cells by active TrkB.
suppressed the metastatic potential of the tumor cells (see figures). Furthermore, we
found that the transcriptome that was associated with the activity of one of these
transcription factors had strong predictive value on the clinical outcome of human
breast cancer. These results reveal a key role for this gene in breast cancer metastasis
and indicate that its transcriptome can be exploited prognostically. Moreover, these
data merit efforts to inactivate the components of this signaling pathway for clinical
intervention of human breast cancer.
We are also in the process of identifying cancer-associated mutations of TrkB,
which we will analyze for biological significance. Finally, we have engineered TrkB
transgenic mouse models, to further explore the role of TrkB in cancer and metastasis,
and to serve as a model system for TrkB in cancer, enabling the development and
testing of TrkB-inhibitory therapeutic drugs.
Oncogene-Induced cellular Senescence (OIS) as a tumor-suppressing
mechanism Currently, we are using various functional genomic approaches to
identify novel OIS pathways whose deregulation contributes to cancer. In one of
these screens we identified a novel oncogene, LRF, which bypasses RAS V12 -induced
senescence via upregulation of cyclin E. In a related study we found that SUMOylation
of DRIL1, a gene we previously identified in this setting, directs its transcriptional
activity towards leukocyte lineage-specific genes. In addition to taking a candidate
gene approach, we are combining gene expression analysis and RNAi in combination
with function-based genomic screens. For example, microarray expression analysis
revealed the induction of various signaling networks that are induced specifically in
BRAF E600 -expressing, senescent human cells. Among the top outliers was interleukin
6 (IL-6). RNAi-mediated knockdown of IL-6 abrogated the senescence response and
led to continuous proliferation of BRAF E600 -expressing cells. Similar findings were
made for other interleukins, which prompted us to search for a common
denominator of BRAF E600 -regulated interleukins. We found that C/EBPb acts as a
major player in this pathway. Consistently, knockdown of C/EBPb effectively
bypassed BRAF E600 -induced senescence. In collaboration with prof. Lucien Aarden
(Sanquin) we found that in OIS IL-6 had a cell-autonomous role, being required in
an autocrine fashion both for the induction and maintenance of cell cycle arrest of
cells exposed to oncogenic stress. Upon IL-6 depletion, the inflammatory network
collapsed and cells bypassed senescence. C/EBP� was identified as an OIS integrator,
acting with IL-6 to amplify the activation of the inflammatory network, including
IL-8. In collaboration with prof. Wolter Mooi (VUmc), we observed that in human
colon adenomas, IL-8 specifically 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 inflammatory phenotype and cancer. We
anticipate that this integrative genomics approach will identify novel tumor
suppressors involved in melanoma and other (BRAF E600 -associated) cancers.
We noted that the activation of the inflammatory transcriptome in cellular senescence
resulted in the secretion of several factors, including cytokines and chemokines.
This Senescence-Messaging Secretome, or SMS, as we propose to call this, plays an
important role in the communication between senescent cells and their microenvironment.
Although the selective advantage of OIS to the organism seems
obvious, it is less straightforward to explain why the SMS contributes to this. We
believe that the evolutionary advantage to utilize the SMS messaging system is that it
does just that: messaging, allowing for communication both within and between
cells. One might hypothesize that, for example, through SMS signals, a senescent cell
sends a ‘danger signal’ to its microenvironment. This could be advantageous early in
life in several ways, but this system could also have adverse effects for the organism,
at later age.

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 identified, while this is essential for the
development of more effective novel therapeutic strategies. The aim of our laboratory
is to identify and study novel genes involved in breast cancer by using mouse
mammary tumor virus (MMTV) induced insertional mutagenesis in mouse models
as paradigms for human breast cancer.
Identification 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 flanking proto-oncogenes
leading to mammary tumor induction. In recent years, we identified over 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 deficient for Tp53 in the
mammary gland and MMTV-NeuNT transgenic mice. Many of the CISs affect genes
not previously implicated in cancer or breast cancer in particular. To identify the
proviral MMTV insertion sites, we have used until recently a technique based on the
splinkerette PCR that was originally developed in the Berns Lab. This method is
laborious, introduces biases due to the use of restriction enzyme digestions, does not
provide information about the clonality of the insertions and reveals at most 50% of
the insertions. Together with Marco Koudijs in the Jonkers lab, part of the collected
MMTV induced mammary tumors was reanalyzed using the new parallel sequencing
platforms. The results indeed show additional proviral insertion sites and some novel
CISs, urging us to reanalyze the complete tumor panels.
The proviral insertion mutagenesis screen performed to date, revealed that Wnt and
Fgf genes are activated in almost all wild type BALB/c tumors, less frequently in Tp53
deficient 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, were also among
the frequent MMTV tagged genes in the tumors from wild type and conditionally
deficient Tp53 mice but rarely activated in tumors from ErbB2 transgenic mice. In
contrast, three genes Irs4, Eras and Igf2 were found to be more frequently tagged in
tumors from MMTV infected ErbB2 transgenic mice then in wild type mice.
Analysis of genes collaborating with ErbB2 We have studied Irs4, Eras and Igf2
in more detail. We found that Irs4 as well as Eras over-expression in immortalized
normal mammary epithelial cells (NMuMG cells) renders these cells tumorigenic in
nude mice. Moreover, Irs4 and Eras strongly accelerated tumorigenesis when coexpressed
with ErbB2 in NMuMG cells (figure 7). We showed that Irs4 and Eras
specifically activate the PI3-kinase pathway but not the MAP kinase pathway in
NMuMG cells. Igf2 is a known strong activator of the PI3-kinase pathway. ERBB2 in
human and mouse mammary tumors is usually present in a heterodimer consisting
of ERBB2 and ERBB3; ERBB2 in this complex mainly activates the MAPK pathway,
while ERBB3 activates the PI3K pathway. We found that ErbB3 is not expressed in a
large proportion of the tumors from ErbB2 transgenic mice while none of the tumors
derived from NMuMG cells transduced with ErbB2 (or ErbB2 + Irs4) express ErbB3.
Since ErbB2 driven mammary tumorigenesis is highly dependent on PI3-kinase
activity, we hypothesize that genes like Irs4 and Eras are targeted by MMTV in ErbB2
tumors that lack ErbB3 mediated PI3K activation. Activation of Eras, Irs4 and Igf2 is
likely an alternate way to activate the PI3-kinase pathway normally accomplished by
ERBB3 activation, PTEN inactivation or PIK3CA mutations.
Group leader John Hilkens
John Hilkens PhD Group leader
Annabel Zwaagstra MSc PhD student
Mandy Boer Technical staff
Publications
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MOLECULAR GENETICS
Van Slambrouck S, Hilkens J, Bisoffi M,
Steelant WF. AsialoGM1 and integrin
alpha2beta1 mediate prostate cancer
progression. Int J Oncol. 2009;35:693-699
Zhao Q, Guo X, Nash GB, Stone PC,
Hilkens J, Rhodes JM, Yu LG. Circulating
galectin-3 promotes metastasis by modifying
MUC1 localization on cancer cell surface.
Cancer Res. 2009; 69:6799-806
Figure 7: Collaboration of ErbB2 (Neu/
Her2) with Irs4 and Eras genes. NMuMG
cells derived from normal mammary
epithelial cells were retrovirally transduced
with NeuNT, Irs4 or Eras and by
combinations of these genes. Cells were
injected subcutaneously and tumor growth
of ErbB2, Irs4,ErbB2-Irs4, Eras, ErbB2-Eras
transduced NMuMG Cells
(+/- SD) was assessed at least biweekly.
The results show that the combined
expression of ErbB2 with Eras or Irs4 in
NMuMG cells strongly accelerates tumor
growth as compared with NMuMG cells
transduced with ErbB2 only. The Irs4 and
Eras transduced NMuMG cells give rise to
tumors after much longer latencies.

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MOLECULAR GENETICS
Junior group leader Jan-Hermen Dannenberg
Jan-Hermen Dannenberg PhD Junior
group leader
Inès Bejaoui MSc PhD student
Roel Wilting MSc PhD student
Eva Yanover Technical Staff
Publications
Farhana L, Dawson MI, Xu L,
Dannenberg JH, Fontana JA. SHP and
Sin3A expression are essential for
adamantyl-substituted retinoid-related
molecule-mediated nuclear factor-b B
activation, c-Fos/c-Jun expression, and
cellular apoptosis. Mol. Cancer Ther.
2009;8:1625-35
Guan JS a , Haggarty SJ a , Giacometti E a ,
Dannenberg JH a , Joseph N, Gao J,
Nieland T, Zhou Y, Wang X, Mazitschek R,
Bradner JE, DePinho RA, Jaenisch R,
Tsai LH. HDAC2 negatively regulates
memory formation and synaptic plasticity.
Nature 2009;459:55-63 ( a authors equally
contributed)
Figure 8: Hdac1 plays a specific role in
differentiation of the erythro-megakaryocytic
lineage. A. Bone marrow sections of
indicated genotypes stained with
hematoxylin-eosin (left panels) and stained
for activated caspase 3 (right panel).
B. Erythrocyte and thrombocyte numbers
in peripheral blood of indicated genotypes.
THE ROLE OF CLASS I HDACS IN TRANSCRIPTION,
DEVELOPMENT AND TUMORIGENESIS
Epigenetic control of gene expression is known to play a prominent role in the
suppression and development of cancer. Histone deacetylases (HDACs), enzymes
involved in removing acetyl groups from lysine residues, are promising drug targets
in cancer therapy. The molecular mechanisms underlying the selective antitumorigenic
effect of HDAC-inhibitors (HDACi) are unclear. HDACi are relatively
non-specific, targeting multiple HDACs, of which only a subset may be important in
tumor biology, resulting in undesirable side-effects. Furthermore, HDAC-function in
normal and tumor cells is not fully understood, hampering understanding of the
inhibitor’s anti-tumor selectivity and identification of critical HDAC targets
responsible for anti-tumor effects. Complete knowledge of HDAC function will aid in
designing more efficacious inhibitors, understanding mechanisms of resistance and
the identification of responders within a patient population.
Our research group wishes to address these issues in a systematic, genetic approach
using conditional knockout alleles of two class I HDACs, Hdac1 and Hdac2. By
deleting these genes in normal cells as well as tumor cells we wish to discover antitumor
relevant targets of these HDACs and study their specific and redundant
functions in transcription regulation, normal and tumor development and tumor
maintenance. Studies aimed at unraveling the transcriptome of these transcriptional
co-repressors in combination with the identification 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 Whereas deletion of
Hdac1 results in early embryonic lethality, we found that Hdac2 ablation results in
viable mice, which are born with a two-fold lower Mendelian frequency and display a
25% reduction in total bodyweight. In collaboration with the group of Dr. Li-Huei
Tsai (Picower Institute for Learning and Memory, MIT, Cambridge, USA), it was
shown that Hdac2 deficiency results in increased synapse number and memory
facilitation, similar to chronic treatment with HDACi in mice. Correspondingly,
treatment with HDACi failed to further facilitate memory formation in Hdac2deficient
mice. Furthermore, analysis of promoter occupancy revealed an association
of Hdac2 with the promoters of genes implicated in synaptic plasticity and memory
formation. Taken together, these results suggest that Hdac2 functions in modulating
synaptic plasticity and long-lasting changes of neural circuits, which in turn
negatively regulates learning and memory. These observations encourage the
development and testing of HDAC2-selective inhibitors for human diseases
associated with memory impairment.
Hdac2 -/- MEFs displayed no obvious phenotype, probably due to compensatory up
regulation of Hdac1 protein levels. Indeed, ablation of both Hdac1 and Hdac2 or their
catalytic activity in MEFs, results in a senescence-like G1 cell cycle arrest
accompanied by increased p21 Cip protein levels. Surprisingly, concomitant genetic
inactivation of the p53-p21 Cip pathway does not allow a bypass of this cell cycle arrest.
In transformed cells harboring Ras V12 and inactivated p53, deletion of Hdac1 and
Hdac2 still activated a senescence-like cell cycle arrest. These data suggest that Hdac1
and Hdac2 collectively suppress a senescence-inducing pathway, which is critical for
the cell cycle progression of normal and tumor cells.
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 offtarget
effects or due to inhibition of HDACs we inactivated Hdac1 and Hdac2 in bone
marrow using MxCre transgenic mice. While ablation of Hdac2 does not affect
hematological differentation, Hdac1 deficiency results in severe anemia and
thrombocytopenia. Hdac1-deficient megakaryocytes display abnormal nuclear
morphology and increased mitotic figures. Surprisingly, concomitant inactivation of
Hdac1 and Hdac2 results in complete absence of megakaryocytes due to induction of
apoptosis. These data indicate that Hdac1 has a specific role in the differentiation of
the erythro-megakaryocytic lineage, while Hdac2 suppresses apoptosis in Hdac1deficient
megakaryocytes. Therefore, hematological related side-effects observed in
HDACi treated patients are very likely related to on-target Hdac1 inhibition.

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 and how telomere maintenance is controlled.
Telomere-induced cellular senescence Loss of telomere function triggers a DNA
damage response that causes cells to undergo apoptosis or to enter an irreversible
growth arrest called senescence. 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 eliminates potentially cancerous cells. We have
designed RNAi loss-of-function genetic screens in order to identify novel factors
involved in (the activation of) telomere-induced senescence. As a frequent tool in
these ongoing screens or in subsequent validation experiments we mimic replicative
telomere shortening by rendering telomeres dysfunctional through removal of the
telomere capping protein TRF2 from telomeres.
As an alternative genome-wide approach 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, gene expression analysis revealed that
genes involved in inflammatory/immune responses represented gene groups with
the most significant changes in expression upon telomere dysfunction. We have
subsequently focused on common upstream transcriptional regulators of these genes
and are investigating whether these regulators respond directly to telomere
dysfunction and to what extend they contribute to telomere-induced senescence.
Telomere maintenance and telomere-induced chromosome instability If cells
with uncapped telomeres fail to undergo senescence or apoptosis 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 ‘repair’
events occur and how these events are controlled. To identify novel factors that
contribute to telomere-induced genome instability we have developed an RNAi lossof-function
genetic screen in mouse cells in which we can instantly and reversibly
uncap telomeres by inactivating TRF2 to induce telomere fusion (see figure 9). The
degree of telomere fusion induced in this system is so severe that cells can’t divide or
will die. From this ongoing screen we have isolated multiple cell clones that survived
and retained the ability to divide while experiencing severe telomere uncapping. The
isolation and characterization of the shRNA vectors responsible for the observed
rescue 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 very
recently been shown by others to indeed contribute to the ‘repair’ of uncapped
telomeres by non-homologous end-joining (see figure 9), and which validates our
screen. The other genes identified so far have not previously been shown to be
involved in the response to uncapped telomeres and their mechanism of action is
now being investigated.
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MOLECULAR GENETICS
Junior group leader Jacqueline Jacobs
Jacqueline Jacobs PhD Junior 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 9: Schematic representation of a
loss-of-function genetic screen aimed at
identifying genes that contribute to telomereinduced
genome instability. Mouse embryo
fibroblasts with a genetic disruption of the
TRF2 gene, but expressing a temperature
sensitive TRF2 allele, lose telomere
protection on all their chromosome ends
when cultured at a non-permissive
temperature that causes inactivation of
TRF2. This results in severe fusion of
chromosome ends and subsequently cells are
unable to divide or will die. However
inhibition of NBS1, by an shRNA isolated
in this screen, allows cells to survive and to
divide in the presence of uncapped
telomeres.

92
MOLECULAR GENETICS
Junior group leader Anna-Pavlina Haramis
Anna-Pavlina Haramis PhD Junior group Leader
Yme van der Velden MSc PhD student
Liqin (Bruce) Wang Research assistant
Figure 10: Zebrafish 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).
THE DEVELOPMENTAL ROLES OF ONCOGENES
AND TUMOR SUPPRESSORS IN ZEBRAFISH
Our research focuses on understanding the role of signaling pathways that regulate
vertebrate development and disease using the zebrafish as a genetic model. Cancer is
a multifactorial disease and exhibits many features of embryonic development.
Deregulation of the genetic programs that guide growth and differentiation of cells
and tissues during organogenesis can contribute to carcinogenesis.
We are following two main lines of research:
Analysis of energy metabolism and hypoxia during normal 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 organism survival. Tumor cells employ
several of the mechanisms that coordinate nutrient availability with growth during
development to ensure 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.
The zebrafish is an ideal model system to dissect molecular mechanisms and
pathways. The large progeny size (females typically lay 200 eggs at a time) and
availability of readily accessible transparent embryos, combine to render the zebrafish
an excellent model for dissecting developmental pathways and for high-throughput
genetic and chemical screens in the intact organism. The major metabolic pathways
are very well conserved between humans and zebrafish.
Mutations in the serine-threonine kinase LKB1 in humans lead to a gastrointestinal
polyposis disorder with highly increased predisposition to 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. To
study control of metabolism during development and cancer, we have generated two
mutant lines in the single zebrafish LKB1 ortholog. Both mutations are stop codons
in the kinase domain and one of them has also been identified in human patients.
Importantly, the zebrafish lkb1 mutants survive gastrulation - unlike their mouse
counterparts - and exhibit deregulated metabolism.
Lkb1 mutant zebrafish 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 (figure 10). Microarray experiments show that genes
involved in lipid and glucose metabolism are differentially expressed in lkb1 mutants
compared to wild-type siblings and that some hypoxia-associated genes are
overexpressed in lkb1 mutants. 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. Fortuitously, zebrafish embryos in which Ring1b function has been
reduced survive gastrulation. Transient, morpholino-mediated knock-down of Ring1b
function in zebrafish leads to multiple developmental abnormalities including heart
and fin defects. 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 and
are currently characterizing the phenotype in detail.

DIVISION OF PSYCHOSOCIAL
RESEARCH AND EPIDEMIOLOGY
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.
Comparing higher order factor models for the EORTC QLQ-C30 Quality of
Life questionnaire The objective of this project is to identify/define higher order
components (or factors), summarizing the HRQL profile (composed of 15 outcomes)
generated by the QLQ-C30 questionnaire. The analyses are based on a large data set
including more than 9,000 baseline (i.e., pre-treatment) QLQ-C30 v3.0
questionnaires completed by cancer patients from 48 countries. Seven factor models
have been compared by means of confirmatory factor analysis. These models build
upon a ‘standard’ 14 dimensional QLQ-C30 model, and include a 1 dimensional (1D)
higher order factor model, a 2D ‘symptom burden and function’ model, two 2D
‘physical and mental health’ models, two models with ‘function’ and a ‘formative’ (or
‘causal’) formulation of ‘symptom burden’, and a ‘bi-factor’ model. One of the 2D
Physical/Mental models exhibits an adequate fit to the data, is the best fitting
summary of the ‘standard’ model, and enjoys empirical and theoretical support based
on other HRQL instruments and applications. This model will be further tested for
measurement equivalence over sub-populations and over time, for (known groups)
validity, and for responsiveness to change over time.
Methods and measures for assessing the HRQL of mid- to long-term
survivors of testicular and prostate cancer This study is a collaboration between
the EORTC Quality of Life and Genito-Urinary Cancer Group. It has two primary
objectives: (1) to test the logistics required to conduct survivorship studies within the
context of the EORTC, with specific 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). This study will generate
practical and ethically acceptable procedures for recruiting patients from EORTC
clinical trials for long-term HRQL survivorship studies and thereby lay the
groundwork for a series of future, substantive studies. 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-specific (the EORTC QLQ-C30 plus condition-specific
modules and (3) cancer survivor-specific (the Impact of Cancer questionnaire).
In 2009, the protocol was finalized and submitted for institutional review board
approval. In 2010, the study will be implemented in all participating countries.
A randomized controlled trial of exercise training in hematological cancer
patients following peripheral blood stem cell transplantation This RCT is being
carried out by R. Knols, a PhD student of ours in Zurich, Switzerland. Adult
hematologic cancer patients who have undergone peripheral blood stem cell
transplantation are randomly assigned to a 12-week, ambulatory, supervised endurance
and repetitive strength exercise program or to a usual care control group. Primary
outcomes include musculoskeletal performance and self-reported physical functioning.
Secondary outcomes include self-reported fatigue and HRQL, fatigue, self-reported
and objectively assessed physical walking activity, whole body composition and
hematological values. Patient accrual and follow-up has been completed. In total,
131 patients were randomized into the study, of whom 114 completed the immediate
post-treatment assessment, and 105 the 6 month follow-up. Preliminary results
indicate that the physical exercise program had a significant, positive effect on
93
PSYCHOSOCIAL RESEARCH
Division head, 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
Senno Verhoef MD PhD Academic staff
Saskia Duijts PhD Post-doc
Marieke van Leeuwen PhD Post-doc
Eric Vermeulen PhD Post-doc
Kirsten Douma MSc PhD student
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 MD PhD student
Chad Gundy MSc Senior statistical analyst
Miranda Gerritsma MSc Research assistant
Marianne Kuenen Research assistant
Jacoline Melis MSc Research assistant
Tanja Nagtegaal MSc Research assistant
Publications
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.
Psycho-oncology 2009
Douma KFL, Aaronson NK, Vasen HFA,
Verhoef S, Gundy CM, Bleiker EMA.
Attitudes towards genetic testing in
childhood and reproductive decision-making
for familial adenomatous polyposis (FAP).
Eur J Human Genetics 2009
Gehring G, Sitskoorn MM, Gundy CM,
Sikkes SAM, Klein M, Postma TJ,
van den Bent MJ, Beute GN, Enting RH,
Kappelle AC, Boogerd W, Veninga T,
Twijnstra A, Boerman DH, Taphoorn MJB,
Aaronson NK. Cognitive rehabilitation in
glioma patients: A randomized, controlled trial.
J Clin Oncol 2009;27:3712-22

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PSYCHOSOCIAL RESEARCH
Publications (continued)
Hoopman R, Terwee CB, Devillé W,
Aaronson NK. Evaluation of the
psychometric properties of the SF-36 Health
Survey for use among Turkish and
Moroccan ethnic minority populations in
the Netherlands. Quality of Life Res
2009;18:753-764
Hoopman R, Terwee CB, Muller MJ,
Ory F, Aaronson NK. Methodological
challenges in quality of life research among
Turkish and Moroccan ethnic minority
cancer patients: Translation, recruitment
and ethical issues. Ethnicity Hlth
2009;14:237-53
Knols RH, de Bruin ED, Aufdemkampe G,
Uebelhart D, Aaronson NK. Reliability
of ambulatory walking activity in patients
with hematological malignancies. Arch
Phys Med Rehab 2009;90:58-65
Lammens CRM, Bleiker EMA, Aaronson NK,
Vriends AHJT, Ausems MGEM,
Jansweijer MCE, Wagner A, Sijmons RH,
van den Ouweland AMW, van der Luijt RB,
Spruijt L, Gómez García EB, Ruijs MWG,
Verhoef S. Attitudes towards pre-implantation
genetic diagnosis for hereditary cancer. Familial
Cancer 2009;8:457-464
Scott NW, Fayers PM, Aaronson NK,
Bottomley A, de Graeff A, Groenvold M,
Gundy C, Koller M, Petersen MA,
Sprangers MAG. A simulation study
provided sample size guidance for differential
item functioning (DIF) studies using short
scales. J Clin Epidemiol 2009;62:288-295
Snyder CF, Aaronson NK. Using patientreported
outcomes in clinical practice.
Lancet 2009;374:370-371
Vermeulen E, Schmidt MK, Aaronson
NK, Kuenen M, van der Valk P, Sietses C,
van den Tol PMP, van Leeuwen FE. Optout
plus: the patients’choice: preferences of
cancer patinets concerning information and
consent regimen for future research with
biological samples archived in the context of
treatment. J Clin Pathol 2009;62:275-278
Vermeulen E, Schmidt MK, Aaronson
NK, Kuenen M, Baas-Vrancken Peters
MJ, van der Poel H, Boot H, Verwaal VJ,
Cats A, van Leeuwen FE. A trial of consent
procedures for future research with
clinically-derived biological samples. Br J
Cancer 2009:101:1505-12
physical performance, as assessed by a number of objective measures. Effect sizes
range from 0.53 to 0.62 at 3 months, and .043 to 0.50 at 6 months. Data analysis and
preparation of manuscripts will continue in 2010.
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 treatment-induced
menopause. Consenting women, younger than 50 years of age, with histologically
confirmed primary breast cancer, are randomized to one of the three intervention
groups or to a usual care, ‘waiting list’ control group. All participating women are
asked to complete a battery of questionnaires prior to randomization (T0), at
12 weeks (T1) and at 6 months (T2) post-entry study. Primary outcome measures are
overall levels of vasomotor symptoms. Secondary outcomes are urinary symptoms,
sexuality, body- and self-image and psychological distress. Patient recruitment was
completed in September 2009, with 422 women randomized to one of the four study
groups. Interventions will continue until December 2009. To date, 317 women have
completed the first follow-up questionnaire (T1) and 257 the 6 month follow-up
questionnaire. Data collection will be completed by mid-2010.
Physical exercise during chemotherapy to improve physical fitness and
reduce fatigue (PACES) This multicenter, randomized trial is part of a larger
program, the Alp D’Huzes Cancer Rehabilitation Research Program (A-CaRe), being
carried out in collaboration with the EMGO Institute.
The PACES study will evaluate the effectiveness of two physical exercise interventions
in maintaining or enhancing physical fitness and in minimizing fatigue of patients
undergoing adjuvant chemotherapy for breast or colon cancer: (1) a low intensity,
home-based, self-management physical activity program (Onco-Move) and (2) a
moderate intensity, structured, supervised exercise program (OnTrack). In total,
360 patients will be randomized to one of the two intervention groups or to a usual
care control group. All participants will be asked to undergo performance tests and to
complete self-report questionnaires at baseline, at the completion of chemotherapy,
and at 6 month follow-up. Primary outcomes are physical fitness (cardiorespiratory
fitness and muscle strength), and self-reported fatigue. Secondary outcome are selfreported
physical activity and physical functioning, mood state, HRQL, sleep quality
and chemotherapy completion rates. The study is in the preparatory phase. Patient
recruitment will start in the first quarter of 2010.
Behavioral and psychosocial effects of rapid genetic counseling and testing
(RGCT) in newly diagnosed breast cancer patients This multicenter, randomized
trial, carried out in collaboration with Dr. M. Ausems of the University Medical
Center Utrecht, is investigating the uptake of RGCT when offered routinely to newly
diagnosed breast cancer patients who, prior to receiving primary treatment, are
identified 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 12 hospitals in the Amsterdam and Utrecht regions of the
Netherlands are randomized to either the RGCT group or a usual care group
(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 cancer-related distress,
(4) knowledge of genetic aspects of breast cancer, (5) decisional satisfaction,
(6) HRQL and (7) satisfaction with RGCT. Questionnaires are administered at study
entry, and at 6 and 12 month follow-up. A subset of women will be interviewed to
obtain supplementary, qualitative data. In 2009, final ethical approval was obtained
and patient accrual was initiated in all hospitals. To date, 94 patients have been
randomized into the study.

COGNITIVE FUNCTION IN CANCER PATIENTS
Significant 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.
Long-term effects of high-dose chemotherapy on the brain: hyporesponsiveness
of grey matter and microstructural and biochemical alterations of white
matter (NKI-AMC) We compared breast cancer survivors who had received adjuvant
high-dose chemotherapy 9.5 years ago (n=18, 4 cycles of FEC followed by one cycle of
high-dose CTC) with cancer survivors who had not received chemotherapy (n=15).
At 3 Tesla field strength, we acquired whole-brain fMRI, whole-brain Diffusion
Tensor Imaging and H-MR spectroscopy. fMRI: the CT group showed task-specific
hyporesponsiveness of dorsolateral prefrontal cortex and parahippocampal gyrus,
generalized hyporesponsiveness of posterior parietal cortex and impaired performance
compared to the control group. DTI: the CT group showed lower FA than the control
group in several white matter tracts, indicating reduced microstructural integrity of
white matter tracts. H-MR spectroscopy: the CT group showed significantly lower
NAA/Creatine than the control group, whereas Choline/Creatine was similar for both
groups, indicating reduced axonal viability in the absence of acute demyelination.
Neuropsychological testing showed a relatively stable pattern of cognitive impairment
in the chemo group over time. This study indicates that the FEC+CTC adjuvant
regimen is associated with hyporesponsiveness of grey matter and microstructural
and biochemical alterations of white matter, and is associated with impaired cognitive
function.
Endocrine treatment and cognitive function In the context of a prospective
multicenter study examining the neuropsychological sequelae of exemestane and
tamoxifen in postmenopausal breast cancer (BC) patients, we analysed the baseline
data with respect to the cognitive functioning of these patients before adjuvant
systemic therapy, and its association with medical and psychological factors. 205
postmenopausal BC patients underwent pre-treatment neuropsychological tests and
provided medical and psychological data. 124 healthy controls underwent the same
assessment. We found that ‘treatment for diabetes mellitus’ and/or ‘hypertension’,
‘less hours spent on cognitively stimulating activities’, ‘fewer days since surgery’ and
‘more reproductive years’ were associated with worse cognitive performance in the
BC patients, independent of age and IQ. Cognitive differences between BC patients
and healthy controls could partly be explained by the evaluated variables. Our results
underscore the need for adjustment for pre-treatment cognitive differences between
study groups.
Animal studies exploring the underlying mechanisms of cognitive impairment
(NKI-RUG) Previous experiments by our group revealed short- and long-term, dosedependent
adverse effects of methotrexate (MTX) on learning behaviour and
hippocampal cell proliferation in rats. We have now investigated effects of MTX on
hippocampal cell proliferation in tumor-bearing animals. MTX significantly decreased
the number of proliferating cells in the hippocampus, regardless of the presence of a
tumor. In tumor-bearing animals, a decrease in hippocampal cell proliferation was
seen although this observation failed to reach statistical significance, and the tumor
furthermore failed to potentiate the effect of MTX. We also investigated a number of
possible mechanisms and found that MTX, next to reduced hippocampal neurogenesis,
affects brain vascularization, glucose metabolism, white matter density, and microglia
activation. Our findings further suggest that the microglial activation was not a
marker for neuroinflammation, as no effects were seen in [(11)C]PK11195 PET tracer
uptake and no increased hippocampal cytokine levels were found.
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PSYCHOSOCIAL RESEARCH
Group leader Sanne Schagen
Sanne Schagen PhD Group leader
Frits Van Dam PhD Academic staff
Willem Boogerd MD PhD Academic staff
Sabine Linn MD PhD Academic staff
Jose Belderbos MD PhD Academic staff
Rita Roodbergen MSc Academic staff
Michiel de Ruiter PhD Post-doc
Christien Schilder MSc PhD student
Vincent Koppelmans MSc PhD student
Riejanne Seigers MSc PhD student
Sanne Menning MSc PhD student
Roy Cox MSc PhD student
Chad Gundy MSc Senior statistical analyst
Marianne Kuenen Research assistant
Marion Wevers Research assistant
Astrid Klinkenberg Research assistant
Publications
Schagen SB, Das E, van Dam FS. The
influence of priming and pre-existing
knowledge of chemotherapy-associated
cognitive complaints on the reporting of
such complaints in breast cancer patients.
Psycho-oncology 2009;18:674-8
Schilder CM, Eggens PC, Seynaeve C,
Linn SC, Boogerd W, Gundy CM, Beex LV,
Van Dam FS, Schagen SB.
Neuropsychological functioning in
postmenopausal breast cancer patients
treated with tamoxifen or exemestane after
AC-chemotherapy: cross-sectional findings
from the neuropsychological TEAM-side
study. Acta Oncol 2009;48:76-85
Schilder CM, Seynaeve C, Linn SC,
Boogerd W, Gundy CM, Beex LV,
van Dam FS, Schagen SB. The impact of
different definitions and reference groups on
the prevalence of cognitive impairment: a
study in postmenopausal breast cancer
patients before the start of adjuvant systemic
therapy. Psycho-oncology 2009

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PSYCHOSOCIAL RESEARCH
Publications (continued)
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
2009 (in press)
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 2009 (in press)
Seigers R, Schagen SB, Coppens CM,
van der Most PJ, van Dam FS,
Koolhaas JM, Buwalda B. Methotrexate
decreases hippocampal cell proliferation
and induces memory deficits in rats. Behav
Brain Res 2009;12;201:279-84
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 2009
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 2009
Understanding cognitive problems after chemotherapy: the added value of
proxy ratings In numerous previous studies self-reported cognitive complaints and
objectively assessed cognitive performance have not shown strong correlations.
We examined the added value of a proxy rating for predicting the outcome of
objective neuropsychological assessment. Data were collected from 240 breast cancer
patients treated with adjuvant chemotherapy. Each patient was asked to provide a
proxy respondent. A measure of pre-morbid IQ, self-reported depression, anxiety, and
fatigue, and a battery of neuropsychological measures were administered. A cognitive
complaints interview and a questionnaire were also administered to patients and
proxies. Patient complaints had small, non-significant relations with domain scores.
Proxy complaints added an occasionally significant – yet weak- improvement in
explained variance. Adding pre-morbid IQ and age resulted in sizeable and significant
improvements in explained variance. Patient complaints did exhibit a significant and
substantial association with depression, anxiety, and fatigue, while proxy measurements
added little. Proxy measurements of patient cognitive complaints do not substantially
improve the prediction of objective neuropsychological assessments.
Late effects of chemotherapy on brain functioning in the elderly (NKI-Erasmus
MC) As support is growing for the persistency of cognitive dysfunction after
chemotherapy, we are investigating whether chemotherapy is associated with an
increased risk for cognitive decline and dementia. Breast cancer patients who received
CMF chemotherapy between 1975 and 1985 and who are currently >60 years of age
are being tested with a neuropsychological examination and neuroimaging techniques.
Patients are being compared with participants of the Rotterdam study, a prospective
population-based cohort study investigating the prevalence and incidence of, and risk
factors for chronic diseases in the elderly. The data collection is completed.
204 breast cancer survivors are included in the study.

PSYCHOSOCIAL 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.
The long term psychosocial impact of genetic testing among familial
adenomatous polyposis (FAP) families This 4 year, cross-sectional study began
in 2004, in collaboration with the Netherlands Foundation for the Detection of
Hereditary Tumors (STOET; H. Vasen). The aim of the study is to evaluate the
psychosocial impact of the (clinical or genetic) diagnosis of FAP among individuals
from high-risk families. In total, 525 FAP-family members (response 64%) and
129 partners of individuals with a FAP-diagnosis have completed the questionnaire,
and 60 respondents have been interviewed. Results indicate that: 1) 20% of
individuals from FAP-families have moderate to severe levels of distress, 2) only
approximately one-third of the moderate and severely distressed individuals received
psychosocial support, 3) psychosocial variables, such as risk perception, family
functioning, and knowledge about FAP, explain significantly more of the variability
in distress levels than do sociodemographic and clinical variables (42% versus 7%,
respectively), 4) partners suffer from at least as much distress as patients, 5) based on
medical record data, 20% of the at-risk group is less than fully compliant with
surveillance advice, 6) under-compliance is associated significantly with low perceived
self-efficacy, non-use of sedatives during surveillance, pain after surveillance and low
perceived benefits of surveillance, 7) FAP-patients who underwent surgery have lower
quality of life than FAP-patients who had not yet undergone surgery, and 8) about
one-third of respondents express a positive attitude toward prenatal diagnosis and
pre-implantation genetic diagnosis.
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, cross-sectional
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 self-report questionnaire. Here, a selection of the results regarding the
LFS families is reported. In total, 18 families with a p53 germline mutation were
identified. Eligible family members were invited to complete a self-report
questionnaire assessing motives for (not) undergoing genetic testing, LFS-related
distress and worries, and health-related 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 significantly 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.
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 significant 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. This questionnaire is now being evaluated for
its reliability, validity, sensitivity, specificity and positive predictive value for detecting
psychosocial problems and psychosocial support needs. For this validation study, new
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PSYCHOSOCIAL RESEARCH
Group leader Eveline Bleiker
Eveline Bleiker PhD Group leader
Annemieke Cats MD PhD Academic staff
Daniela Hahn MSc Academic staff
Irma Kluijt MD Academic staff
Senno Verhoef MD PhD Academic staff
Leonie Woerdeman MD PhD Academic staff
Kirsten Douma MSc PhD student
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 scientific researcher
Publications
Bleiker EMA, Hahn DEE, Smets, EMA.
Familiaire Tumoren. In: JCJM de Haes,
LM Gualthérie van Weezel, R Sanderman,
HBM van de Wiel (Ed.): Psychologische
patiëntenzorg in de oncologie. Van Gorcum
Uitgeverij 2009;89-106
Douma KFL, Aaronson NK, Vasen HF,
Verhoef S, Gundy CM, Bleiker EMA.
Attitudes towards genetic testing in
childhood and reproductive decision-making
for familial adenomatous polyposis (FAP).
European Journal of Human Genetics 2009
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 2009
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. Psycho-oncology
2009
Douma KFL, Bleiker EMA, Vasen HF A,
Gundy CM, Gerritsma MA, Aaronson NK.
Psychological distress and quality of life of
partners of individuals with familial
adenomatous polyposis. Psycho-oncology
2009 (in press)

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PSYCHOSOCIAL RESEARCH
Publications (continued)
Lammens C, Bleiker E, Aaronson N,
Vriends A, Ausems M, Janswijer M,
Wagner A, Sijmons R, van den Ouweland A,
van der Luijt R, Spruijt L, Gomez Garcia E,
Ruijs M, Verhoef S. Attitude toward preimplantation
genetic diagnosis for hereditary
cancer. Familial Cancer 2009
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. Clinical Genetics 2009 (in press)
Smets EMA, Bleiker EMA, van Esch SCM.
Erfelijkheidsadvisering. In AA Kaptein,
JB Prins, EH Colette, RL Hulsman (Ed.):
Leerboek Medische Psychologie. Uitgeverij
Bohn, Stafleu, Van Loghum (in press)
Tan MB, Bleiker EMA, Menke-Pluymers MB,
Van Gool AR, Van Dooren S, Van Geel BN,
Tilanus-Linthorst MM, Bartels KC, Klijn JG,
Brekelmans CT, Seynaeve C. Standard
psychological consultation and follow up
for women at increased risk of hereditary
breast cancer considering prophylactic
mastectomy. Hereditary Cancer in Clinical
Practice 2009;7:6
counselees (N=180) 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
final 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. The
availability of this screening instrument will set the stage for future intervention
studies aimed at 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 influence 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 have been identified 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 2009, 26 of the 32 (81%) carriers of a
CDH1 mutation completed self-report questionnaires, and 19 participated in a
face-to-face interview. Data collection of the comparison group and data analyses
are on-going.

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) targeted SNPs
are associated with the risk of breast cancer among BRCA1/2 carriers.
We examined the association between body weight and breast cancer risk among 908
BRCA1/2 carriers in our Dutch retrospective cohort (GEO-HEBON), starting
follow-up 10 years before date of questionnaire completion. When compared to
having a normal weight (BMI between 18.5 and 22.5 kg/m 2 ) at age 18, being
overweight (BMI > 25kg/m 2 ) reduced the risk of premenopausal breast cancer
(Hazard Ratio (HR) = 0.23. Risk of premenopausal breast cancer was not associated
with current BMI >25 kg/m 2 nor with adult weight gain of >15kg. For
postmenopausal breast cancer overweight seemed to increase the risk of breast
cancer (HR=1.70 among the carriers. Thus, being overweight appears to have a
protective effect on the risk of premenopausal breast cancer, while it seems to
increase the risk of postmenopausal breast cancer among the BRCA1/2 carriers.
Similar associations are observed in the general population.
We coordinated a collaborative European retrospective cohort study (GENE-RAD-
RISK) of 1,993 BRCA1/2 mutation carriers in which GEO-HEBON is one of the
collaborators. The main aim of the study was to investigate the association between
self-reported exposure to diagnostic radiation and risk of breast cancer. Risk of breast
cancer was analyzed using a time-varying weighted Cox proportional hazards model
with a 5 year time lag. A unique feature of this study is the individually estimated
cumulative breast dose score. When compared to no exposure, having had more than
2 fluoroscopies or 4 chest X-rays before age 20 increased the risk of breast cancer
(HR fluoroscopies=2.46, P trend=0.10; HR X-rays=1.53, P trend=0.04). Ever having had a
mammogram before age 30 increased the risk of breast cancer 1.5-fold (HR=1.54) and
a dose-response trend (HR >4mammograms=2.41, P trend=0.04) of greater risk with
higher number of mammograms was observed, which could not be attributed to
confounding by indication because of family history of breast cancer . Other exposure
types were not significantly associated with breast cancer risk. Exposure to any
diagnostic radiation before age 30 increased the risk of breast cancer (HR=1.73).
The risk of breast cancer was greater with increasing cumulative radiation dose from
diagnostic exposures: the risks for a cumulative dose score 0.002-
0.0066 Gy, > 0.0066-0.0174 Gy, and > 0.0174 Gy were 1.45, 1.70, 1.74, and 3.29,
respectively, when compared to no exposure. In conclusion, exposure to diagnostic
radiation before age 30 was associated with at least a 1.5-fold increase in breast cancer
risk in BRCA1/2 mutation carriers. This relative risk was observed for doses that are
at least an order of magnitude lower than those for which in previous studies
significant associations were observed. The data supported a dose-response model.
A potential difference in mechanism between BRCA1 and BRCA2 needs to be
investigated in future studies. If confirmed in prospective studies, these results have
clinical implications for the use of diagnostic X rays in BRCA carriers.
We continued the coordination of the data collection of the International BRCA1/2
Carrier Cohort Study (IBCCS), a follow-up study on 3,757 BRCA1/2 mutation carriers,
and the risk factor data collection for the Consortium of Investigators of Modifiers of
BRCA1/2 (CIMBA). The CIMBA risk factor database includes 6,946 BRCA1/2
carriers. The association between four breast cancer risk susceptibility loci (common
single nucleotide polymorphisms, SNPs) and risk of breast cancer was examined in
9,442 BRCA1 and 5,665 BRCA2 mutation carriers, participating in CIMBA. The LSP1
and 2q35 SNPs appeared to interact multiplicatively on breast cancer risk for BRCA2
mutation carriers, whereas SNP rs13281615 at 8q24 and GATA3 rs570613 were not
associated with breast cancer for either BRCA1 or BRCA2 mutation carriers.
We validated a number of additional SNPs in part of our cohort breast cancer cohort
of sporadic breast cancer patients aged

100
EPIDEMIOLOGY
Publications
Ahmed S, Thomas G, Schmidt MK,
Broeks A, Easton DF and 136 other
authors. Newly discovered breast cancer
susceptibility loci on 3p24 and 17q23.2.
Nat Genet 2009;41:585-90
Antoniou AC, Rookus M, Andrieu N,
Brohet R, Chang-Claude J, Peock S,
Cook M, Evans DG, Eeles R; EMBRACE,
Nogues C, Faivre L, Gesta P; GENEPSO,
van Leeuwen FE, Ausems MG, Osorio A;
GEO-HEBON, Caldes T, Simard J,
Lubinski J, Gerdes AM, Olah E,
Fürhauser C, Olsson H, Arver B,
Radice P, Easton DF, Goldgar DE.
Reproductive and hormonal factors, and
ovarian cancer risk for BRCA1 and BRCA2
mutation carriers: results from the
International BRCA1/2 Carrier Cohort
Study. Cancer Epid Bio Prev 2009;18:601
Antoniou AC, Sinilnikova OM, Rookus M,
Chenevix-Trench G; CIMBA and 140 other
authors. Common variants in LSP1, 2q35
and 8q24 and breast cancer risk for BRCA1
and BRCA2 mutation carriers. Hum Mol
Genet 2009;18:4442-56
Van den Belt-Dusebout AW, Aleman BM,
Besseling G, De Bruin ML, Hauptmann M,
Van ’t Veer MB, De Wit R, Ribot JG,
Noordijk EM, Kerst JM, Gietema JA,
Van Leeuwen FE. Roles of radiation dose
and chemotherapy in the etiology of
stomach cancer as a second malignancy.
Int J Rad Onc Biol Physics 2009;75:1420-9
De Bruin ML, Dorresteijn LDA,
Van ’t Veer MB, Noordijk EM,
Van den Berg H, Kapelle AC, Boogerd W,
Aleman B, Van Leeuwen FE. Increased risk
of Stroke and Transient Ischemic Attack
(TIA) in 5-year survivors of Hodgkin’s
lymphoma. J Natl Cancer Inst
2009;101:928 -937
De Bruin ML, Burgers S, Baas P,
Van ’t Veer MB, Noordijk EM,
Louwman MWJ, Zijlstra JM,
Van den Berg H, Aleman BPM,
Van Leeuwen FE. Malignant mesothelioma
following treatment for Hodgkin’s
lymphoma. Blood 2009;113:3679-81
with other groups in the international Breast Cancer Association Consortium. These
loci explain an additional ~5% of familial breast cancer besides the ~25% explained by
BRCA1 and BRCA2 (see also section Experimental Therapy). We analyze DNA
(obtained from paraffin-embedded tissue blocks) in our study under a strict coding
protocol, on which we published in 2009.
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 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 = 13,000), we collected detailed data regarding IVF treatment (number of
cycles, drug doses, number of oocytes retrieved) for 11 of the 12 collaborating IVFclinics
through computerized databases and medical charts (in one IVF clinic). For
the 12th clinic the data will be available soon. We are enlarging the comparison group
with 4,000 subfertile women not treated by IVF comprising women who underwent
tubal surgery or intra-uterine insemination, or withdrew from the IVF waiting list.
Medical data from all clinics have been pooled, also with the data from the existing
cohort of 25,152 women. Addresses have been updated for about 30% of the cohort
and two pilot studies of 250 women have been performed to optimize the response
rate using on-line questionnaires and/or paper versions, according to the responders’
preference. Preliminary results show that 61% of responders prefer on-line
questionnaires, whereas 39% prefer paper versions. Data collection regarding lifestyle
and reproductive factors and health outcomes through (on-line and paper)
questionnaires is ongoing.
In our prospective cohort of 12,091 DES daughters (DESnet project), followed from
December 1992 till June 2008 the mean detection rate of severe dysplasia (CIN3) was
assessed through linkage with the Dutch pathology database (PALGA). The rate in
the cohort was compared with age-, and period-specific detection rates of the
screened general population. In the cohort a total of 124 incident CIN3s occurred at a
mean age of 40.1 (± 4.9) years. A significantly increased risk of CIN3 was found
(Detection Rate Ratio=2.27), which could not be explained by the more intensive
screening of the DES daughters.
Use of stored human materials in clinical research Significant medical progress
has been made based using tissue samples, especially in cancer research. In the
Netherlands and many other countries, the use of residual tissues for research is
regulated by an ‘opt-out’ procedure. Patients can declare that they do not agree with
the use of their tissue on the basis of information which in most hospitals is available
in a general information leaflet. At present, the extent of information about research
with residual tissue in the hospital leaflets differs widely in Dutch hospitals. Our
earlier research, in limited cancer patient groups, shows that patients are unaware of
Figure 1: Cumulative incidence of breast cancer (invasive and ductal carcinoma in situ) according to age
at first treatment of Hodgkin Lymphoma

Figure 2: Cumulative incidence of contralateral testicular cancer following non-seminoma
testicular cancer according to treatment with or without chemotherapy
residual tissue storage for their own clinical benefit and possible use in research, and
are unsatisfied with the current information procedure (i.e. opt-out), though only a
minority wished to refuse research with their residual tissue. The current procedure
with ‘opt out’ does not secure that patients are informed and therefore many patients
wish to complement the current opt out procedure with short verbal information by a
member of hospital staff. Patients indicated indeed that ‘opt-out’ would suffice if it
was ascertained that patients were well informed. In our most recent study, 146
patients were offered the ‘one-time general consent’ procedure (verbal and written
information including a leaflet and a consent form) or the ‘opt-out plus’ procedure
(verbal and written information with a similar leaflet and the opportunity to opt out).
A control group (n=131) with the standard procedure received no active/extra
information (information about research with tissue and opting-out is included in
the general hospital leaflet). Patients preferred ‘opt-out plus’ (43%) over ‘one-time
consent’ (34%) or the ‘opt-out’ without actively being informed (16%). Results of this
study were also included in a report of the Rathenau Institute, which gained
nationwide attention. We will investigate the new opt-out plus procedure in larger
populations from other hospitals, including various tumor types, in future studies.
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 2009 we published on treatment-related risk factors for the strongly increased risk
of breast cancer following Hodgkin’s lymphoma. Among 1122 female 5-year survivors,
treated for Hodgkin lymphoma before age 51 (median follow-up time of 17.8 years),
we identified 120 cases of breast cancer. The overall risk was 5.6-fold increased
compared with the general population and the 30-year cumulative incidence of breast
cancer, accounting for death as a competing risk, was 19%. The cumulative incidence
in the youngest group (treated before age 21) 30 years after treatment was as high as
26%, with an attained age of 51 years or less at that time. Importantly, mantle field
irradiation was associated with a 2.7-fold significantly increased risk of breast cancer
compared to similarly dosed radiation to the mediastinum alone. This finding is
reassuring since present-day radiotherapy for Hodgkin lymphoma employs smaller
radiation volumes. Alkylating CT significantly reduced the risk for BC among women
treated with radiotherapy, with an overall risk reduction of 50%. Chemotherapy
regimens with higher cumulative procarbazine doses seemed to be associated with a
greater risk reduction, with 40% and 60% risk reductions for regimens with less
than 8.4 g/m2 procarbazine and more than 8.4 g/m2 procarbazine, respectively.
These reduced risks could be attributed to CT-induced premature menopause. Thirty
percent of all women reached menopause before age 41; such an early menopause
was associated with a 60% reduced risk of breast cancer. Women with less than
Publications (continued)
101
EPIDEMIOLOGY
De Bruin ML, Sparidans J, Van ’t Veer MB,
Noordijk EM, Louwman MJ, Zijlstra JM,
Van den Berg H, Russell N, Broeks A,
Baaijens MHA, Aleman B, Van Leeuwen
FE. Long-term breast cancer risk in female
survivors of Hodgkin; lower risk after
smaller radiation volumes. J Clin Oncol
2009;27:4239
Marees T, Van Leeuwen FE, De Boer MR,
Imhof SM, Ringens, PH, Moll AC. Cancer
mortality in long-term survivors of
retinoblastoma. Eur J Cancer
2009;45:3245-53
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 2009 (in press)
Schmidt MK, Vermeulen E, Tollenaar RA,
van ’t Veer LJ, Van Leeuwen FE.
Regulatory aspects of genetic research with
residual human tissue: Effective and
efficiënt data coding. Eur J Cancer
2009;45:2376-82
Vermeulen E, Schmidt MK, Aaronson NK,
Kuenen M, van Leeuwen FE. Obtaining
‘fresh’ consent for genetic research with
biological samples archived ten years ago.
Eur J Cancer 2009;45:1168-74
Vermeulen E, Schmidt MK, Aaronson NK,
Kuenen M, Baas-Vrancken Peters MJ,
van der Poel H, Boot H, Verwaal VJ,
Cats A, van Leeuwen FE. A trial of consent
procedures for future research with
clinically-derived biological samples. Br J
Cancer 2009;101:1505-12
Vermeulen E, Geesink I, Schmidt MK,
Steegers C, Verhue D, Brom FWA,
Aaronson NK, van Leeuwen FE. Nader
gebruik van lichaamsmateriaal:
zeggenschap en betere informatie nodig
NTvG 2009 (in press)
Broeks A, Van Leeuwen FE, Van ’t Veer LJ,
et al. Radiation-associated breast tumors
display a distinct gene expression profile
frequently correlated with the basal
molecular subtype. Int J Rad Onc Biol Phys
2009 (In press)

102
EPIDEMIOLOGY
Publications (continued)
Ceelen M. Van Weisssenbruch MM,
Vermeuden JP, Van Leeuwen FE,
Delemarre-van de Waal HA. Pubertal
development in children and adolescents
born after IVF and spontaeous
conception. Human Reprod
2009;23;2791-8
10 years of intact ovarian function after radiotherapy had a 70% decreased risk of
breast cancer compared with women with 10-20 years of ovarian function after
irradiation, while those with more than 20 years of intact ovarian function after
radiotherapy had 5.3-fold increased risk of breast cancer. These risk reductions were
observed both among women treated before age 21, and among those treated
between ages 21 and 30. Among women treated between ages 31 and 40 cumulative
exposure to endogenous estrogens was not associated with risk for breast cancer,
possibly because these women were closer to natural menopause at time of
treatment. These results show that stimulation by ovarian hormones is crucial in
radiation-induced breast cancer.
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 Annegien Broeks and Laura van
’t Veer from the Division Experimental Therapy). So far we have identified 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.
Testicular cancer patients are known to have a strongly increased risk of developing
contralateral testicular cancer. It is still unclear whether treatment for the primary
cancer, especially chemotherapy, alters the risk of developing a contralateral testicular
cancer. In a previous report from our group published in 1993 we found no increased
risk of contralateral testicular cancer for patients treated with chemotherapy, possibly
due to eradication of foci of in situ tumour in the contralateral testis. Recently we
expanded the cohort and updated follow-up. In this nationwide cohort, currently
comprising 3,745 testicular cancer patients treated in the Netherlands during 1965-
1995 – approximately 48% of all Dutch patients treated in this period – we studied
risk factors for developing contralateral testicular cancer with emphasis on the role of
prior chemotherapy. With a median follow-up of 15 years 85 contralateral testicular
cancers were diagnosed; 10 within 6 months of the primary cancer (synchronous)
and 75 thereafter (metachronous contralateral testicular cancer). The risk to develop a
metachronous contralateral testicular cancer was 21 times higher than in the general
male population. The risk was 28 times higher following a seminoma testicular
cancer and 17 times higher following a non-seminoma testicular cancer. For nonseminoma
patients treated with chemotherapy we found significantly lower risks
compared to non-seminoma patients treated without chemotherapy (standardized
incidence ratio 10.7 versus 24.5, p=0.015). In period specific Cox analysis this effect
was limited to patients treated prior to 1986. Non-seminoma patients treated since
1986 with courses consisting of bleomycin, etoposide and cis-platin had no decreased
risk of contralateral testicular cancer compared to patients who did not receive
chemotherapy.
We also evaluated the long term risk of leukaemia and myelodysplastic syndrome in
our nationwide cohort. Eight cases of leukaemia, 3 acute and 5 chronic leukaemias,
and 3 cases of myelodysplastic syndrome were observed, which translated into a nonsignificantly
2.0-fold increased risk. Leukaemia risk was not associated with type and
doses of standard chemotherapy. Survival was very poor for patients diagnosed with
acute leukaemia or MDS. Nonetheless, in retrospect, risk of leukaemia and
myelodysplastic syndrome does not pose a major problem for patients treated for
testicular cancer.
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 recently started two new large cohort
studies. The first is a population-based cohort of patients with invasive breast cancer
(n=58,000) and ductal carcinoma in situ (n=14,000) of the breast diagnosed between
1989 and 2004 in the Netherlands. 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.

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 RASTER-study).
This study is being continued as a side study of the European MINDACT-study. 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 finalised 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 will be completed, including real life scenarios. A comparison between
cost-effectiveness in France and the Netherlands is foreseen. Additionally, in
cooperation with the University of Twente, we have initiated an early stage
technology assessment of TIL-transfer technology in advanced melanoma.
Operations improvement in oncology Translating operations management and
research (OM/OR) principles into oncologic care is likely to improve both quality and
efficiency of hospital processes. A series of international benchmarking projects has
been performed comparing performance of three Comprehensive Cancer Centers,
three ambulatory chemotherapy treatment centers (ACT), six fundamental research
organisations and four radiotherapy departments. Using the experience of earlier OR
simulation techniques (such as a verification of the efficiency 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.
In cooperation with the University of Twente, Peter van Berkel, a PhD student is
working on a mathematical analysis and scheduling of care pathways within the
oncologic hospital setting, and the effect of increased focus on efficient capacity use.
A study on contingency of OM interventions in hospitals is currently on-going. In
early 2010, a study will be initiated comparing characteristics of colorectal surgery
pathways using structured efficiency measures and the national colorectal quality
registry.
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 rehabilitation 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
to be rolled out in early 2010. Finally, an Alpe d’Huzes program of research has been
proposed and submitted for funding, focusing on patient empowerment, return to
work, tele-monitoring and implementation of relevant findings and programs related
to physical exercise and supported by innovative IT.
103
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
Marloes Sanders Research Assistant
Linda Timmer-Arents Research Assistant
Publications
Retèl VP, Bueno de Mesquita JM,
Hummel MJM, Van de Vijver MJ,
Douma KFL, Karsenberg K,
Van Dam FSAM, Van Krimpen C,
Bellot FE, Roumen RMH, Linn SC,
Van Harten WH. Constructive Technology
Assessment (CTA) as a tool in coverage
with evidence development: the case of the
70-gene prognosis signature for breast
cancer diagnostics. International Journal
of Technology Assessment in Health Care
2009;25:73-83
Retèl VP, Hummel MJM, Van Harten WH.
Review on Early Technology Assessment of
nanotechnologies in oncology. Molecular
Oncology 2009
Van Harten WH, Van Bokhorst L,
Van Luenen HGAM. Benchmarking
Biology Research Organisations Using a
new Dedicated Tool. Molecular
Oncology 2009
Van Lent WA, Goedbloed N,
van Harten WH. Improving the efficiency of
a chemotherapy day unit: applying a
business approach to oncology. Eur J
Cancer. 2009;45:800-6
Ploem MC, Retèl VP, Linn SC,
Van de Vijver MJ, Van Boven HH,
Schmidt MK, de Jong JP, Gevers JKM,
Van Harten WH. Tumour tissue: who is in
control? A Guideline on tissue banking for
clinical purposes. The Lancet Oncology
2009

104
DIAGNOSTIC ONCOLOGY
Division head Laura van ’t Veer (at interim)
DEPARTMENT OF CLINICAL CHEMISTRY
Willem Nooijen PhD Head
Hans Bonfrer PhD Academic staff
Olaf van Tellingen PhD Academic staff
Nienke van den Brink-de Vries PhD student
Lin Fan PhD student
Levi Buil Technical staff in training
Tiny Korse Technical staff
Dorothé Linders Technical staff
Marian Buning-Kager Technical staff
DEPARTMENT OF NUCLEAR MEDICINE
Cornelis Hoefnagel MD PhD Head
Philippe Baars MD Academic Staff
Fijs van Leeuwen PhD Academic Staff
Saar Muller PhD Academic Staff
Michiel Sinaasappel PhD Academic Staff
Ferida Sivro-Prndelj MD Academic Staff
Renato Valdés Olmos MD PhD Academic Staff
Wouter Vogel MD PhD Academic Staff
Tjeerd Aukema MD Clinical Research Fellow
Lenka Vermeeren MD Clinical Research Fellow
Ly Tran PhD Research Fellow
Jan Willem Postema MD Registrar
Chau Le 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
Lyandra Rooze Technical Staff
Mariska Sonneborn Technical Staff
Colinda Vroonland Technical Staff
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
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
Douwe Atsma Technical staff
Lucie Boerrigter-Barendsen Technical staff
Michael Knauer MD Academic staff
DIVISION OF
DIAGNOSTIC ONCOLOGY
DEPARTMENT OF CLINICAL CHEMISTRY
Pharmacological studies in mice
Nienke van den Brink-de Vries, Lin Fan, Levi Buil, Mark de Gooijer, Olaf van Tellingen
High-grade gliomas, in particular glioblastoma multiforme (GBM), are among the
deadliest and most devastating of human cancers. Considerable efforts to better
understand the aberrant pathways that drive glioma progression are ongoing and the
hope is that this will help to identify more effective (targeted) therapies. Our work is
aimed to develop and implement state-of-the-art preclinical models in order to select
the most appropriate (combination of) candidate agents for further clinical
evaluation. This year on April 15, Nienke de Vries defended her thesis entitled
Preclinical models to study the impact of the blood-brain barrier in brain tumor
chemotherapy, describing much of the work performed in recent years on this subject.
A major complication in the treatment of glioma is that glioma cells infiltrate deeply
into normal surrounding brain where they are hidden behind an intact blood-brain
barrier (BBB). Several ABC transporters (e.g. ABCB1, ABCG2, ABCC1-5) are located
at the BBB. The importance of Abcb1 and Abcg2 in reducing the brain penetration of
substrate drugs has been well established, while the role of Abcc4 is now emerging.
Important lessons learned of previous work, is that because the overlapping substrate
specificities of these transporters, it requires the use compound knockouts to really
appreciate the impact of the less abundantly expressed ones. We have now created
quadruple Abcg2;Abcb1a/b;Abcc4;Abcc5 compound knockout mice that will allow us to
interrogate the role of Abcc5 at the BBB.
Activation of the EGFR-PI3K-Akt-mTOR signaling pathway is a central event in
glioma. By using our ABC transporter knockout mouse models, we have been able to
show that a novel dual mTORC1/mTORC2 inhibitor Palomid 529 is not a substrate
of any of these efflux pumps and demonstrates a favorable brain distribution making
it a candidate for studies in glioma patients. Similar studies with other mTOR / PI3K
inhibitors are underway.
Importantly, we have now firmly demonstrated the applicability of our spontaneous
murine high-grade glioma tumor model for use in therapy-intervention studies. In
this genetically engineered Cre/LoxP conditional mouse model, tumors are induced
after localized inactivation of glioma relevant suppressor genes (Ink4a/Arf, P53 and/
or Pten) and activation of Kras V12 by stereotactic intracranial injection of lentiviral
vectors mediating the expression of Cre-recombinase. The tumors arise within
2-3 weeks and can be followed non-invasively by bioluminescence. Therapy with
temozolomide, currently the standard agent for this disease demonstrated a modest
but significant efficacy, in line with the clinical results with this agent. This model
will now be used for testing other modalities, in particular combination therapies
with agent that may enhance the activity of temozolomide (e.g. PARP inhibitors).
Moreover, we have generated a set of neurosphere cultured tumor cell lines from
these tumors. These cells can be grafted into our nude mice that are proficient or
deficient for Abcb1 and/or Abcg2, which will allow us to assess the impact of these
drug transporters in the BBB on the efficacy of investigational therapeutics.
A different approach to increase the brain penetration of drugs is by use of targeted
delivery systems that shuttle drug over the blood-brain barrier. This year we
embarked on collaboration with the Dutch biotech company to-BBB in order to
evaluate their G-technology for delivery of the cytotoxic drug doxorubicin. This
G-technology encompasses the use of glutathione-tagged pegylated liposomes that
have earlier been shown to improve delivery of their cargo over the BBB by up to
5-fold relative to naked pegylated liposomes. Efficacy studies using our intracranial
U87luc5 xenograft have now confirmed that this also translates into a significant
treatment benefit of G-Doxil over Doxil, thus paving the road for clinical evaluation.
We will further scrutinize this delivery system in our models also for the delivery of
other substances.

DEPARTMENT OF NuCLEAR MEDICINE
Clinical Nuclear Medicine
Joke Baars, Axel Bex, Hans Bonfrer, Jan Paul de Boer, Sjaak Burgers, Paul Baas, Kenneth Gilhuijs,
Niels Graafland, Jos van der Hage, Michel van den Heuvel, Alwin Huitema, Simon Horenblas,
Ingrid Kappers, Houke Klomp, Bin Kroon, Charlotte Lange, Willem Meinhardt, Omgo Nieweg,
Hester Oldenburg, Iris van der Ploeg, Willem Prevoo, Henk van der Poel, Emiel Rutgers,
Marieke Straver, Marcel Verheij, Marie Jeanne Vrancken-Peters, Babs Taal, Jelle Teertstra, Lin Tran,
Michel Wouters, Tjeerd Aukema, Philippe Baars, Kees Hoefnagel, Saar Muller, Ferida Sivro,
Michiel Sinaasappel, Renato Valdés Olmos, Lenka Vermeeren, Wouter Vogel
New SPECT-CT applications for sentinel node localization were evaluated. Although
PET-CT activities were focused on the clinical use of 18 F-FDG PET-CT, new PET
tracers were introduced for various malignancies. The validation of new small
nuclear medicine devices has continued and real time intraoperative radioguided
imaging with a portable gamma camera was extended to head and neck. Also the
evaluation of a mini PET ring device for dedicated breast molecular imaging was
started. All these activities resulted at the Annual European Nuclear Medicine
Congress held In Barcelona in the designation of the NKI-AVL as the best European
institution concerning quantity and quality of original work.
SPECT-CT for sentinel node identification SPECT-CT based on both twodimensional
and three-dimensional display was further evaluated for the anatomical
localization of sentinel nodes in various malignancies.
The feasibility of ultrasound or CT guided intratumoral injection of 99mTcnanocolloid
in renal cell carcinoma resulted in sentinel node detection in 6 out of
8 patients. SPECT-CT anatomically localized retroperitoneal sentinel nodes
(2 retrocaval and 4 interaortocaval). In one patient a sentinel node along the internal
mammary chain was visualized. All sentinel nodes could be mapped and sampled.
In 38 patients with head and neck melanoma SPECT-CT led to the anatomical
localization of 100 sentinel nodes (average 2.6). In comparison with planar images
SPECT-CT depicted additional sentinel nodes in 16% of the patients. The surgical
approach was adjusted in 11 patients (55%).
In 40 patients with previous penile tumor resection lymphoscintigraphy based on
planar scintigraphy and SPECT-CT visualized sentinel nodes in 56 of 60 (93%)
clinically node-negative groins. This led to the detection of metastases in 7 groins
(12%) No inguinal recurrences were seen in patients with tumor-free sentinel nodes
during a median follow-up of 13 months.
A multicenter study aimed to evaluate lymphatic drainage from multifocal breast
cancer was started including the first 5 patients. The protocol concerns tracer
injection into the tumors in two consecutive days and SPECT-CT for sentinel node
localization.
Intraoperative sentinel node detection using a portable gamma camera The
use of a portable CsI(Na) gamma camera using an innovative device for simultaneous
99m Tc/ 125 I detection for intraoperative sentinel node localization by laparoscopy or
open surgery contributed to retrieve para-aortic sentinel nodes in 18 patients with
different urological malignancies. Para-aortic drainage was preoperatively evaluated
using SPECT-CT and subsequently the portable gamma camera in combination with
a gamma probe led to surgical localization and removing of the sentinel nodes.
In 25 patients with head and neck melanoma or oral cavity carcinoma both SPECT-CT
and the portable gamma camera led to retrieve 70 sentinel nodes (average 2.8) in
various levels of the neck. The portable gamma camera visualized the sentinel nodes
in all patients and was particularly useful to identify nodes near the injection area
around the primary tumor and post-excision monitoring led to retrieve additional
sentinel nodes in 6 patients.
The effect of an increase in tracer particle concentration on pre-and intraoperative
sentinel node depiction was evaluated in 50 consecutive prostate cancer patients
scheduled for laparoscopic sentinel node biopsy. Sentinel node visualization was
100% (against 88%) in the 25 patients receiving more particles with significant more
counts/pixels in comparison with the patients of the group receiving less concentrated
105
DIAGNOSTIC ONCOLOGY
Stella Mook MD PhD student
Aafke Wieringa-Ariaens Technical staff
Henrique Ruijter-Schippers Technical staff
Esther Scheerman Technical staff
Marjanka Schmidt PhD Post-doc
Carla Schippers-Gillissen Technical staff
Ivon Tielen Technical staff
Miranda van Dongen Technical staff
THE NETHERLANDS CANCER INSTITUTE
FAMILY CANCER CLINIC
Senno Verhoef MD PhD Head
Frans Hogervorst PhD Academic staff, head
Diagnostic Laboratory
Efraim Rosenberg Academic staff
Laura van ’t Veer PhD Academic staff
Irma Kluijt MD Academic staff
Priscilla Axwijk MD Academic staff
Marielle Ruijs MD Academic staff
Lizet van der Kolk MD PhD Academic staff
Eveline Bleiker 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
Marije Olsthoorn - Ooms Research assistant
Daoud Ait Moha Research assistant
Mohamed Achachah Technical staff
Rob Plug Technical staff - Quality 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
Fijs van Leeuwen PhD 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
Patrick Chin PhD Post-doc
Saske Hoving PhD Post-doc
Joeri Kuil PhD Post-doc
Edoardo Pasca PhD Post-doc
Wen Qi PhD Post-doc
Christian Siedschlag PhD Post-doc
Wei Chen PhD student
Kenneth Pengel PhD student
Annemarie Schmitz PhD student
Ingar Seemann PhD student
Lotte Elshof Graduate student

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DIAGNOSTIC ONCOLOGY
Lotte Lutkenhaus Graduate student
Alexander Schmitz Graduate student
Kirsten Zuurmond Graduate student
Nienke van der Berg Graduate student
Lukas Batteau Technical staff
Chantal Beekman Technical staff
Tessa Buckle Technical staff
Anita Paape Technical staff
Maddalena Rossi Technical staff
Publications
Abraham J, Edgerly M, Wilson R, Chen C,
Rutt A, Bakke S, Robey R, Dwyer A,
Goldspiel B, Balis F, van Tellingen O,
Bates SE, Fojo T. A phase I study of the
P-glycoprotein antagonist tariquidar in
combination with vinorelbine. Clin Cancer
Res 2009;15:3574-3582
Alderliesten T, Loo CE, Schlief ATEF,
Paape A, van der Meer M, Gilhuijs KG,
Application of an image-guided navigation
system in breast cancer localization. SPIE.
2009;7261:1111-1118
Aukema TS, Russell NS, Wesseling J,
Rutgers EJ. Extensive soft tissue resection
with autologous tissue closure for locally
recurrent breast cancer: lasting local control
and acceptable morbidity. Eur J Surg
Oncol. 2009;35:469-74
Aukema TS, Rutgers EJT, Vogel WV,
Valdés Olmos RA. Staging of breast cancer
with 18F-FDG PET and PET-CT. Curr
Med Imag Rew 2009 (in press)
Aukema TS, Straver ME, Valdés Olmos RA,
Vogel WV. A different role for FDG PET/CT
in axillary l lymph node staging in breast
cancer. Eur J Nucl Med Mol Imaging.
2009;36:1896-7
Aukema TS, Teunissen JJ, Burgers SA,
van Pel R, Vogel WV. Extensive soft-tissue
metastases from malignant pleural
mesothelioma. J Clin Oncol. 2009;27:e24-5
Aukema TS, Valdés Olmos RA, Klomp HM,
Teertstra HJ, Belderbos JS, Vogel WV,
Baas P, Burgers SA, Van den Heuvel MM.
Evaluation of 18F-FDG PET-CT for
Differentiation of Pulmonary Pathology in an
Approach of Outpatient Fast Track
Assessment. J Thorac Oncol. 2009
tracer. This led to a more efficient intraoperative sentinel node detection with the
portable gamma camera (100% vs 84%).
PET-CT in lung cancer Sequential 18 F-FDG PET-CT was used to early predict tumor
response in operable non-small cell lung cancer patients receiving preoperative
erlotinib 150 mg daily for 3 weeks. PET-CT performed before and within one week of
initiation of treatment were compared and patients showing decreases in maximal
standardized uptake values (SUVmax) in tumor of ≥25% were classified as metabolic
responders. Necrosis of ≥50% on histopathologic examination of the resected
specimen was considered as response. The median percentage of the early metabolic
responder group was 70% against 40% for non metabolic responders (p=0.009).
The kappa agreement between the metabolic and the histopathologic responders was
0.55 (p=0.008).
PET-CT in melanoma and penile cancer 18 F-FDG PET-CT detected additional
metastases in 26 of 70 melanoma patients (37%) with palpable lymph node
metastases. PET-CT was false positive in one patient and false negative in four. This
resulted in a sensitivity of 87% and a specificity of 98%. Instead of the planned node
dissection 10 patients received palliative chemotherapy, two underwent palliative
radiotherapy and eight no further treatment. The overall survival of the patients
without additional metastases on PET-CT was 84% which was better than the 56% of
patients presenting no additional metastases (p3cm and/or lymph node
involvement PET-CT was performed before neoadjuvant chemotherapy. The
sensitivity and specificity of PET-CT in detecting axillary metastases were respectively
97% and 100%. No difference existed between the SUV max of the primary tumour
and that from the related most intense lymph node metastasis. Moreover, the mean
tumour-to-background ratio was 90% higher in the lymph nodes compared to the
primary tumour (P=0.006). Ninety-three percent of the patients had sufficient
uptake in the lymph nodes to qualify for subsequent response monitoring of the
axilla. A considerable distinction in metabolic activity was observed between the
different subtypes of breast cancer. The mean SUV max in lymph node metastases of

ER-positive, triple-negative and HER2-positive tumours was 6.6, 11.6 and 6.6,
respectively.
The incidence of extra-axillary lymph node involvement on baseline FDG PET-CT in
patients with stage II-IV breast cancer, scheduled for neo-adjuvant chemotherapy,
was evaluated. In 17 of 60 patients (28%) increased FDG extra-axillary lymph node
uptake was visualized by PET-CT. Ultrasound guided cytology was able to detect extraaxillary
lymph node involvement in 7 of these patients. Lymph nodes outside the
axilla on PET-CT were localized in the intra mammary chain, mediastinal stations,
internal mammary chain, intra- and interpectoral, subclavicular, infraclavicular and
in the contra-lateral axilla.
First experience in breast cancer with a small PET ring camera Following the
adjustment of the design of a newly dedicated mini-PET ring camera aimed for
screening and biopsy of FDG avid lesions (European project MAMMI EU-037555) the
first clinical studies were performed. The first prototype of this dedicated breast PET
demonstrated to acquire images with a spatial resolution near 1.5 mm. The mini-PET
reconstructed images using a Maximum Likelihood Expectation Maximization 3D
algorithm in about 15 min for a transaxial FOV of 170 mm in diameter and 40 mm
axial in one shoot. However, an expanded 2D algorithm has also been developed to
reconstruct the same volume within just 1 min. The whole axial FOV varies as a
function of the breast length. In 15 breast cancer patients aimed to receive
neoadjuvant chemotherapy mini-PET images were obtained after 18 F-FDG
administration in order to standardize display and for comparison with PET-CT
(figure 1). Validation of therapy response in breast cancer using SUVmax calculation
will be included in the second part of the project. In 2010 a second prototype with
improved detector sensitivity will be installed at the NKI-AVL.
Figure 1: First prototype of a mini-PET ring camera for dedicated hanging breast imaging and detection
of malignant tumors (European project MAMMI EU-037555).
New PET tracers In 2009 several new molecular pathways became available for
clinical evaluation of tumor characteristics in vivo, because of the introduction of
novel radiolabeled tracer molecules for imaging with PET/CT. An anti-CD20
chimeric antibody (Rituximab) was labeled with the positron emitting nuclide
iodine-124, and was applied for detection and localization of CD20 expressing
leukocytes. In seven patients with reumatoid arthritis, infiltration of CD20
expressing B cells in the synovia of inflamed joints could be demonstrated (figure 2).
A new trial was opened in the field of hypoxia imaging (using 18-fluor-FAZA), to
determine the stability of hypoxia in tumors in vivo in a test-retest approach. Several
other radiolabeled tracer molecules were acquired and validated for detection and
characterization of tumors in humans, including the proliferation marker Fluor-18choline
(for well-differentiated tumors, e.g. prostate cancer), the somatostatin
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DIAGNOSTIC ONCOLOGY
Publications (continued)
Axwijk PH, Kluijt I, De Jong D, Gille JJ,
Teertstra HJ, Horenblas S. Erfelijke
oorzaken van niertumoren. Ned. Tijdschrift
voor oncologie 2009;6:99-107
Balague O, Mozos A, Martinez D,
Hernandez L, Colomo L, Mate JL,
Teruya-Feldstein J, Lin O, Campo E,
Lopez-Guillermo A, Martinez A.
Activation of the endoplasmic reticulum
stress-associated transcription factor x
box-binding protein-1 occurs in a subset of
normal germinal-center B cells and in
aggressive B-cell lymphomas with prognostic
implications. Am J Pathol. 2009;174:2337-46
Balague Ponz O, Ott G, Hasserjian RP,
Elenitoba-Johnson KS, de Leval L,
de Jong D. Commentary on the WHO
classification of tumors of lymphoid tissues
(2008): aggressive B-cell lymphomas.
J Hematop. 2009
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, 2009
Beumer JH, Lopez-Lazaro L, Schellens JH,
Beijnen JH, Van Tellingen O. Evaluation of
human plasma protein binding of
trabectedin (Yondelis, ET-743). Curr Clin
Pharmacol 2009;4:38-42
Borovski T, Verhoeff JJ, ten Cate R,
Cameron K, de Vries, NA, van Tellingen O,
Richel DJ, van Furth WR, Medema JP,
Sprick MR. Tumor microvasculature
supports proliferation and expansion of
glioma-propagating cells. Int J Cancer,
125:1222-1230, 2009
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. 2009
Bueno-de-Mesquita JM, Linn SC, Keijzer R,
Wesseling J, Nuyten DS, van Krimpen C,
Meijers C, de Graaf PW, Bos MM,
Hart AA, Rutgers EJ, Peterse JL,
Halfwerk H, de Groot R, Pronk A,
Floore AN, Glas AM, Van ‘t Veer LJ,
van de Vijver MJ. Validation of 70-gene
prognosis signature in node-negative breast
cancer. Breast Cancer Res Treat.
2009;117:483-95

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DIAGNOSTIC ONCOLOGY
Publications (continued)
Bueno-de-Mesquita JM, Nuyten DS,
Wesseling J, van Tinteren H, Linn SC,
van de Vijver MJ. The impact of interobserver
variation in pathological
assessment of node-negative breast cancer
on clinical risk assessment and patient
selection for adjuvant systemic treatment.
Ann Oncol. 2009
Courrech Staal EF, van Coevorden F, Cats A,
Aleman BM, van Velthuysen ML, Boot H,
Vrancken Peeters MJ, van Sandick JW.
Outcome of Low-Volume Surgery for
Esophageal Cancer in a High-Volume
Referral Center. Ann Surg Oncol. 2009
De Jong D, de Boer JP. Predicting
transformation in follicular lymphoma.
Leuk Lymphoma. 2009:1-6
De Jong D, Koster A, Hagenbeek A,
Raemaekers J, Veldhuizen D,
Heisterkamp S, de Boer JP, van Glabbeke M.
Impact of the tumor microenvironment on
prognosis in follicular lymphoma is
dependent on specific treatment protocols.
Haematologica. 2009;94:70-7
De Jong D, Xie W, Rosenwald A,
Chhanabhai M, Gaulard P, Klapper W,
Lee A, Sander B, Thorns C, Campo E,
Molina T, Hagenbeek A, Horning S, Lister A,
Raemaekers J, Salles G, Gascoyne RD,
Weller E. Immunohistochemical prognostic
markers in diffuse large B-cell lymphoma:
validation of tissue microarray as a
prerequisite for broad clinical applications
(a study from the Lunenburg Lymphoma
Biomarker Consortium). J Clin Pathol.
2009;62:128-38
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. 2009
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. 2009
receptor ligand Gallium-68-octreotate (for neuro-endocrine tumors), and iodine-124sodiumchloride
(for differentiated thyroid cancer). These facilities will lead to new
fields of research in 2010.
Figure 2: 124 Iodine-Rituximab PET/CT imaging of CD20 positive B cells in the synovia of an inflamed
knee in a patient with rheumatoid arthritis.
MOLECuLAR IMAGING NuCLEAR MEDICINE
Combined pre- and intraoperative imaging of the sentinel lymph node using a
self assembled multimodal complex
Tessa Buckle, Anne van Leeuwen, Bert Pool, Gavin Bendle, Jos Jonkers, Michiel Sinaasappel,
Renato Valdés Olmos, Henk van der Poel, Fijs van Leeuwen
Preoperative lymphoscintigraphy using radiolabeled albumin aggregates is a standard
procedure in sentinel lymph node (SLN) detection. To increase the accuracy of
intraoperative SLN detection, new methods with a higher sensitivity and specificity
than that of dyes such as the visible patent blue or the fluorescent indocyanine green
(ICG) are required. In order to facilitate this, we developed a self-assembled
multimodal complex in which the NIR fluorescent dye ICG is non-covalently bound
to radioactive labeled albumin aggregates. After intratumoral injection, the selfassembled
multimodal complex provides identical dynamics of the radioactive and
optical component. Hence this allows us to depict identical features in the pre- and
intra- operative situation. While SLN specificity of this multimodal complex is similar
to conventional lymphoscintigraphy, the fluorescent signal-to-noise ratio was improved
by 77% compared to ICG alone and the sensitivity and the specificity nearly doubled.

Figure 3: Multi modal imaging of the SLN: a.) Preoperative SPECT/CT of the SLN in breast cancer using
the radioactive component of the complex and b.) intraoperative visualization of the SLN using the
fluorescent component of the complex.
Improved fluorescent dyes for intraoperative imaging purposes
Patrick Chin, Tessa Buckle, Joeri Kuil, Chantal Beekman, Albert Ruggi1 , Peter Steunenberg1 ,
Aldrik Velders1 , Arantxa Aguirre de Miguel2 , Stefan Meskers2 , René Janssen2 and Fijs van Leeuwen
Fluorescence molecular imaging is rapidly increasing its popularity in image guided
surgery applications. To help develop its full surgical potential it remains a challenge
to: 1) generate dual emissive imaging agents that allow for combined visible assessment
and sensitive camera based imaging; 2) enable improved signal to background ratios
in deep tissue imaging, and 3) allow for depth estimation. To this end, we develop
new fluorescent imaging agents. For example we have developed multispectral InP/
ZnS quantum dots (QDs) that exhibit a bright visible green/yellow exciton emission
combined with a long lived far red afterglow. The latter allows for deep tissue
detection by eliminating the interference of autofluorescence. We demonstrated that
this can help provide guidance to the The difference in tissue penetration of these
emissions can also be used to determine the tissue depth of the QDs. We use the
latter property to help advance the fluorescent detection of multimodal marker seeds.
Alternatively, we have developed inorganic dyes that give a bright fluorescence. Other
than organic dyes, these dyes do not suffer from quenching and can thus be used to
increase the fluorescent intensity on e.g. a targeting moiety.
Figure 4: Schematic representation of lifetime dependant multispectral imaging of the SLN using dual
emissive InP/ZnS QDs.
1University Twente, Enschede;
2TU Eindhoven, Eindhoven
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DIAGNOSTIC ONCOLOGY
Publications (continued)
De Vreeze RS, de Jong D, Tielen IH,
Ruijter HJ, Nederlof PM, Haas RL,
van Coevorden F. Primary retroperitoneal
myxoid/round cell liposarcoma is a
nonexisting disease: an immunohistochemical
and molecular biological
analysis. Mod Pathol. 2009;22:223-31
De Vries NA, Beijnen JH, van Tellingen O.
High-grade glioma mouse models and their
applicability for preclinical testing. Cancer
Treat Rev, 2009;35:714-723
Geurts TW, Balm AJ, van Velthuysen ML,
van Tinteren H, Burgers JA,
van Zandwijk N, Klomp HM. Survival
after surgical resection of pulmonary
metastases and second primary squamous
cell lung carcinomas in head and neck
cancer. Head Neck. 2009;31:220-6
Geurts TW, van Velthuysen ML,
Broekman F, van Huysduynen TH,
van den Brekel MW, van Zandwijk N,
van Tinteren H, Nederlof P, Balm AJ,
Brakenhoff RH. Differential diagnosis of
pulmonary carcinoma following head and
neck cancer by genetic analysis. Clin Cancer
Res. 2009;15:980-5
Gómez García EB, Oosterwijk JC,
Timmermans M, van Asperen CJ,
Hogervorst FB, Hoogerbrugge N,
Oldenburg R, Verhoef S, Dommering CJ,
Ausems MG, van Os TA, van der Hout AH,
Ligtenberg M, van den Ouweland A,
van der Luijt RB, Wijnen JT, Gille JJ,
Lindsey PJ, Devilee P, Blok MJ,
Vreeswijk MP. A method to assess the
clinical significance of unclassified variants
in the BRCA1 and BRCA2 genes based on
cancer family history. Breast Cancer Res.
2009;11:R8
Graafland NM, Leijte JA, Valdés Olmos RA,
Hoefnagel CA, Teertstra HJ, Horenblas S.
Scanning with 18F-FDG-PET/CT for
detection of pelvic nodal involvement in
inguinal node-positive penile carcinoma.
Eur Urol. 2009;56:339-45
Graafland NM, Leijte JA, Valdés Olmos RA,
Van Boven HH, Nieweg OE, Horenblas S.
Repeat dynamic sentinel node biopsy in
locally penile carcinoma. BJU Int. 2009

110
DIAGNOSTIC ONCOLOGY
Publications (continued)
Haas RL, Girinsky T, Aleman BM,
Henry-Amar M, de Boer JP, de Jong D.
Low-dose involved-field radiotherapy as
alternative treatment of nodular lymphocyte
predominance Hodgkin’s lymphoma. Int J
Radiat Oncol Biol Phys. 2009;74:1199-202
Hambach L, Ling KW, Pool J, Aghai Z,
Blokland E, Tanke HJ, Bruijn JA, Halfwerk H,
van Boven H, Wieles B, Goulmy E.
Hypomethylating drugs convert HA-1negative
solid tumors into targets for stem
cell-based immunotherapy. Blood.
2009;113:2715-22
Hasserjian RP, Ott G, Elenitoba-Johnson KS,
Balague-Ponz O, de Jong D, de Leval L.
Commentary on the WHO classification of
tumors of lymphoid tissues (2008): ‘Gray
zone’ lymphomas overlapping with Burkitt
lymphoma or classical Hodgkin lymphoma.
J Hematop. 2009
Holm C, Kok M, Michalides R, Fles R,
Koornstra RH, Wesseling J, Hauptmann M,
Neefjes J, Peterse JL, Stål O, Landberg G,
Linn SC. Phosphorylation of the oestrogen
receptor alpha at serine 305 and prediction
of tamoxifen resistance in breast cancer.
J Pathol. 2009;217:372-9
Holstege H, Joosse SA, van Oostrom CT,
Nederlof PM, de Vries A, Jonkers J. High
incidence of protein-truncating TP53
mutations in BRCA1-related breast cancer.
Cancer Res. 2009;69:3625-33
Hughes BE, Leijte JA, Kroon BK, Shabbir MA,
Swallow TW, Heenan SD, Corbishley CM,
van Boven HH, Perry MJ, Watkin NA,
Horenblas S. Lymph Node Metastasis in
Intermediate-Risk Penile Squamous Cell
Cancer: A Two-Centre Experience. Eur
Urol. 2009
Hugo M. Horlings, Carmen Lai,
Dimitry S.A. Nuyten, Hans Halfwerk,
Petra Kristel, Erik van Beers, Simon A.
Joosse, Christiaan Klijn, F. Reyal, Petra M.
Nederlof, Marcel J.T. Reinders, Lodewyk F.A.
Wessels, Marc J. van de Vijver. Integration
of DNA copy number alterations and
prognostic gene expression signatures in
breast cancer patients. Clin cancer Res
2009
Combined pre-, intra-, and postoperative detection of tumor tissue based on
the targeting of biomarkers over expressed in breast tumors
Tessa Buckle, Nynke van den Berg, Joeri Kuil, Chantal Beekman, Ed Roos, Jos Jonkers, Shinya Oishi1 ,
Nobutaka Fujii1 , Hideo Saji1 , Emiel Rutgers, Fijs van Leeuwen
Tumor cells can show overexpression of certain biomarkers and specific targeting of
these tumor cells wil advance both pre- and intraoperative diagnostics. To this end we
currently investigate a number of biomarkers e.g. Her2 and the chemokine receptor
CXCR4. Different labels on ‘large’ Her2 targeted antibodies allow us to specifically
detect Her2 expressing tumors in both a pre- and intraoperative setting. Alternatively,
biomarkers viz. CXCR4 can also be targeted using ‘small’ peptides that give a better
biodistribution. To this end we radiolabel a CXCR4 targeted peptide antagonist with
indium (111-In) and used this for pre-operative SPECT/CT based detection in
mammary mouse tumor models o.a. a DCIS model. For intra-operative detection we
use the same peptide functionalized with a NIR fluorescent label. Post operative
pathology, immunohistochemistry (IHC) is generally considered ‘the golden
standard’ in the assessment of the expression of molecular tumor markers.
Therefore, we study the feasibility of direct fluorescent detection using the same
CXCR4 targeted peptide antagonists an compare their accuracy to commercially
available CXCR4 antibodies.
Overall, we are able to detect Her2 and CXCR4 positive breast tumors using both
radioactivity (preoperative) and fluorescence (intraoperative) imaging. Furthermore
our results indicate that the peptides have a similar CXCR4 affinity as the best
commercial antibodies And can also be applied for fluorescence IHC.
Figure 5: Combined pre-, intra-, and postoperative imaging of CXCR4 using peptides.
Tumor response measurement using radiolabeled 99mTc Annexin V
and 18F FDG imaging
Chantal Beekman, Tessa Buckle, Sven Rottenberg, Renato Valdes-Olmos, Marcel Verheij,
Jack van Asten2 , Tone Bathen3 , Arend Heerschap2 , Fijs van Leeuwen
When treating cancer, tumor response monitoring after therapeutic intervention is
crucial to evaluate treatment efficacy and can help reduce the amount of miss- or
over-treatment. One method to predict response is to determine the degree of cell
death via apoptotic pathways. In the clinic, radiolabeled annexin V (99mTc-AnxV ) is
used to noninvasively determine the degree of apoptosis. Using genetically similar
docetaxel sensitive and -resistant tumors in a ‘spontaneous’ mouse model for breast
cancer we set out to check the predictive value of annexin V to noninvasively
1Kyoto University, Kyoto, Japan
2UMC Radboud, Nijmegen;
3Norwegian University of Science and Technology, Trondheim

determine apoptosis levels in vivo. While TUNEL and cleaved caspase 3 staining at
IHC show a clear increase in apoptotic activity one day after the treatment, we cannot
detect this using SPECT/CT with 99mTc-AnxV. In our opinion, using in vivo 99mTc-
AnxV imaging requires vigorous control experiments to asses its prognostic value.
A second method to predict response is to determine the metabolic activity of a
tumor. The cytotoxic effect of chemotherapy reduces the uptake of the PET-tracer
18F-fluorodeoxyglucose (FDG). Although 18F-FDG imaging has shown good
prognostic potential in the clinic, predictive values vary widely. Using the same
mouse model mentioned before, we measured 18F-FDG uptake before and after
doxorubicin treatment using a gamma probe. This way we can precisely determine
the kinetic of 18F-FDG uptake in the tumor and possibly correlate this to tumor size
and response to chemotherapy. We observe a clear correlation between tumor size
and tracer uptake, however, a correlation between uptake and tumor response has
not yet been detected in these mice.
Thirdly, we are investigating the potential of choline based (N)MR spectroscopy to
predict the response to therapy.
Figure 6: Tumor volume corrected 99mTc-AnxV uptake percentages in mice with sensitive and resistant
tumors.
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
Gefitinib (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. However, a large portion
of the patients does not respond to this agent. Recent publications show a correlation
between the presence of a mutation (small in-frame deletion or point mutation) in
exon 18, 19, 20 or 21 of the EGFR gene and response to therapy. We have now
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 amplification (CISH).
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DIAGNOSTIC ONCOLOGY
Publications (continued)
Joosse SA, van Beers EH, Tielen IH,
Horlings H, Peterse JL, Hoogerbrugge N,
Ligtenberg MJ, Wessels LF, Axwijk P,
Verhoef S, Hogervorst FB, Nederlof PM.
Prediction of BRCA1-association in
hereditary non-BRCA1/2 breast carcinomas
with array-CGH. Breast Cancer Res Treat.
2009;116:479-89
Kluijt I, de Jong D, Teertstra HJ,
Axwijk PH, Gille JJ, Bell K, van Rens A,
van der Velden AW, Middelton L,
Horenblas S. Early onset of renal cancer in
a family with Birt-Hogg-Dubé syndrome.
Clin Genet. 2009;75:537-43
Kok M, Koornstra RH, Margarido TC,
Fles R, Armstrong NJ, Linn SC,
van ‘t Veer LJ, Weigelt B. Mammospherederived
gene set predicts outcome in patients
with ER-positive breast cancer. Journal of
Pathology 2009;218:316-26
Kok M, Linn SC, Van Laar RK,
Jansen MPHM, Van Den Berg TM,
Delahaye LJMJ, Glas AM, Peterse JL,
Hauptmann M, Foekens JA, Klijn JGM,
Wessels LFA, van ‘t Veer LJ, Berns EMJJ.
Comparison of gene expression profiles
predicting progression in breast cancer
patients treated with tamoxifen. Breast
Cancer Research and Treatment
2009;113:275-83
Koski TA, Lehtonen HJ, Jee KJ, Ninomiya S,
Joosse SA, Vahteristo P, Kiuru M, Karhu A,
Sammalkorpi H, Vanharanta S,
Lehtonen R, Edgren H, Nederlof PM,
Hietala M, Aittomäki K, Herva R,
Knuutila S, Aaltonen LA, Launonen V.
Array comparative genomic hybridization
identifies a distinct DNA copy number
profile in renal cell cancer associated with
hereditary leiomyomatosis and renal cell
cancer. Genes Chromosomes Cancer.
2009;48:544-51
Leijte JA, Graafland NM, Valdés Olmos RA,
Van Boven HH, Hoefnagel CA,
Horenblas. Prospective evaluation of
hybrid 18F-fluorodeoxyglucose positron
emission tomography/computed
tomography in staging clinically nodenegative
patients with penile carcinoma.
BJU Int. 2009;104:640-4

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DIAGNOSTIC ONCOLOGY
Publications (continued)
Leijte JA, Hughes B, Graafland NM,
Kroon BK, Valdés Olmos RA, Nieweg OE,
Corbishley C, Heenan S, Watkin N,
Horenblas S. Two-center evaluation of
dynamic sentinel node biopsy for squamous
cell carcinoma of the penis. J Clin Oncol.
2009;27:3325-9
Leijte JA, Van der Ploeg IM,
Valdés Olmos RA, Nieweg OE,
Horenblas S. Visualization of tumor
blockage and rerouting of lymphatic
drainage in penile cancer patients by use of
SPECT/CT. J Nucl Med. 2009;50:364-7
Linn SC, van ‘t Veer LJ. Clinical relevance
of the triple-negative breast cancer concept:
Genetic basis and clinical utility of the
concept. European Journal of Cancer
2009;45:11-26
Linn SC, Wesseling J. Molecular tests for
breast-cancer diagnosis? Lancet Oncol.
2009;10:314-5
Loo CE, van Pel R. Diagnostic image.
A woman with an abnormal screening
mammogram. Ned Tijdschr Geneeskd.
2009;153
Mann RM, Loo CE, Wobbes T, Bult P,
Barentsz JO, Gilhuijs KG, Boetes C. The
impact of preoperative breast MRI on the
re-excision rate in invasive lobular
carcinoma of the breast. Breast Cancer
Res Treat. 2009
Mohammadi L, Vreeswijk MP,
Oldenburg R, van den Ouweland A,
Oosterwijk JC, van der Hout AH,
Hoogerbrugge N, Ligtenberg M,
Ausems MG, van der Luijt RB,
Dommering CJ, Gille JJ, Verhoef S,
Hogervorst FB, van Os TA,
Gómez García E, Blok MJ, Wijnen JT,
Helmer Q, Devilee P, van Asperen CJ,
van Houwelingen HC. A simple method
for co-segregation analysis to evaluate the
pathogenicity of unclassified variants;
BRCA1 and BRCA2 as an example.
BMC Cancer. 2009;9:211
Gene Expression Profiling to identify the histogenetic origin of metastatic
adenocarcinomas of unknown primary
Daphne de Jong, Martijn Kerst, Helgi Helgason, Jelle Wesseling, Annuska Glas1 , Laura van ’t Veer
Patients with adenocarcinoma of unknown primary origin (ACUP) constitute
approximately 4% of all malignancies. For effective treatment of these patients, it is
considered optimal to identify the primary tumor origins. Currently, the success rate
of the diagnostic work-up is only 20% to 30%.
We previously established that the gene expression microarray test CUPPrint can
classify correctly from FFPE samples the majority of tumor classes both in patients
with known primary and in patients with ACUP and this method may be an attractive
additional analytic approach to assist with the histogenetic diagnosis of patients with
ACUP. We now evaluated the use of CUPPrint prospectively in 34 carcinoma of
unknown primary CCUPS patients diagnosed at NKI-AvL between 2005 and 2009.
In 14/34 patients CUPPrint was fully consistent with clinical presentation and
histopathological interpretation of the diagnosis. In 5/34 patients integration of the
CUPPrint result had direct treatment consequences. In all other cases the diagnosis
was somewhat helped, but could not yet provide additional information for clinical
management beyond CUP.
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-defined disease entities, characterized by
morphology and in part by genetic alterations as well-differentiated (WDLS), dedifferentiated
(DDLS), myxoid, round cell (MRLS), and pleomorphic (PLS). WLS
and DDLS are characterized by amplification 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. The metastatic or second primary nature of
MRLS, defined as tumor presentation in at least two separate sites before manifestation
in the lungs, is a matter of debate with essential clinical consequences. Using the
detailed molecular composition of the translocation and loss-of-heterozygozity (LOH)
analysis, we were able to show that apparent multifocality actually represents metastatic
disease in all instances. Moreover, LOH patterns showed that MRLS is characterized
by extensive and ongoing mutation over time, resulting in a relatively large spectrum
of coexisting subclones as compared to other solid tumors.
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 files 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 findings support a stem cell model
with different levels of maturation within specific disease entities and expands the
morphological spectrum of MRLS. This stem cell model with tumor progression
from intermediate stages of development is further supported by finding of 12q
amplification in WDLS, DDLS, but also in a subset of PLS.
Tumor microenvironment in malignant lymphoma
Jan Paul de Boer, Martijn Kerst, Joke Baars, Colette van de Bogaard, Martine van Glabbeke,
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 nonmalignant
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
1 Agendia BV, Amsterdam

via components of the adaptive and innate immune response. We have been mostly
involved in studies in this field 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 significance are highly conflicting. 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. Especially in variation
range was systematically overestimated. 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 fludarabine to CVP chemotherapy. Most
importantly, some T-cell and accessory cell populations showed an homogeneous
prognostic impact, while others had a different and sometimes opposite effect in the
treatment arms. FoxP3 regulatory T-cells and CD68 positive macrophages seem to be
key players in the growth regulation of FL cells. Within the NKI-AVL, this feature is
now further explored by FACS analysis on pre- and post treatment cytological
aspirates in FL patients.
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.
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 profile 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.
Figure 7: Five-year breast cancer specific survival by treatment within the 70-gene signature groups
(70-gene low risk on the left, high risk on the right). Abbreviations: BCSS, breast cancer specific survival;
n, number; ET, endocrine therapy, ET+CT, endocrine+chemotherapy; HR, univariate hazard ratio.
1University Medical Center Groningen
2Nijmegen Medical Center, the Netherlands
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DIAGNOSTIC ONCOLOGY
Publications (continued)
Mook S, Schmidt MK, Viale G, Pruneri G,
Eekhout I, Floore A, Glas AM, Bogaerts J,
Cardoso F, Piccart-Gebhart MJ,
Rutgers ET, van ‘t Veer LJ. The 70-gene
prognosis-signature predicts disease outcome
in breast cancer patients with 1-3 positive
lymph nodes in an independent validation
study. Breast Cancer Research and
Treatment 2009;116:295-302
Mozos A, Royo C, Hartmann E, De Jong D,
Baró C, Valera A, Fu K, Weisenburger DD,
Delabie J, Chuang SS, Jaffe ES,
Ruiz-Marcellan C, Dave S, Rimsza L,
Braziel R, Gascoyne RD, Solé F,
López-Guillermo A, Colomer D, Staudt LM,
Rosenwald A, Ott G, Jares P, Campo E.
SOX11 expression is highly specific for
mantle cell lymphoma and identifies the
cyclin D1-negative subtype. Haematologica.
2009;94:1555-62
Niers TM, Bruggemann LW, Klerk CP,
Muller FJ, Buckle T, Reitsma PH, Richel DJ,
Spek CA, van Tellingen O, Van Noorden CJ.
Differential effects of anticoagulants on
tumor development of mouse cancer cell
lines B16, K1735 and CT26 in lung. Clin
Exp Metastasis 2009;26:171-178
Noordzij JG, Wulffraat NM, Haraldsson A,
Meyts I, van ‘t Veer LJ, Hogervorst FBL,
Warris A, Weemaes CMR. Ataxiatelangiectasia
patients presenting with
hyper-IgM syndrome. Archives of Disease in
Childhood 2009;94:448-9
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. 2009
Ott G, Balague-Ponz O, de Leval L,
de Jong D, Hasserjian RP,
Elenitoba-Johnson KS. Commentary on
the WHO classification of tumors of
lymphoid tissues (2008): indolent B cell
lymphomas. J Hematop. 2009
Patriarca C, Colombo P, Pio Taronna A,
Wesseling J, Franchi G, Guddo F,
Naspro R, Macchi RM, Giunta P, Di
Pasquale M, Parente M, Arizzi C,
Roncalli M, Campo B. Cell discohesion and
multifocality of carcinoma in situ of the
bladder: new insight from the adhesion
molecule profile (e-cadherin, Ep-CAM, and
MUC1). Int J Surg Pathol. 2009;17:99-106

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DIAGNOSTIC ONCOLOGY
Publications (continued)
Pengel KE, Loo CE, Teertstra HJ, Muller SH,
Wesseling J, Peterse JL, Bartelink H,
Rutgers EJ, Gilhuijs KG. The impact of
preoperative MRI on breast-conserving
surgery of invasive cancer: a comparative
cohort study. Breast Cancer Res Treat.
2009;116:161-9
Pijpe A, Manders P, Brohet RM,
Collée 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
Research and Treatment 2009 (in press)
Ploem MC, Retèl VP, Linn SC, van Boven HH,
Schmidt MK, de Jong JP, Gevers JK,
van Harten WH. Tumour tissue: who is in
control? Lancet Oncol. 2009
Pluim D, Beijnen, JH, Schellens, JH,
van Tellingen O. Simultaneous
determination of AZD1152 (prodrug)
and AZD1152-hydroxyquinazoline
pyrazol anilide by reversed phase liquid
chromatography. J Chromatogr B Analyt
Technol Biomed Life Sci, 2009;877:
3549-3555
Prevoo W, van den Munckhof MP,
Meinhardt W, Horenblas S,
van den Bosch MAAJ. Radiofrequency
Ablation of kidney tumors in patients with
a Solitary kidney, Clinical Radiology 2009
(in press)
Puppe J, Drost R, Liu X, Joosse SA, Evers B,
Cornelissen-Steijger P, Nederlof P, Yu Q,
Jonkers J, van Lohuizen M, Pietersen AM.
BRCA1-deficient mammary tumor cells are
dependent on EZH2 expression and
sensitive to Polycomb Repressive Complex
2-inhibitor 3-deazaneplanocin A. Breast
Cancer Res. 2009;11:R63
Robertus JL, Harms G, Blokzijl T,
Booman M, de Jong D, van Imhoff G,
Rosati S, Schuuring E, Kluin P,
van den Berg A. Specific expression of
miR-17-5p and miR-127 in testicular and
central nervous system diffuse large B-cell
lymphoma. Mod Pathol. 2009;22:547-55
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) benefit 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 significant HR
of 0.21(95%CI 0.07-0.59;p

Using Array CGH for hereditary breast/ovarian cancer we study whether the genomic
aberrations in breast tumors are similar to the specific aberrations found in BRCA1
tumors (in collaboration with Petra Nederlof, Division of Experimental Therapy and
Diagnostic Oncology). More than 200 a-CGH analyses have been completed, for high
risk families and unclassified variant analysis mostly. A BRCA2 classifier 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 specific
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), a national study of families with Li Fraumeni and variant Li
Fraumeni(-like) syndrome, DNA-profiling by classic cGH and array cGH of breast
and ovarian cancer patients (see Division of Experimental Therapy), the Breast
Cancer Linkage Consortium, the BCAC and CIMBA consortiums, a study into the
biological significance of so called unclassified variants (DNA changes of which it is
uncertain whether they be pathogenic mutations or polymorphisms) in a national
collaboration with other DNA-diagnostic labs, coordinated from LUMC by Dr Peter
Devilee cs and by participating in the IARC Unclassified Genetic Variants Working
Group and ENIGMA (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 current number of included patients
approaches 100 where 255 are needed.
DEPARTMENT OF RADIOLOGY
Laboratory for Diagnostic Imaging Research
Kenneth Gilhuijs, Christian Siedschlag, Wen Qi, Edoardo Pasca, Lukas Batteau, Anita Paape,
Maddalena Rossi, Kenneth Pengel, Wei Chen, Alexander Schmitz, Annemarie Schmitz, Lotte Elshof,
Lotte Lutkenhaus, Kirsten Zuurmond, Claudette Loo, Eline Deurloo, Saar Muller, Michiel Sinaasapel,
Jelle Teertstra
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 specificity of
cancer diagnosis, pre-treatment assessment of tumor extent, response monitoring
and image-guided therapy.
MRI decision 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. Ongoing efforts in our research programme on tumor response monitoring
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DIAGNOSTIC ONCOLOGY
Publications (continued)
Rodenhuis S, Mandjes IA, Wesseling J,
van de Vijver MJ, Vrancken Peeters MJ,
Sonke GS, Linn SC. A simple system for
grading the response of breast cancer to
neoadjuvant chemotherapy. Ann Oncol.
2009
Scheenstra RJ, Muller SH, Vincent A,
Sinaasappel M, Zuur JK, Hilgers FJ.
Endotracheal temperature and humidity
measurements in laryngectomized patients:
intra- and inter-patient variability. Med
Biol Eng Comput. 2009;47:773-82
Schmitz AC, van den Bosch MA,
Gilhuijs KG. Re: ‘MR-guided focused
ultrasound for malignant tumors: the
importance of margins’. J Am Coll Radiol.
2009;6:814;author reply 814-5
Siedschlag Ch, van Loon J, van
Baardwijk A, Rossi MM, van Pel R,
Blaauwgeers JL, van Suylen RJ, Boersma L,
Stroom J, Gilhuijs KG. Analysis of the
relative deformation of lung lobes before and
after surgery in patients with NSCLC. Phys
Med Biol. 2009;54:5483-92
Straver ME, Aukema TS, Valdes Olmos RA,
Rutgers EJT, Gilhuijs KGA, Vogel WV,
Vrancken Peeters MJTFD. 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
2009 (in press)
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. 2009
Straver ME, Loo CE, Rutgers EJT,
Oldenburg HSE, Wesseling,
Vrancken Peeters MT, Gilhuijs KG, MRImodel
to guide the surgical treatment in
breast cancer patients after neoadjuvant
chemotherapy, Annals of Surgery, 2009
(in press)
Straver ME, Rutgers EJ, Oldenburg HS,
Wesseling J, Linn SC, Russell NS,
Vrancken Peeters MJ. Accurate axillary
lymph node dissection is feasible after
neoadjuvant chemotherapy. Am J Surg.
2009;198:46-50

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DIAGNOSTIC ONCOLOGY
Publications (continued)
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;45:2284-92
Stroom J, Schlief A, Alderliesten T,
Peterse H, Bartelink H, Gilhuijs K. Using
histopathology breast cancer data to reduce
clinical target volume margins at
radiotherapy. Int J Radiat Oncol Biol Phys.
2009;74:898-905
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.
2009
Toracchio S, Ota H, de Jong D,
Wotherspoon A, Rugge M, Graham DY,
Samani A, El-Zimaity HM. Translocation
t(11;18)(q21;q21) in gastric B-cell
lymphomas. Cancer Sci. 2009;100:881-7
Valdés Olmos RA, Vidal-Sicart S,
Nieweg OE. SPECT/CT and real-time
intraoperatieve imaging: new tools for
sentinel node localization and radioguided
surgery? Eur J Nucl Med Mol Imaging.
2009;36:1-5
Valdés Olmos RA, Vermeeren L,
Hoefnagel CA, Aukema TS, Vogel WV.
New dimensions of nuclear medicine in
oncology. Almenara 2009 (in press)
Van den Bosch MA, Prevoo W,
van der Linden EM, Meijerink MR,
van Delden OM, Mali WP, Reekers JA.
The radiologist as the treating physician
for cancer: interventional oncology.
Ned Tijdschr Geneeskd. 2009;153
Van den Broek GB, Wildeman M, Rasch CR,
Armstrong N, Schuuring E, Begg AC,
Looijenga LH, Scheper R, van der Wal JE,
Menkema L, van Diest PJ, Balm AJ,
van Velthuysen ML, van den Brekel MW.
Molecular markers predict outcome in
squamous cell carcinoma of the head and
neck after concomitant cisplatin-based
chemoradiation. Int J Cancer.
2009;124:2643-50
focused on establishing an MRI-based interpretation model to facilitate the selection
of breast-conserving 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 crossvalidation
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 unjustified 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).
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.
using histopathology breast cancer data to reduce treatment margins at
radiotherapy Our MARGINS research programme to fine-tune the balance between
local control and cosmetic outcome in breast-conserving therapy (BCT) resulted in
quantification of the incidence and extension of microscopic disease around primary
breast tumors. An extensive pathology tumor-distribution study was performed using
38 wide-local excision specimens of patients who underwent BCT. Specimen
orientation was recorded and microscopic findings reconstructed to assess the
incidence of microscopic disease around the macroscopic tumor. A model of disease
spread was build, giving likelihood of disease extension outside a treated volume (i.e.,
P out,vol). Application of the model was explored in 10 new BCT patients. The clinical
target volume (CTV) margin at radiotherapy was defined as the safety margin of
healthy tissue to account for microscopic disease. Taking asymmetry of tumor
excision into account, new asymmetric CTV boost margins were evaluated that
minimize CTV boost without increasing P out,TTV (TTV being total-treated-volume:
V surgery + CTV boost). Potential reductions in CTV boost and TTV were evaluated.
Microscopic disease beyond the tumor boundary occurred isotropically at distances
>1 cm (intended surgical margin) and >1.5 cm (intended TTV margin) in 53% and
36% of the excision specimens, respectively. In the 10 prospective patients the
average P out,TTV was, however, only 16% due to larger surgical margins than
intended in some directions. Asymmetric CTV boost margins reduced the CTV boost
and TTV by 27% (20 cc) and 12% (21 cc) on average, without compromising tumor
coverage. We conclude that microscopic disease may occur beyond the current
CTV boost in about one-sixth of patients. An asymmetric CTV boost that corrects for
asymmetry of the surgical excision has the potential to reduce boost volumes while
maintaining tumor coverage.
Analysis of the relative deformation of lung lobes before and after surgery in
patients with NSCLC An accurate assessment of the extent of the tumor is critical for
successful local treatment of lung cancer by surgery and/or radiotherapy. A-prior
guidelines to establish the extent of treatment margins may be derived from correlation
studies between pre-treatment imaging and histopathology. Deformations occur,
however, between in-vivo CT imaging and ex-vivo pathology due to the softness of lung
tissue and pathology processing. We conducted a study to quantify these deformations
in tissue around non-small-cell lung cancer. We explored factors associated with the
magnitude of the deformations in 25 patients who underwent lobectomy after
preoperative CT. Nonrigid registration was employed to evaluate tissue deformations
around the gross tumor volume (GTV), taking into account potential differences in
elasticity between tumor and healthy lung tissue. Tissue was compacted by
approximately 60% depending on circularity of the tumor and orientation of the
specimen on the pathology table during processing. The deformations give rise to
potential underestimation of the treatment margins in pathology studies that do not
take this aspect into account. The findings of this study will be incorporated into our
prediction models aimed at pretreatment assessment of the extent of lung cancers.

Breast tomosynthesis in clinical practice: initial results Aiming to assess the
potential complementary value of tomosynthesis over conventional breast imaging
and MRI, mammography and tomosynthesis investigations of 513 woman with an
abnormal screening mammogram or with clinical symptoms were prospectively
classified according to the ACR BIRADS criteria. Sensitivity and specificity of both
techniques for the detection of cancer were calculated. In 112 newly detected cancers,
tomosynthesis and mammography were each false-negative in 8 cases (7%). In the
false-negative mammography cases, the tumour was detected with ultrasound, (n=4),
MRI (n=2), by recall after breast tomosynthesis interpretation (n=1), and after
prophylactic mastectomy (n=1). Combining the results of mammography and
tomosynthesis detected 109 cancers. Therefore in 3 patients, both mammography
and tomosynthesis missed the carcinoma. The sensitivity of both techniques for the
detection of breast cancer was 93%, the specificity of mammography and tomosynthesis
was 86% and 84%, respectively. Tomosynthesis can therefore be used as an
additional technique to mammography in patients referred with an abnormal
screening mammogram or with clinical symptoms. Additional lesions detected by
tomosynthesis are, however, also likely to be detected by other techniques used in the
clinical workup of these patients.
Survey of uncertainites in image-guided navigation for breast cancer
localization Image-guided navigation on the basis of pre-treatment MRI in a highly
deformable organ, such as the breast, requires a survey of the factors that cause
uncertainties. A deformable breast-tissue-mimicking phantom with simulated
tumors was employed to investigate the accuracy of lesion localization with a needle
instrument coupled to an optical image-navigation system. The RMS deviation was
1.1 mm with errors ≤ 2.0 mm in 96% of the procedures. Ultrasonography data
acquired during needle localization of breast tumors were analyzed in 20 patients
(23 tumors; 12 benign, 11 malignant) to investigate the deformation due to presence
of instruments. The overall RMS tumor shift was 2.3 mm after release of pressure on
the needle. These results are encouraging to proceed with image-guided bracketing
of breast cancer for local surgical resection.
Radiofrequency ablation
Willem Prevoo, MP van den Munckhof, Wim Meinhardt, Simon Horenblas, MAAJ van den Bosch,
EM von Meyenfeldt, MW Wouters, Nathalie Lai A Fat, Sjaak Burgers, Johanna van Sandick,
Houke Klomp
Radiofrequency ablation (RFA) induces cancer cell death using electrical currents at
radio frequencies which produce frictional heat. The heat is conducted through a
needle, inserted into the tumor and connected to an RF generator.
At the NKI-AVL, this technique was only applied to liver metastases during open
surgical procedures. More recently, several advances have been made: 1) more
indications for RFA, 2) shift from open surgical procedures to minimally invasive
image-guided procedures. 3) improved needles to allow larger ablated regions, 4)
microwave ablation for larger regions.
The number of RFA procedures performed at the Radiology department of NKI-AVL
is increasing, and our institute is profiling itself in this field. Because the technique
is relatively new, research to establish the proper indications for RFA compared to
those for other treatment modalities is still ongoing. Long-term results for survival
are starting to come into focus. It is, however, still challenging to initiate randomized
clinical trials to compare RFA with other techniques. Hence, RFA is primarily
applied in patients for whom other forms of therapy are no longer an option.
The treatment of kidney tumors are, however, an exception. We performed an RFA
study (Prevoo et al, 2009) in mono kidneys with or without nephron-sparing therapy,
and in kidney tumors that were not eligible for kidney-sparing resection. The results
of this study are currently further analyzed together with results obtained at the
LUMC. We also performed a study to evaluate lung RFA of lung to target lung
metasteses (von Meyenfeldt et al.).
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DIAGNOSTIC ONCOLOGY
Publications (continued)
Van den Ouweland AM, Dinjens WN,
Dorssers LC, van Veghel-Plandsoen MM,
Brüggenwirth HT, Withagen-Hermans CJ,
Collée JM, Joosse SA, Terlouw-Kromosoeto JN,
Nederlof PM. Deletion of exons 1a-2 of
BRCA1: a rather frequent pathogenic
abnormality. Genet Test Mol Biomarkers.
2009;13:399-406
Van der Ploeg IM, Kroon BB, Antonini N,
Valdés Olmos RA, Nieweg OE.
Comparison of three micromorphometric
pathology classifications of melanoma
metastases in the sentinel node. Ann Surg.
2009;250:301-4
Van der Ploeg IM, Kroon BB, Antonini N,
Valdés Olmos RA, Nieweg OE. Is completion
lymph node dissection needed in case of
minimal melanoma metastasis in the sentinel
node? Ann Surg. 2009;249:1003-7
Van der Ploeg IM, Kroon BB, Valdés Olmos RA,
Nieweg OE. Evaluation of lymphatic drainage
patterns to the groin and implications for the
extent of groin dissection in melanoma patiënts.
Ann Surg Oncol. 2009;16:2994-9
Van der Ploeg IM, Nieweg OE, Kroon BB,
Rutgers EJ, Baas-Vrancken Peeters MJ,
Vogel WV, Hoefnagel CA, Valdés Olmos RA.
The yield of SPECT/CT for anatomical
lymphatic mapping in patients with breast
cancer. Eur J Nucl Med Mol Imaging.
2009;36:903-9
Van der Ploeg IM, Russell NS, Nieweg OE,
Oldenburg HS, Kroon BB, Olmos RA,
Rutgers EJ. Lymphatic drainage patterns in
breast cancer patients who previously
underwent mantle field radiation. Ann Surg
Oncol. 2009;16:2295-9
Van der Ploeg IM, Valdés Olmos RA,
Kroon BB, Rutgers EJ, Nieweg OE. The
hidden sentinel node and SPECT/CT in
breast cancer patients. Eur J Nucl Med Mol
Imaging. 2009;36:6-11
Van der Ploeg IM, Valdés Olmos RA,
Kroon BB, Wouters MW, Van den Brekel MW,
Vogel WV, Hoefnagel, Nieweg OE. The yield
of SPECT/CT for anatomical lymphatic
mapping in patients with melanoma. Ann
Surg Oncol. 2009;16:1537-42

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Publications (continued)
Van der Vegt B, de Bock GH, Bart J,
Zwartjes NG, Wesseling J. Validation of
the 4B5 rabbit monoclonal antibody in
determining Her2/neu status in breast
cancer. Mod Pathol. 2009;22:879-86
Van der Vegt B, de Bock GH, Hollema H,
Wesseling J. Microarray methods to
identify factors determining breast cancer
progression: potentials, limitations, and
challenges. Crit Rev Oncol Hematol.
2009;70:1-11
Van Laar RK, Ma XJ, de Jong D,
Wehkamp D, Floore AN, Warmoes MO,
Simon I, Wang W, Erlander M,
van ‘t Veer LJ, Glas AM. Implementation
of a novel microarray-based diagnostic
test for cancer of unknown primary.
Int J Cancer. 2009;125:1390-7
Van Leeuwen FW, Buckle T, Kersbergen A,
Rottenberg S, Gilhuijs KG. Noninvasive
functional imaging of P-glycoproteinmediated
doxorubicin resistance in a mouse
model of hereditary breast cancer to predict
response, and assign P-gp inhibitor
sensitivity. Eur J Nucl Med Mol Imaging.
2009;36:406-12
Van ‘t Veer MB, de Jong D, MacKenzie M,
Kluin-Nelemans HC, van Oers MH,
Zijlstra J, Hagenbeek A, van Putten WL.
High-dose Ara-C and beam with autograft
rescue in R-CHOP responsive mantle cell
lymphoma patients. Br J Haematol.
2009;144:524-30
Verduijn GM, Bartels LW, Raaijmakers CP,
Terhaard CH, Pameijer FA, van den Berg CA.
Magnetic resonance imaging protocol
optimization for delineation of gross tumor
volume in hypopharyngeal and laryngeal
tumors. Int J Radiat Oncol Biol Phys.
2009;74:630-6
Verhagen MMM, Abdo WF,
Willemsen MAAP, Hogervorst FBL,
Smeets DFCM, Hiel JAP, Brunt ER,
Van Rijn MA, Majoor Krakauer D,
Oldenburg RA, Broeks A, Last JI,
van ‘t Veer LJ, Tijssen MAJ, Dubois AMI,
Kremer HPH, Weemaes CMR,
Taylor AMR, Van Deuren M. Clinical
spectrum of ataxia-telangiectasia in
adulthood. Neurology 2009;73:430-7
Verhave G, Lettinga KD, van de Wiel BA,
Vasmel WL. Castleman’s disease, a fatal
cause of lymph node enlargement and
fever. Ned Tijdschr Geneeskd. 2009;153.
pii:A485
Verhoeff JJC, van Tellingen O, Claes A,
Stalpers LJA, van Linde ME, Richel DJ,
Leenders WPJ, van Furth WR. Concerns
about anti-angiogenic treatment in
patients with glioblastoma multiforme.
BMC Cancer 2009 (in press).
Vermeeren L, Klop WMC,
Van de Brekel MWM, Balm AJM,
Nieweg OE, Valdés Olmos RA.
Sentinel node detection in head and neck
malignancies: innovations in radioguided
surgery. J Oncol 2009 (in press)
Vermeeren L, Meinhardt W,
Valdés Olmos RA. Prostatic lymphatic
drainage with sentinel nodes near the
ventral abdominal wall visualized with
SPECT-CT: a case series. Clin Nucl Med
2009 (in press)
Vermeeren L, Valdés Olmos RA,
Meinhardt W, Bex A, Van der Poel HG,
Vogel WV, Sivro F, Hoefnagel CA,
Horenblas S. Value of SPECT-CT for
detection and anatomic localization of
sentinel lymph nodes before laparoscopic
sentinel node lymphadenectomy in
prostate carcinoma. J Nucl Med.
2009;50:865-70
Vermeeren L, Valdés Olmos RA,
Meinhardt W, Bex A, Van der Poel HG,
Vogel WV, Sivro F, Hoefnagel CA,
Horenblas S. Intraoperative
radioguidance with a portable gamma
camera: a novel technique for
laparascopic sentinel node technique for
laparoscopic sentinel node localization in
urological malagnancies. Eur J Nucl Med
Mol Imaging. 2009;36:1029-36
Vermeeren L, Van der Ploeg IMC,
Valdés Olmos RA, Meinhardt W,
Klop WMC, Kroon BBR, Nieweg OE.
SPECT/CT for preoperative sentinel
node localization. J Surg Oncol 2009
(in press)
Vermeeren L, Tanis P.J, Nieweg O.E,
Valdés Olmos R.A. Lymphoscintigraphy
of a breast tumor showing focal tracer
accumulation along the falciform
ligament of the liver. Clin Nucl Med
2009 (in press)
Vogel WV, Verheij M, Teertstra HJ.
Hercules op het kruispunt. Tijdsch Nuc
Geneesk 2009;31:326-8
Von Meyenfeldt EM, Wouters MW,
Lai A Fat N, Prevoo W, Burgers JA van
Sandick JW, Klomp HM, Local
Treatment of pulmonary metastases: from
open resection to minimaly invasive
approach? Submitted Eur J of Onc
Surgery
Vrieling A, Voskuil DW, Bosma A,
Majoor DM, van Doorn J, Cats A,
Depla AC, Timmer R, Witteman BJ,
Wesseling J, Kampman E, Van ‘t Veer LJ.
Expression of insulin-like growth factor
system components in colorectal tissue
and its relation with serum IGF levels.
Growth Horm IGF Res. 2009;19:126-35
Wildeman MA, Gibcus JH, Hauptmann M,
Begg AC, van Velthuysen ML, Hoebers FJ,
Mastik MF, Schuuring E, van der Wal JE,
van den Brekel MW. Radiotherapy in
laryngeal carcinoma: can a panel of
13 markers predict response?
Laryngoscope. 2009;119:316-22
Yang TI, Aukema TS, Van Tinteren H,
Burgers S, Valdés Olmos RA, Verhey M.
Predicting Early Chemotherapy
Response with Technetium-99m
Methoxyisobutylisonitrile SPECT/CT in
Advanced Non-Small Cell Lung Cancer.
Mol Imaging Biol. 2009
Zuur JK, Muller SH, Vincent A,
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The influence of a heat and moisture
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environment. Med Eng Phys.
2009;31:852-7

diVision of mediCal onCology
CliniCal pharmaCology of antiCanCer drugs
Jan schellens, Jos Beijnen, Henk Boot, Annemieke Cats, Alwin Huitema, Serena Marchetti,
Bastiaan Nuijen, Hilde Rosing, Neeltje Steeghs
Background and objectives The division of Clinical Pharmacology is involved in
research in different phases of anticancer drug development concerning: 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 2009 we continued clinical research fully compliant with ICH-GCP guidelines,
previously certified by the Dutch Ministry of Health. The department of Pharmacy &
Pharmacology successfully continued its official governmental GLP (in the field of
analytical chemistry), GMP (formulation and manufacturing of investigational cytotoxic
drugs) and GDP (ISO9002 for worldwide distribution of clinical supplies) licenses.
research performed in the year 2009
I Pharmaceutical formulation
Formulation research and clinical manufacturing of the anti-metastatic Rutheniumcontaining
experimental anticancer agent NAMI-A was successful and resulted in a
suitable pharmaceutical formulation and manufacturing of the first batches for
clinical trial use.
Clinical manufacturing (Thalidomide tablets) of oral formulations of anticancer
agents continued. A pharmaceutical development program of oral taxanes resulted in
a first formulation composed of a solid dispersion of docetaxel that entered the clinic
(ModraDoc). A similar formulation of paclitaxel (ModraPac001) was developed and is
clinically tested. Additionally, formulation research was started to develop a
controlled-release dosage form of capecitabine.
A first formulation project aimed at the GMP-manufacture of ready-to-label
molecular imaging agents is started with bevacizumab, which is now available as an
on-the-shelf product for imaging studies.
The Amsterdam BioTherapeutics Unit (AmBTU) is fully operational for the
manufacture of GMP-grade DNA-plasmid. A lyophilized pharmaceutical formulation
of pDERMATT (plasmid DNA Encoding Recombinant Mart-1 and Tetanus Toxin
fragment-c) for the intradermal administration was developed for a phase I trial,
which started in our institute. The next project, the pharmaceutical development and
manufacture of HPV E6 and E7 pDNA vaccines for clinical trial use is initiated.
Clean room facilities are in place to enable GMP-preparation of T-lymphocytes for
TIL and T-cell receptor gene therapy, for which the development of the preparation
processes is ongoing. 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 and implementation in PK studies
In 2009, our Bioanalytical Laboratory extended their triple quadrupole mass
spectrometry (MS) equipment with the purchase of a highly sensitive API4000
(Applied Biosystems). The instrument was validated and new methods for the
quantification of AS703026, an inhibitor of MEK 1 and MEK2 were developed.
Carbon-14 labelled samples obtained from our three mass balance studies (E7389,
E7080 and bendamustine) are being analyzed. E7389 is a synthetic analog of
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
Bastiaan nuyen phd Academic staff
sjoerd rodenhuis md phd Head
hilde rosing phd Academic staff
Jan schellens md phd Academic staff
marianne smits md phd Academic staff
gabe sonke md 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
sandra Bakker Temporary staff
marja Bonarius Temporary staff
melanie Bos Temporary staff
rogier Boshuizen Temporary staff
emile Kerver md Temporary staff
peter Kunst md phd Temporary staff
alet mager Temporary staff
serena marchetti Temporary staff
Boelo poppema Temporary staff
Quirine van rossum-schornagel
Temporary staff
neeltje steeghs md phd Temporary staff
Catrien tromp-van driel Temporary staff
suzan Vrijaldenhoven Temporary staff
Karin Beelen PhD student
david Boss PhD student
nienke Brink-de Vries PhD student
Carola damen PhD student
maarten deenen PhD student
lot devriese PhD student
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mediCal onCology

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mediCal onCology
thomas dorlo PhD student
anne Charlotte dubbelman PhD student
Corinne ekhart PhD student
geert frederikx PhD student
marie-Christine gast PhD student
andrew goey PhD student
Claudia heijens PhD student
helgi helgason PhD student
robert Jansen PhD student
tine Janssens PhD student
eefje Jong PhD student
ron Keizer PhD student
heinz Josef Klümpen PhD student
stijn Koolen PhD student
Jurjen lagas PhD student
nienke lankheet PhD student
Jelte meulenaar PhD student
Johannes moes PhD student
ruud van de noll PhD student
roos oostendorp PhD student
susanne Quaak PhD student
rick stuurman PhD student
rob ter heine PhD student
Bas teunissen PhD student
linda tulner PhD student
ly tran 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
robert Van Waterschoot 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 roelvink Clinical trial manager
sandra adriaansz Nurse practitioner
roel Blanken Nurse practitioner i.o.
annelies Boekhout Nurse practitioner
Karina Bucholtz Nurse practitioner i.o.
ria dubbelman Nurse practitioner
Joke foekema Nurse practitioner
emmy harms Nurse practitioner
marjo holtkamp Nurse practitioner
marianne Keessen Nurse practitioner
annemieke Koenen Nurse practitioner i.o.
maria Kuiper Nurse practitioner
henk mallo Nurse practitioner
annemarie nol Nurse practitioner
albert olijve Nurse practitioner
suzanne onderwater Nurse practitioner
margaret schot Nurse practitioner
Wilma uyterlinde Nurse practitioner
Jana Van der sar Nurse practitioner
dick Visser Nurse practitioner i.o.
Halichondrin B (HalB), a natural product, acting by interfering with mitotic spindle
formation. Assays have been validated in whole blood, plasma, urine, and faeces for
the quantification of the parent drug. E7389 was slowly metabolised and no major
metabolites were formed. Several minor monooxygenated metabolites were detected
using our novel linear ion trap LTQ XL MS instrument.
E7080 is an experimental tyrosine kinase inhibitor and bioanalytical assays were set
up for the quantification of parent drug and its four metabolites to support the mass
balance study. Excretion data reveal that around 25% and 64% of the total radioactivity
were found in urine and faeces, respectively. Using LC-MS/MS, low concentrations of
unchanged drug were found in these matrices, indicating that the drug is highly
metabolised.
For bendamustine a bifunctional mechlorethamine alkylating drug and metabolite
analysis of mass balance study samples is ongoing employing MSn.
A method for the simultaneous quantification of emtricitabine and tenofovir
nucleotides in PBMCs using weak anion-exchange liquid chromatography coupled
with tandem mass spectrometry has been developed and validated.
Sample analysis were performed for clinical trials within and outside the Institute
concerning topotecan, irrinotecan and its active metabolite SN-38, trabectedin,
indibulin, tamoxifen, gemcitabine and dFdU, gemcitabine triphosphate, capecitabine
and its metabolites, and cyclophosphamide in combination with fludarabine.
The determination of platinum, originating from cisplatin, carboplatin and oxaliplatin,
in plasma ultrafiltrate using ICP-MS, can now be performed in a GLP environment,
and first study samples have been received for analysis.
For trastuzumab, a humanized monoclonal antibody (MAb), an assay has been
developed to quantify the drug in human serum by precipitate-enhanced ellipsometry
(PEIA). Sensitivity was improved when compared to the same assay in enzyme-linked
immunosorbent assays (ELISA) format.
In September 2009 our laboratory was inspected by the Dutch Authorities for
compliance with Good Laboratory Practice (GLP). The audit was successful and the
endorsement of compliance in analytical chemistry testing was obtained.
III Early clinical phase I/II studies
III-1 Novel approaches to improve oral bioavailability
We have made significant steps forward in the development of an oral treatment
strategy with docetaxel. In combination with the ‘boosting’ agent ritonavir, an
efficacious inhibitor of gut wall and hepatic CYP3A4, we tested an oral capsule
formulation of docetaxel, denoted ModraDoc001, in patients administered together
with ritonavir (figure 1). The systemic exposure to oral docetaxel was on average 75%
compared with the systemic exposure after intake of the drinking solution of
docetaxel applied previously. However, the interpatient variation of systemic exposure
to docetaxel was substantially lower after intake of the capsule. In addition, the
intrapatient variability in systemic exposure to docetaxel after intake of ModraDoc001
Figure 1: Pharmacokinetics of the novel drug formulation ModraDoc.

plus ritonavir was low. This represents an excellent basis for further clinical
development of this concept. Other boosting agents have been tested in combination
with ModraDoc001, especially ketoconazole, clarithromycin and grapefruit juice. All
these inhibitors enhanced systemic exposure to oral docetaxel. Ritonavir was selected
as the best booster drug as it resulted in the highest systemic exposure to coadministered
oral docetaxel and the lowest Cmax value. The novel oral paclitaxel
capsule formulation (ModraPac001) showed excellent systemic exposure to paclitaxel
and has therefore been taken further into the clinic.
III-2 Pharmacology (PK/PD, ADME, mass balance) of novel anticancer drugs
Currently, we perform twenty-nine phase I/II, pharmacological and proof of concept
studies, which number has increased compared with last year. We recruited over
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 AZD2281
We continued the 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. We demonstrated promising
anticancer activity in a range of tumor types, but especially patients with tumors
harboring BRCA1/2 mutations benefited most. Prolonged myelosuppression is the
main toxicity of the combination.
b. Phase I study with oral gemcitabine prodrug
The phase I study with the oral gemcitabine produg was finished. As deamination of
oral gemcitabine presystemically was encountered in our previous phase I study with
oral gemcitabine, a produg was developed in which the amino group of gemcitabine
is protected from deamination by the linker valproic acid. Clinical and laboratory
studies with this novel prodrug formulation of gemcitabine revealed relevant systemic
exposure to gemcitabine after oral administration of the prodrug. We embarked on a
new phase I study with another gemcitabine prodrug (CP4126—111), in which a C18
group is attached at the 5 position of the sugar moiety of the nucleoside molecule.
Pharmacokinetic results are promising.
c. Studies with angiogenesis inhibitors
Two phase I studies with the orally available angiogenesis inhibitors E7080 and Bay
57-9352 were finished. In dose-rising studies E7080 was applied as single agent and
Bay 57-9352 in combination with irinotecan and capecitabine. Hypertension and
proteinuria were main toxicities of these agents. We continued the trial with the
angiogenesis agent pazopanib given in combination with topotecan. In heavily
pretreated patients with advanced mesothelioma, melanoma, rectal cancer,
osteosarcoma, soft tissue sarcoma and head and neck cancer significant antitumor
activity including long lasting partial remissions was noted in all three studies.
d. Other phase I studies
This year we started new first-in-man phase I studies, including the novel bFGF
receptor inhibitor AZ4547. This is an orally available drug given on a continuous
daily schedule. We also started the first-in-man study with the oral MEK inhibitor
(BO21189). Preliminary results indicate promising pharmacokinetics. Main toxicity is
EGFR-inhibitor-like skin toxicity. Furthermore, we started the phase I study of the
combination of erlotinib and the PI3Kinase inhibitor GDC0941. Main toxicity is skin
toxicity. This study is especially of interest for patients progressing but previously
responding to erlotinib.
III-3 Pharmacokinetic and pharmacodynamic modelling and simulation
Pharmacokinetic and pharmacodynamic (PK/PD) modelling and simulation was used
to develop a (semi)-mechanistic model relating drug exposure of the experimental
VEGFR inhibitor E7080 to changes in blood pressure and proteinuria. The model
consisted of an indirect effect model for hypertension and a Markov model describing
proteinuria and was used to study the optimal management of these dose-limiting
toxicities in order to optimize treatment with this compound. Further studies are in
publications
121
mediCal onCology
Amant F, Van Calsteren K, Halaska MJ,
Beijnen JH, Lagae L, Hanssens M,
Heyns L, Lannoo L, Ottevanger NP,
Vanden Bogaert W, Ungar L, Vergote I,
du Bois A. Gynecologic cancers in
pregnancy: guidelines of an international
consensus meeting. Int J Gynecol Cancer
2009;19(Suppl 1):S1-12
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. 2009 (in press)
Aukema TS, Valdés Olmos RA, Klomp HM,
Teertstra HJ, Belderbos JS, Vogel WV,
Baas P, Burgers SA, van den Heuvel MM.
Evaluation of 18F-FDG PET-CT for
Differentiation of Pulmonary Pathology
in an Approach of Outpatient Fast Track
Assessment. J Thorac Oncol. 2009
(in press)
Aukema TS, Teunissen JJ, Burgers SA,
van Pel R, Vogel WV. Extensive soft-tissue
metastases from malignant pleural
mesothelioma. J Clin Oncol. 2009;27:e24-5
Baas P. Eds: Pass HI, Carbone DP,
Johnson DH, Mina JD, Turrisi AT,
Scagliotti GV. Management of
mesothelioma. Principles and Practice of
Lung Cancer, 4e. 2009
Bendle GM, Haanen JB, Schumacher TN.
Preclinical development of T cell receptor
gene therapy. Curr Opin Immunol.
2009;21:209-14
Van den Berg JH, Nujien B, Beijnen JH,
Vincent A, van Tinteren H, Kluge J,
Woerdeman LA, Hennink WE, Storm G,
Schumacher TN, Haanen JB.
Optimization of intradermal vaccination
by DNA tattooing in human skin. Hum
Gene Ther. 2009;20:181-9
Van den Berg JH, Nuijen B,
Schumacher TN, Haanen JB, Storm G,
Beijnen JH, Hennink WE. Synthetic
vehicles for DNA vaccination. J Drug
Target. 2009

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mediCal onCology
publications (continued)
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.
2009
Van den Berg JH, Quaak SG, Beijnen JH,
Hennink WE, Storm G, Schumacher TN,
Haanen JB, Nuijen B. Lipopolysaccharide
contamination in intradermal DNA
vaccination: Toxic impurity or adjuvant?
Int J Pharm. 2009
Beumer JH, Lopez-Lazaro L, Schellens JH,
Beijnen JH, van Tellingen O. Evaluation of
Human Plasma Protein Binding of
Trabectedin (Yondelistrade mark, ET-743).
Curr Clin Pharmacol 2009;4:38-42
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
2009 (in press)
Bex A, van der Veldt AA, Blank C,
van den Eertwegh AJ, Boven E, Horenblas S,
Haanen J; Neo-adjuvant sunitinib for
surgically complex advanced renal cell
cancer of doubtful resectability: initial
experience with downsizing to reconsider
cytoreductive surgery. World J Urol.
2009;27:533-9
Boparai KS, Mathus-Vliegen EMH,
Koornstra JJ, Nagengast FM, van Leerdam M,
van Noesel CJM, Houben M, Cats A,
van Hest L, Fockens P, Dekker E;
Increased colorectal cancer risk during
follow-up in patients with hyperplastic
polyposis syndrome: a multicentre cohort
study. Gut 2009 (in press)
Borst GR, Belderbos J, Burgers S, Sonke JJ,
Lebesque J. Re: Dyspnea evolution after
high-dose radiotherapy in patients with
non-small cell lung cancer. Radiother
Oncol. 2009;91:461;author reply 461-2
progress to investigate the applicability of this model for other VEGF inhibitors.
A two-stage PK/PD model based dose escalation strategy for cytotoxic drugs has been
developed and retrospectively validated using data from phase I studies. Furthermore,
the integration of preclinical data in PK/PD models to support early clinical trials has
been explored.
III-4 Pharmacogenetic screening of patients treated with 5-FU/Capecitabine
DYPD*2 genotyping in routine clinical practice was continued. To date more than
1000 patients have been genotyped prior to start of capecitabine therapy, amongst
which fourteen heterozygotes were identified. Dose-adaptation could successfully be
performed in these patients. Currently, we perform a cost-effectiveness analysis of
this genotyping approach.
IV Supportive care studies in patients with breast cancer
We finished recruitment of the planned 100 patients in the intervention study in
female patients with menopausal complaints induced by treatment for breast cancer
with placebo, venlafaxine and clonidine hydrochloride. The primary endpoint is
reduction in intensity and number of flushes.
We continued the multicenter double blind randomized placebo-controlled cardiac
protection trial with the angiotensine II receptor antagonist candesartan in patients
with Her2-positive breast cancer who receive adjuvant treatment with trastuzumab.
The number of study centers has increased from 18 last year to 21 this year and in
total 115 of in total 200 planned patients have been registered. The recruitment rate
has significantly increased over the last 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 over 130 patients are available for this analysis. Left ventricular
ejection fraction data show that about 20% of the patients who started with normal
LVEF (>50%) drop to values < 50% with 15% reduction in LVEF. This confirms that
trastuzumab induces significant cardiac toxicity.
V Collaboration with the Dutch Medicines Evaluation Board
We prepared a scientific report as European (co-)rapporteur for Opaxio (paclitaxel
poliglumex) in NSCLC, Zactima (vandetanib) in NSCLC, Docefrez (generic
docetaxel), Teva (generic oxaliplatin), Bosatria (mepolizumab), Clofarabine (evoltra),
Tarceva (Type II variation in NSCLC, i.e. expansion of the labelled indication), Erbitux
(Type II variation in NSCLC), Pemetrexed (Type II variation in NSCLC) and Fastuterc
(Resburicase).
We continued active support of the Scientific Advisory Group Oncology (SAG-O) of
the EMEA as vice-chairperson. The working relationship with the MEB has expanded
by appointment of a second medical oncologist.
We continued the scientific advices to the minister of Health and the Dutch Health
Insurance Authority (NZA) as chairperson of the Committee for Pharmaceutical Help.
CliniCal immunotherapy and targeted therapy
John haanen, Christian Blank, Sandra Adriaansz, Willem Boogerd, Henk Mallo
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-infiltrating lymphocytes (TIL) for melanoma and DNA and
peptide vaccination studies for HPV-related squamous cell cancers. For renal cell cancer
our group invests 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 anti-angiogenesis drugs.
melanoma
Translational research with DNA vaccination in melanoma patients:
In January 2009 we started a first in man study with DNA tattoo vaccination. In this
dose-escalating phase I clinical study advanced-stage HLA-A*0201-posiitve 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 × 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 2009 the first dose cohort
(4 cm 2 ) with three patients was finished and the first patient in the second dose
cohort was treated as well. 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-specific
T cells, indicating that a cellular immune response had been induced. We are
awaiting the results from peripheral blood samples (both MART-specific and TTFCspecific
responses).
Translational research with tumor infiltrating lymphocytes in melanoma patients:
We are preparing for a clinical phase II study using tumor-infiltrating 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 pretreated stage IV melanoma
patients. Recently, these impressive response rates were confirmed 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 pretreated stage IV melanoma
patients. Ours will be the first trial in Europe and 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. We expect to enroll patients in this study in 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 affinity 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 2009,
still 4 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 define the optimal time point for tumor
nephrectomy in primary metastatic renal cell carcinoma patients is ongoing. A total
of 17 patients have been treated in this small proof of principle trial. The idea of
defining the optimal timing for nephrectomy in primary metastatic RCC was adopted
by the EORTC GU group and has now been approved as a randomized controlled
multicenter phase III trial that will start enrollment 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 investigator initiated study 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.
123
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publications (continued)
Borst GR, Ishikawa M, Nijkamp J,
Hauptmann M, Shirato H, Onimaru R,
van den Heuvel MM, Belderbos J,
Lebesque JV, Sonke JJ. Radiation
pneumonitis in patients treated for
malignant pulmonary lesions with
hypofractionated radiation therapy.
Radiother Oncol. 2009;91:307-13
Boss DS, Siegel-Lakhai WS,
van Egmond-Schoemaker NE, Pluim D,
Rosing H, Ten Bokkel Huinink WW,
Beijnen JH, Schellens JH. Phase I
pharmacokinetic and pharmacodynamic
study of Carboplatin and topotecan
administered intravenously every 28 days to
patients with malignant solid tumors. Clin
Cancer Res 2009;15:4475-83
Boss DS, Beijnen JH, Schellens JH.
Clinical experience with aurora kinase
inhibitors: a review. Oncologist
2009;14:780-93
Boss DS, Schwartz GK, Middleton MR,
Amakye DD, Swaisland H, Midgley RS,
Ranson M, Danson S, Calvert H,
Plummer R, Morris C, Carvajal RD,
Chirieac LR, Schellens JH, Shapiro GI.
Safety, tolerability, pharmacokinetics and
pharmacodynamics of the oral cyclindependent
kinase inhibitor AZD5438
when administered at intermittent and
continuous dosing schedules in patients
with advanced solid tumours. Ann Oncol
2009 (in press)
Brouwers EE, Söhne M, Kuipers S,
van Gorp EC, Schellens JH, Koks CH,
Beijnen JH, Huitema AD. Ciprofloxacin
strongly inhibits clozapine metabolism:
two case reports. Clin Drug Investig
2009;29:59-63
Brouwers EE, Huitema AD, Boogerd W,
Beijnen JH, Schellens JH. Persistent
neuropathy after treatment with cisplatin
and oxaliplatin. Acta Oncol 2009;48:832-41
De Bruin ML, Burgers JA, Baas P,
van ‘t Veer MB, Noordijk EM,
Louwman MW, Zijlstra JM,
van den Berg H, Aleman BM,
van Leeuwen FE. Malignant mesothelioma
after radiation treatment for Hodgkin
lymphoma. Blood. 2009;113:3679-81

124
mediCal onCology
publications (continued)
Bueno-de-Mesquita JM, Sonke GS,
van de Vijver MJ, Linn SC. Additional
value and potential use of the 70-gene
prognosis signature in node-negative breast
cancer in daily clinical practice. Ann
Oncol. 2009 (in press)
Bueno-de-Mesquita JM, Nuyten DS,
Wesseling J, van TH, Linn SC,
van de Vijver MJ. The impact of interobserver
variation in pathological
assessment of node-negative breast cancer
on clinical risk assessment and patient
selection for adjuvant systemic treatment.
Ann Oncol. 2009
Courrech Staal EF, van Coevorden F,
Cats A, Aleman BM, van Velthuysen ML,
Boot H, Vrancken Peeters MJ,
van Sandick JW. Outcome of low volume
esophageal cancer surgery in a high-volume
referral center. Ann Surg Oncol. 2009
Damen CW, Israëls T, Caron HN,
Schellens JH, Rosing H, Beijnen JH.
Validated assay for the simultaneous
quantification of total vincristine and
actinomycin-D concentrations in human
EDTA plasma and of vincristine
concentrations in human plasma
ultrafiltrate by high performance liquid
chromatography coupled with tandem
mass spectrometry. Rapid Commun
Mass Spectrom 2009;23:763-74
Damen CW, Rosing H, Schellens JH,
Beijnen JH. Application of dried blood spots
combined with high-performance liquid
chromatography coupled with electrospray
ionisation tandem mass spectrometry for
simultaneous quantification of vincristine
and actinomycin-D. Anal Bioanal Chem
2009;394:1171-82
Damen CW, Lagas JS, Rosing H,
Schellens JH, Beijnen JH. The bioanalysis
of vinorelbine and 4-O- deacetylvinorelbine
in human and mouse plasma using highperformance
liquid chromatography
coupled with heated electrospray ionization
tandem mass-spectrometry. Biomed.
Chromatogr 2009;23:1316-25
Damen CW, Derissen EJ, Schellens JH,
Rosing H, Beijnen JH. The bioanalysis of
the monoclonal antibody trastuzumab by
high-performance liquid chromatography
with fluorescence detection after immunoaffinity
purification from human serum.
J Pharm Biomed Anal 2009;50:861-66
Breast CanCer therapy and response prediCtion
sabine linn, sjoerd rodenhuis, gabe sonke, Jos Beijnen, Jorma De Ronde, Marjo Holtkamp,
Alwin Huitema, Esther Lips, Ingrid Mandjes, Lennart Mulder, Paula Nederlof, Margaret Schot,
Laura Van ‘t Veer, Marieke Vollebergh, Jelle Wesseling, Lodewyk Wessels
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 2009, about 175 patients were entered in ten ongoing (neo)adjuvant
medical studies in breast cancer, and in five studies in advanced disease.
preoperative chemotherapy The preoperative chemotherapy program continues
to accrue over 80 patients per year and now includes data and tissues of some 400
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 amplification). In some of these
patients excellent partial remissions can be obtained, in others there is hardly any
response. For this situation, a simple method to quantify the response has been
developed: the neoadjuvant response index (NRI). Highly endocrine responsive
tumors have lower NRI values after chemotherapy than others. Only few of the breast
tumors selected for neoadjuvant chemotherapy have a favorable 70-gene expression
signature. In a study of 167 of such patients this was the case in only 14% and not a
single one of these achieved a pCR. Luminal tumors with an unfavorable signature
did much better. A further study compared the clinical utility of determining the
intrinsic (molecular) subtype of breast cancer in the adjuvant setting employing
microarray technology. For most tumors, the molecular classification added little to
standard immunohistochemistry subtyping. There may, however, exist a subgroup of
tumors that contains a HER2 amplification but is nevertheless of the luminal type
according to microarray analysis. This group may respond poorly to trastuzumabbased
neoadjuvant chemotherapy.
A multicenter phase II study has been initiated with paclitaxel, carboplatin and
trastuzumab to assess the pCR rate in HER2-positive, stage II/III breast cancer
patients (NCT00768859).
adjuvant systemic therapy, prognosis and prediction The primary objective of
the Matador study is to identify predictive gene expression profiles for a disease-free
survival benefit of either a docetaxel-containing regimen (docetaxel-doxorubicincyclophosphamide
(TAC)) or an accelerated doxorubicin-cyclophophamide (ACdd)
regimen (ISRCTN61893718). The study, open since 2004, is running in over
30 centers in the Netherlands and ~360 patients have been included. Safety data of
the first 200 patients revealed that both regimens were generally well tolerated.
Significantly more patients suffered from vomiting in the ACdd arm, probably due to
less dexamethasone use. Significantly more patients in the TAC arm developed
edema, as was expected. Neutropenic fever occurred in 7% in the ACdd arm, and
14% in the TAC arm.
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 will be extended to other centers
(NCT00738777). Blood and tumor tissue is collected pre and post treatment for
translational research, including gene expression profiling. 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.
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
benefit of three years adjuvant oral ibandronate in addition to standard adjuvant
systemic therapy in postmenopausal patients with hormone-receptor positive early
breast cancer (currently ~325 patients included) (ISRCTN17633610).

thoraCiC onCology
paul Baas, sjaak Burgers, Wieneke Buikhuisen, michel van den heuvel, Jose Belderbos,
Houke Klomp, Peter Kunst, Petra Nederlof, Michel Wouters, Jan Schellens
The department’s clinical research focuses on immunological studies, combined
modality approaches and mesothelioma treatment.
non-small Cell lung Cancer (nsClC) Patients with early stage NSCLC and who
are candidates for surgical resection are included in a neo-adjuvant study with erlotinib
given for 3 weeks. This study is led by the department of thoracic surgery (Houke
Klomp) and pathological examination of the specimen indicate that this short course
of chemotherapy already induces significant apoptosis.
In patients with advanced NSCLC, a pharmacogenomics study has now included
100 patients who have received cisplatin and pemetrexed. The correlation between
metabolism of the two drugs and the presence of Single Nucleotide Polymorphisms
(SNP’s) has been performed and published. Currently, correlative studies on available
tumor material are being planned and will be compared to data obtained from the
cisplatin gemcitabine study (Jan Schellens).
Two vaccination studies are in progress for patients with advanced stages of NSCLC:
A phase IB study on maintenance therapy with Selectikine after a short course of
radiotherapy; a phase II randomized study of Stimuvax with or without radiotherapy
in HLA-A2 positive patients. Both studies build on the postulated synergistic effects
of combined RT and immunotherapy. In addition, patients will be enrolled in a
phase III vaccination study with Lucanix.
In second line NSCLC, a Dutch randomized study of erlotinib vs. erlotinib plus
chemotherapy is ongoing. We will perform additional pharmaco-kinetic and genomic
studies.
small Cell lung Cancer (sClC) In patients with Limited Stage disease, we participate
in the European phase III CONVERT study, which investigates the effects of
concurrent twice-daily radiotherapy and chemotherapy vs. sequential chemotherapy
and radiotherapy. For patients with extensive stage disease, a phase II study with
Sunitinib induction therapy has started. In the first six weeks after registration single
agent Sunitinib is given and patients are closely monitored for response/progression
with PET-CT scan. In second line, we participate in a Dutch study of amrubicine vs.
topotecan in patients with WHO performance score ≤2.
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, who are candidates for drainage are asked to participate and
to consent to biomarker research emplying both frozen serum and pleural fluid
samples. Currently we have patient data and matched samples in over 400 patients.
malignant pleural mesothelioma (mpm) The national randomized phase III
maintenance study of thalidomide after standard chemotherapy in MPM has now
completed accrual. 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 planned for 2010.
The results of the EORTC feasibility phase II study 08931 tested the toxicity of
induction chemotherapy followed by extrapleural pneumonectomy and radiation
have been presented at ASCO and WCLC.
For second and third line therapy we offer patients a randomized phase III study of
Vorinostat vs. placebo. Of the 660 planned patients we included over 40 patients.
We have started a randomized phaseI/II study examining the effect of Axitinib
(a potent oral anti vascular drug) to standard chemotherapy in patients with MPM.
Biopsies and dynamic MRI examination before and after 3 courses of chemotherapy
will be performed to evaluate the effect of the addition of Axitinib. Vascular staining,
apoptosis and circulating endothelial/tumor cells will be measured, in addition to
vascular flow analysis by MRI (figure 2).
125
mediCal onCology
publications (continued)
Damen CW, de Groot ER, Heij M,
Boss DS, Schellens JH, Rosing H,
Beijnen JH, Aarden LA. Development and
validation of an enzyme-linked
immunosorbent assay for the quantification
of trastuzumab in human serum and
plasma. Anal Biochem 2009;391:114-20
Damen CW, Rosing H, Schellens JH,
Beijnen JH. High-performance
liquidchromatography coupled with mass
spectrometry for the quantitative analysis of
vinca-alkaloids in biological matrices: a
concise survey from the literature. Biomed
Chromatogr 2009 (in press)
Damen CW, Speijer H, Hermens WT,
Schellens JH, Rosing H, Beijnen JH. The
bioanalysis of trastuzumab in human
serum using precipitate-enhanced
ellipsometry. Anal Biochem 2009;393:73-79
Damen CW, Chen W, Chakraborty AB,
van Oosterhout M, Mazzeo JR, Gebler JC,
Schellens JH, Rosing H, Beijnen JH.
Electrospray Ionization Quadrupole Ion-
Mobility Time-of-Flight Mass Spectrometry
as a Tool to Distinguish the Lot-to-Lot
Heterogeneity in N-Glycosylation Profile of
the Therapeutic Monoclonal Antibody
Trastuzumab. J Am Soc Mass Spectrom
2009;20:2021-33
Damen CW, Schellens JH, Beijnen JH.
Bioanalytical methods for the quantification
of therapeutic monoclonal antibodies and
their application in clinical pharmacokinetic
studies. Hum Antibodies 2009;18:47-73
Desar IM, Burger DM, Van Hoesel QG,
Beijnen JH, Van Herpen CM,
Van der Graaf WT. Pharmacokinetics of
sunitinib in an obese patient with a GIST.
Ann Oncol 2009;20:599-600
Djoba Siawaya JF, Chegou NN,
van den Heuvel MM, Diacon AH,
Beyers N, van Helden P, Walzl G.
Differential cytokine/chemokines and KL-6
profiles in patients with different forms of
tuberculosis. Cytokine. 2009;47:132-6
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
2009

126
mediCal onCology
publications (continued)
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. 2009
Ekhart C, Kerst JM, Rodenhuis S,
Beijnen JH, Huitema AD. Altered
cyclophosphamide and thiotepa
pharmacokinetics in a patient with
moderate renal insufficiency; Cancer
Chemother. Pharmacol. 2009;63:375-379
Ekhart C, Rodenhuis S, Beijnen JH,
Huitema AD. Pharmacokinetics of
cyclophosphamide and thiotepa in a
conventional fractionated high-dose
regimen compared with a novel simplified
unfractionated regimen. Ther. Drug.
Monit; 2009;31:95-103
Ekhart C, Doodeman VD, Rodenhuis S,
Smits PH, Beijnen JH, Huitema AD.
Polymorphisms of drug-metabolizing
enzymes (GST, CYP2B6 and CYP3A)
affect the pharmacokinetics of thiotepa
and tepa. Br. J. Clin. Pharmacol.
2009;67:50-60
Ekhart C, Rodenhuis S, Smits PH,
Beijnen JH, Huitema AD. Carboplatin
dosing in overweight and obese patients
with normal renal function, does weight
matter? Cancer Chemother. Pharmacol.
2009;64:115–122
Ekhart C, Rodenhuis S, Smits PH,
Beijnen JH, Huitema AD. An overview of
the relations between polymorphisms in
drug metabolising enzymes and drug
transporters and survival after cancer drug
treatment. Cancer Treat. Rev. 2009;35:18-31
Ekhart C, Rodenhuis S, Beijnen JH,
Huitema AD. Carbamazepine induces
bioactivation of cyclophosphamide and
thiotepa. Cancer Chemother. Pharmacol.
2009;63:543-547
Engwegen JY, Mehra N, Haanen JB,
Schellens JH, Voest EE, Beijnen JH.
Identification of two new serum protein
profiles for renal cell carcinoma.
Oncol Rep. 2009;22:401-8
gastroenterology
Figure 2: Staining procedure of
endothelial cells with arrow heads
indicating proliferation.
henk Boot, annemieke Cats, marianne smits, Babs taal, Hans Bonfrer, Maarten Deenen, Luc Dewit,
Edwin Jansen, Tiny Korse, Jan Schellens, Maurits Swellengrebel, Marcel Verheij
neuroendocrine tumours (net) NET are well-known for the production of various
vasoactive peptides in particular serotonin, which is measured as the degradation
product 5-HIAA in the urine. In our study of 39 patients with metastatic NET the
new marker chromogranin A (CgA) showed a significant association with Quality of
Life, physical functioning and survival and is therefore recommended over the 5-HIAA.
In a longitudinal series we found the natriuretic markers ANP and especially BNP a
fair marker to detect carcinoid heart disease (CHD: fibrosis of the tricuspid heart
valve). In a larger study of 102 patients with 15% CHD we found that pro-BNP and
CgA to be independent prognostic variables for overall survival.
gastric cancer An international phase III multicenter study (CRITICS; NCT
00407186) has currently accrued nearly 200 of the 788 patients aimed for. The study
investigates whether postoperative chemoradiotherapy (CRT) with cisplatin and
capecitabine in resectable gastric cancer patients pre-operatively treated with
combination chemotherapy (epirubicine, cisplatin, capecitabine; ECC) improves
overall survival compared with postoperative chemotherapy (ECC). Quality assurance
analyses of surgery and histology in the first 100 patients has been performed and
will be used for further optimalisation of the study’s quality.
Because previous studies have shown that completion of postoperative treatment
modalities in patients with resected gastric cancer is diminished (42-80%), we are
exploring the operability, pCR rate and safety of induction chemoradiotherapy
consisting of 5 weeks concurrent carboplatin, paclitaxel, and radiotherapy in patients
with locally advanced gastric cancer in a multicenter study.
A phase I-II study with an outpatient chemotherapy regimen with docetaxel, oxaliplatin
and capecitabine in patients with metastatic gastric cancer is currently expanding its
maximal tolerable dose (MTD) level. Farmacogenomic, -kinetic and –dynamic studies
of capecitabine, platinum derivatives and/or docetaxel are in full progress.
rectal cancer Between 2004-2008, 147 consecutive patients with cT3-4 (with a
threatened circumferential resection margin or T3 only when

Anastomotic leakage and perineal wound complications developed in 26% and 32%
of LAR and APR patients, respectively. This relatively large series of patients shows
that acute toxicity is acceptable, but that surgical morbidity is high. Due to wide
variation in the use and reporting of a definition of major surgical complications in
other studies, interpretation is difficult. A uniform definition has been proposed.
In a prospective observational study in 9 medically inoperable rectal cancer patients
we evaluated the long-term attachment of two types of endoscopic clips in the human
gastrointestinal tract. Clips were placed as part of a prospective feasibility study
evaluating external beam radiotherapy followed by high dose rate brachytherapy
(HDRBT with an endoluminal applicator, 3x weekly fractions) and followed-up during
endoscopies or using fluoroscopic images (figure 3). The Resolution ® clip (Microvasive)
had a higher overall retention rate than the Quickclips ® (Olympus) (p=0.01). After a
median period of 81 days after placement, long-term retention rates for the
Resolution clip and Olympus clip were 67% and 35%, respectively. One Resolution
clip stayed attached more than 231 days.
Figure 3: Fluoroscopic image as made
for HDRBT planning purposes and in
between HDRBT fractions. Four
Quickclips are still attached (arrows).
127
mediCal onCology
publications (continued)
Van Erp NP, Eechoute K, van der Veldt AA,
Haanen JB, Reyners AK, Mathijssen RH,
Boven E, van der Straaten T,
Baak-Pablo RF, Wessels JA, Guchelaar HJ,
Gelderblom H. Pharmacogenetic pathway
analysis for determination of sunitinibinduced
toxicity. J Clin Oncol.
2009;27:4406-12
Fong PC, Boss DS, Yap TA, Tutt A, Wu P,
Mergui-Roelvink M, Mortimer P,
Swaisland H, Lau A, O’Connor MJ,
Ashworth A, Carmichael J, Kaye SB,
Schellens JH, de Bono JS. Inhibition of
poly(ADP-ribose) polymerase in tumors
from BRCA mutation carriers. N Engl J
Med 2009;361:123-34
Francart J, Vaes E, Henrard S, Legrand C,
Baas P, Gaafar R, van Meerbeeck JP,
Sylvester R, Robert A. A prognostic index
for progression-free survival in malignant
mesothelioma with application to the design
of phase II trials: a combined analysis of
10 EORTC trials. Eur J Cancer.
2009;45:2304-11
Gast MC, Schellens JH, Beijnen JH.
Clinical proteomics in breast cancer: a
review. Breast Cancer Res Treat
2009;116:17-29
Gast MC, van Gils CH, Wessels LF,
Harris N, Bonfrer JM, Rutgers EJ,
Schellens JH, Beijnen JH. Influence of
sample storage duration on serum protein
profiles assessed by surface-enhanced laser
desorption/ionisation time-of-flight mass
spectrometry (SELDI-TOF MS). Clin
Chem Lab Med 2009;47:694-705
Gast MC, Van Gils CH, Wessels LF,
Harris N, Bonfrer JM, Rutgers EJ,
Schellens JH, Beijnen JH. Serum protein
profiling for diagnosis of breast cancer
using SELDI-TOF MS. Oncol Rep
2009;22:205-13
Gast MC, van Dulken EJ, van Loenen TK,
Kingma-Vegter F, Westerga J, Flohil CC,
Knol JC, Jimenez CR, van Gils CH,
Wessels LF, Schellens JH, Beijnen JH.
Detection of breast cancer by surfaceenhanced
laser desorption/ionization timeof-flight
mass spectrometry tissue and serum
protein profiling. Int J Biol Markers
2009;24:130-41

128
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publications (continued)
Geurts TW, Balm AJ, van Velthuysen ML,
van Tinteren H, Burgers JA, van Zandwijk N,
Klomp HM. Survival after surgical resection of
pulmonary metastases and second primary
squamous cell lung carcinomas in head and
neck cancer. Head Neck 2009;31:220-6
Gore ME, Szczylik C, Porta C, Bracarda S,
Bjarnason GA, Oudard S, Hariharan S,
Lee SH, Haanen J, Castellano D, Vrdoljak E,
Schöffski P, Mainwaring P, Nieto A, Yuan J,
Bukowski R. Safety and efficacy of sunitinib
for metastatic renal-cell carcinoma: an
expanded-access trial. Lancet Oncol.
2009;10:757-63
Gross AM, Diacon AH, van den Heuvel MM,
van Rensburg J, Harris D, Bolliger CT.
Management of life-threatening
haemoptysis in an area of high tuberculosis
incidence. Int J Tuberc Lung Dis.
2009;13:875-80
Hadrup SR, Bakker AH, Shu CJ, Andersen RS,
van Veluw J, Hombrink P, Castermans E,
Thor Straten P, Blank C, Haanen JB,
Heemskerk MH, Schumacher TN.
Parallel detection of antigen-specific T-cell
responses by multidimensional encoding of
MHC multimers. Nat Methods.
2009;6:520-6
Harmsen S, Meijerman I, Beijnen JH,
Schellens JHM. Nuclear receptor mediated
induction of cytochrome P450 3A4 by
anticancer drugs: a key role for the
pregnane X receptor. Cancer Chemother
Pharmacol 2009;64:35-43
Ter Heine R, Hillebrand MJ, Rosing H,
van Gorp EC, Mulder JW, Beijnen JH,
Huitema ADR. Quantification of the HIVintegrase
inhibitor raltegravir and detection
of its main metabolite in human plasma,
dried blood spots and peripheral blood
mononuclear cell lysate by means of highperformance
liquid chromatography
tandem mass spectrometry. J Pharm
Biomed Anal 2009;49:451-58
Ter Heine R, Davids M, Rosing H,
van Gorp EC, Mulder JW, van der Heide YT,
Beijnen JH, Huitema ADR. Quantification
of HIV protease inhibitors and nonnucleoside
reverse transcriptase inhibitors in
peripheral blood mononuclear cell lysate
using liquid chromatography coupled with
tandem mass spectrometry. J Chromatogr B
Analyt Technol Biomed Life Sci
2009;877:575-80
Ter Heine R, Rosing H, van Gorp EC,
Mulder JW, Beijnen JH, Huitema AD.
Quantification of etravirine (TMC125) in plasma,
dried blood spots and peripheral blood
mononuclear cell lysate by liquid chromatography
tandem mass spectrometry. J Pharm Biomed Anal
2009;49:393-400
Ter Heine R, Hillebrand M, Rosing H,
van Gorp E, Mulder JW, Beijnen JH,
Huitema ADR. Identification and profiling of
circulating metabolites of atazanavir, a human
immunodeficiency virus protease inhibitor.
Drug Metab Dispos 2009;37:1826-40
Ter Heine R, Huitema ADR, Jansen RS,
Smits PH, van Gorp EC, Wagenaar JF,
Beijnen JH, Mulder JW. Prolonged exposure to
tenofovir monotherapy 1 month after treatment
discontinuation because of tenofovir-related renal
failure. Antivir Ther 2009;14:299-301
Hendrikx JJ, Beijnen JH, Schellens JH.
Methylnaltrexone. Oncologist 2009;14:679-82
Van den Heuvel MM, Burgers SA,
van Zandwijk N. Immunotherapy in non-small-cell
lung carcinoma: from inflammation to vaccination.
Clin Lung Cancer. 2009;10:99-105. Review
Giovannetti E, Zucali PA, et al. Association of
polymorphisms in AKT1 and EGFR with clinical
outcome and toxicity in non-small-cell lung cancer
patients treated with gefitinib. Clin Cancer Res
2009 (in press)
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, 2009
(in press)
Jansen RS, Rosing H, Schellens JH, Beijnen JH.
Retention studies of 2’-2’-difluorodeoxycytidine
and 2’-2’-difluorodeoxyuridine nucleosides and
nucleotides on porous graphitic carbon:
Development of a liquid chromatography-tandem
mass spectrometry method. J Chromatogr A
2009;1216:3168-74
Jansen RS, Rosing H, Schellens JH,
Beijnen JH. Simultaneous quantification of
2’,2’-difluorodeoxycytidine and
2’,2’-difluorodeoxyuridine nucleosides and
nucleotides in white blood cells using porous
graphitic carbon chromatography coupled with
tandem mass spectrometry. Rapid Commun
Mass Spectrom 2009;23:3040-50
Jansen RS, Rosing H, Schellens JH, Beijnen JH.
Protein versus DNA as a marker for peripheral
blood mononuclear cell counting. Anal Bioanal
Chem 2009;395:863-67
Van Kan HJM, de Jonge E, Lie-A-Huen L,
Beijnen JH. Viral encephalitis masking acyclovir
neurotoxicity? A case report. Neth J Crit Care
2009;13:96-98
Kappers I, van Sandick JW, Burgers SA,
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.
2009 (in press)
Kappers I, van Sandick JW, Burgers JA,
Belderbos JS, Wouters MW, van Zandwijk N,
Klomp HM. Results of combined modality
treatment in patients with non-small-cell lung
cancer of the superior sulcus and the rationale for
surgical resection. Eur J Cardiothorac Surg.
2009;36:741-6
Keizer RJ, Zamacona MK, Jansen M, Critchley D,
Wanders J, Beijnen JH, Schellens JHM,
Huitema AD. Application of population
pharmacokinetic modeling in early clinical
development of the anticancer agent E7820.
Invest New Drugs 2009;27:140-52
Kluijt I, Cats A, Fockens P, Nio Y, Gouma DJ,
Bruno MJ. Atypical familial presentation of
FAMMM syndrome with high incidence of
pancreatic cancer. Case finding of asymptomatic
individuals by EUS surveillance. J Clin
Gastroenterol 2009
Koegelenberg CF, Bolliger CT, Plekker D,
Wright CA, Brundyn K, Louw M, Schubert P,
van den Heuvel MM, Diacon AH. Diagnostic
yield and safety of ultrasound-assisted biopsies in
superior vena cava syndrome. Eur Respir J.
2009;33:1389-95
Kok M, Koornstra RH, Margarido TC, Fles R,
Armstrong NJ, Linn SC, Van ‘t Veer LJ,
Weigelt B. Mammosphere-derived gene set predicts
outcome in patients with ER-positive breast cancer.
J Pathol. 2009;218:316-326
Kok M, Holm-Wigerup C, Hauptmann M,
Michalides R, Stal O, Linn S, Landberg G.
Estrogen Receptor-{alpha} Phosphorylation at
Serine-118 and Tamoxifen Response in Breast
Cancer. J Natl Cancer Inst. 2009

Koolen SL, Huitema AD, Jansen RS,
van Voorthuizen T, Beijnen JH, Smit WM,
Schellens JH. Pharmacokinetics of Gemcitabine
and Metabolites in a Patient with Double-Sided
Nephrectomy: A Case Report and Review of the
Literature. Oncologist 2009;14:944-48
Koolen SL, Beijnen JH, Schellens JH.
Intravenous-to-Oral Switch in Anticancer
Chemotherapy: A Focus on Docetaxel and
Paclitaxel. Clin Pharmacol Ther. 2009
Lagas JS, van der Kruijssen CM,
van de Wetering K, Beijnen JH, Schinkel AH.
Transport of diclofenac by breast cancer resistance
protein (ABCG2) and stimulation of multidrug
resistance protein 2 (ABCC2)-mediated drug
transport by diclofenac and benzbromarone. Drug
Metab Dispos 2009;37:129-36
Lagas JS, van Waterschoot RA, van Tilburg VA,
Hillebrand MJ, Lankheet N, Rosing H,
Beijnen JH, Schinkel AH. Brain Accumulation of
Dasatinib Is Restricted by P-Glycoprotein
(ABCB1) and Breast Cancer Resistance Protein
(ABCG2) and Can Be Enhanced by Elacridar
Treatment. Clin Cancer Res 2009;15:2344-51
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 2009 (in press)
Lankheet AG, Hillebrand MJ, Langenberg MH,
Rosing H, Huitema AD, Voest EE, Schellens JH,
Beijnen JH. A validated assay for the quantitative
analysis of vatalanib in human EDTA plasma by
liquid chromatography coupled with electrospray
ionization tandem mass spectrometry.
J Chromatogr B Analyt Technol Biomed Life Sci
2009;877:3625-30
Linn SC, Van ‘t Veer LJ. Clinical relevance of the
triple-negative breast cancer concept: genetic basis
and clinical utility of the concept. Eur J Cancer.
2009;45 Suppl 1:11-26
Linn SC, Wesseling J. Molecular tests for breastcancer
diagnosis? Lancet Oncol. 2009;10:314-315
Lukas A, Perez RSGM. Pulsed radiotfrEquency
treatment for postmastectomy pain. Der Schmerz
2009;291:103
Van Meerbeeck JP, Baas P. Palliative
chemotherapy: do we need yet another end-point?
Eur J Cancer. 2009;45:2234-5
Oostendorp RL, Beijnen JH, Schellens JHM.
The biological and clinical role of drug transporters
at the intestinal barrier. Cancer Treat Rev
2009;35:137-47
Oostendorp RL, Buckle T, Beijnen JH,
van Tellingen O, Schellens JHM. The effect of
P-gp (Mdr1a/1b), BCRP (Bcrp1) and P-gp/BCRP
inhibitors on the in vivo absorption, distribution,
metabolism and excretion of imatinib. Invest New
Drugs 2009;27:31-40
Oostendorp RL, van de Steeg E,
van der Kruijssen CM, Beijnen JH, Kenworthy
KE, Schinkel AH, Schellens JH.
OATP1B1 mediates transport of gimatecan and
BNP1350 and can be inhibited by several classical
ABCB1 and/or ABCG2 inhibitors. Drug Metab
Dispos 2009;37:917-23
Oostendorp RL, Witteveen PO, Schwartz B,
Vainchtein LD, Schot M, Nol A, Rosing H,
Beijnen JH, Voest EE, Schellens JH. Dose-finding
and pharmacokinetic study of orally administered
indibulin (D-24851) to patients with advanced
solid tumors. Invest New Drugs 2009 (in press)
Oostendorp RL, Huitema A, Rosing H,
Jansen RS, Ter Heine R, Keessen M, Beijnen JH,
Schellens JH. Coadministration of ritonavir
strongly enhances the apparent oral bioavailability
of docetaxel in patients with solid tumors. Clin
Cancer Res 2009;15:4228-33
Oostendorp RL, Loftiss J, Goel S, Smith DA,
Dar MM, Witteveen PO, Cohen RB, Lewis LD,
Kurian S, Patnaik A, Rosing H, Beijnen JH,
Voest EE, Burris H, Schellens JHM.
Bioequivalence study of a new oral topotecan
formulation, relative to the current topotecan
formulation, in patients with advanced solid
tumors. Int J Clin Pharmacol Ther 2009;47:
195-206
Ploem MC, Retel VP, Linn SC, van Boven HH,
Schmidt MK, de Jong JP, Gevers JK,
van Harten WH. Tumour tissue: who is in
control? Lancet Oncol. 2009
Pluim D, Beijnen JH, Schellens JH,
van Tellingen O. Simultaneous determination of
AZD1152 (prodrug) and AZD1152hydroxyquinazoline
pyrazol anilide by reversed
phase liquid chromatography. J Chromatogr B
Analyt Technol Biomed Life Sci 2009;877:3549-55
129
mediCal onCology
Poley JW, Kluijt I, Gouma DJ, Harinck F,
Wagner A, Aalfs C, van Eijck CH, Cats A,
Kuipers EJ, Nio Y, Fockens P, Bruno MJ.
The yield of first-time endoscopic
ultrasonography in screening individuals
at high-risk to develop pancreatic cancer.
Am J Gastroenterol 2009;104:2175-81
Quaak SG, van den Berg JH, Oosterhuis K,
Beijnen JH, Haanen JB, Nuijen. DNA
tattoo vaccination: effect on plasmid purity
and transfection efficiency of different
topoisoforms. J Control Release.
2009;139:153-9
Quaak SG, Nuijen B, Haanen JB,
Beijnen JH. Development and validation of
an anion-exchange LC-UV method for the
quantification and purity determination of
the DNA plasmid pDERMATT. J Pharm
Biomed Anal. 2009;49:282-8
Retel VP, Bueno-de-Mesquita JM,
Hummel MJ, van de Vijver MJ,
Douma KF, Karsenberg K, van Dam FS,
van Krimpen C, Bellot FE, Roumen RM,
Linn SC, van Harten WH. Constructive
Technology Assessment (CTA) as a tool in
coverage with evidence development: the
case of the 70-gene prognosis signature for
breast cancer diagnostics. Int J Technol
Assess Health Care. 2009;25:73-83
Rodenhuis S, Mandjes IA, Wesseling J,
van de Vijver MJ, Vrancken Peeters MJ,
Sonke GS, Linn SC. A simple system for
grading the response of breast cancer to
neoadjuvant chemotherapy. Ann Oncol.
2009
Roepman P, Horlings HM, Krijgsman O,
Kok M, Bueno-de-Mesquita JM, Bender R,
Linn SC, Glas AM, van de Vijver MJ.
Microarray-based determination of estrogen
receptor, progesterone receptor, and HER2
receptor status in breast cancer. Clin Cancer
Res. 2009;15:7003-7011
Roepman P, Jassem J, Smit EF, Muley T,
Niklinski J, van de Velde T, Witteveen AT,
Rzyman W, Floore A, Burgers S,
Giaccone G, Meister M, Dienemann H,
Skrzypski M, Kozlowski M, Mooi WJ,
van Zandwijk N. An immune response
enriched 72-gene prognostic profile for earlystage
non-small-cell lung cancer. Clin
Cancer Res. 2009;15:284-90

130
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publications (continued)
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; 2009
Sangha R, Lara PN Jr, Adjei AA, Baas P,
Choy H, Gaspar LE, Goss G, Saijo N,
Schiller JH, Vokes EE, Gandara DR.
Cooperative group research endeavors
in small-cell lung cancer: current and
future directions. Clin Lung Cancer.
2009;10:322-30
Schellens JHM, Grouls R, Guchelaar HJ,
Touw DJ, Rongen GA, de Boer A,
van Bortel LM. The Dutch vision of
Clinical Pharmacology. Clin Pharmacol
Ther 2009;4:366-68
Schellens JH, Beijnen JH. Novel clinical
trial designs for innovative therapies. Clin
Pharmacol Ther 2009;85:212-16
Schellens JH. Phase 0 (zero) clinical trials:
More than zero benefit? Eur J Cancer
2009;45:728-29
Scherpereel A, Astoul P, Baas P,
Berghmans T, Clayson H, de Vuyst P,
Dienemann H, Galateau-Salle F,
Hennequin C, Hillerdal G, Le Péchoux C,
Mutti L, Pairon JC, Stahel R, van Houtte P,
van Meerbeeck J, Waller D, Weder W.
Guidelines of the European Respiratory
Society and the European Society of
Thoracic Surgeons for management of
Malignant Pleural Mesothelioma. Eur
Respir J. 2009 (in press)
Schilder CM, Seynaeve C, Linn SC,
Boogerd W, Gundy CM, Beex LV,
van Dam FS, Schagen SB. The impact of
different definitions and reference groups on
the prevalence of cognitive impairment: a
study in postmenopausal breast cancer
patients before the start of adjuvant
systemic therapy. Psychooncology. 2009
Schilder CM, Seynaeve 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 breast cancer
patients: results from the neuropsychological side
study of the Tamoxifen and Exemestane Adjuvant
Multicenter (TEAM) trial. J Clin Oncol. 2009
(in press)
Schuster K, Gadiot J, Andreesen R,
Mackensen A, Gajewski T, Blank Ch.
Homeostatic proliferation of naïve CD8+
T cells depends on CD62L/L- selectin-mediated
homing to peripheral LN. Eur J Immunol.
2009;39:2981-2990
Sigmond J, Honeywell RJ, Postma TJ,
Dirven CM, de Lange SM, van der Born K,
Laan AC, Baayen JC, Van Groeningen CJ,
Bergman AM, Giaccone G, Peters GJ.
Gemcitabine uptake in gliobalstoma
multiforme: potential as a radiosensitizer.
Ann Oncol. 2009;20:182-7
Smit EF, Burgers SA, Biesma B, Smit HJ,
Eppinga P, Dingemans AM, Joerger M,
Schellens JH, Vincent A, van Zandwijk N,
Groen HJ. A randomized phase II and
pharmacogenetic study of pemetrexed compared
with pemetrexed plus carboplatin in pretreated
patients with advanced non-small-cell lung cancer.
J Clin Oncol. 2009;27:2038-45
Sonke GS, Linn SC. Isolated tumor cells in breast
cancer. N Engl J Med. 2009;361:1995-1996
Sparidans RW, Vlaming ML, Lagas JS,
Schinkel AH, Schellens JH, Beijnen JH. Liquid
chromatography-tandem mass spectrometric assay
for sorafenib and sorafenib-glucuronide in mouse
plasma and liver homogenate and identification of
the glucuronide metabolite. J Chromatogr B
Analyt Technol Biomed Life Sci 2009;877:269-76
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;45:2284-2292
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. 2009
Straver ME, Rutgers EJ, Oldenburg HS,
Wesseling J, Linn SC, Russell NS,
Vrancken Peeters MJ. Accurate axillary lymph
node dissection is feasible after neoadjuvant
chemotherapy. Am J Surg. 2009;198:46-50
Strizzi L, Postovit LM, Margaryan NV,
Lipavsky A, Gadiot J, Blank C, Seftor RE,
Seftor EA, Hendrix MJ. Nodal as a biomarker for
melanoma progression and a new therapeutic
target for clinical intervention. Expert Rev
Dermatol. 2009;4:67-78
Teunissen SF, Rosing H, Koornstra RH,
Linn SC, Schellens JH, Schinkel AH, Beijnen JH.
Development and validation of a quantitative
assay for the analysis of tamoxifen with its four
main metabolites and the flavonoids daidzein,
genistein and glycitein in human serum using
liquid chromatography coupled with tandem mass
spectrometry. J Chromatogr B Analyt Technol
Biomed Life Sci. 2009;877:2519-2529
Tol, J, Koopman M, Cats A, Rodenburg CJ,
Creemers GJM, Schrama JG, Erdkamp FLG,
Vos AH, van Groeningen CJ, Sinnige HAM,
Richel DJ, Voest EE, Dijkstra JR,
Vink- Börger ME, Antonini NF, Mol L,
van Krieken JHJM, Dalesio O, Punt CJA.
Capecitabine, oxaliplatin, bevacizumab with or
without cetuximab in advanced colorectal cancer.
New Engl J Med, 2009;360:563-72
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 2009 (in press)
Tran L, Baars JW, Maessen HJ,
Hoefnagel CA, Beijnen JH, Huitema ADR.
A simple and safe method for 131I radiolabeling
of rituximab for myeloablative high-dose
radioimmunotherapy. Cancer Biother
Radiopharm 2009;24:103-110
Tulner LR, Kuper IM, Frankfort SV,
van Campen JP, Koks CH, Brandjes DP,
Beijnen JH. Discrepancies in reported drug use in
geriatric outpatients: relevance to adverse events
and drug-drug interactions. Am J Geriatr
Pharmacother 2009;7:93-104

Tulner LR, Frankfort SV, Wesselius F,
van Campen JP, Koks CH, Beijnen JH. Do
geriatric outpatients adhere to medication changes
advised after assessment? An exploratory pilot
study. Curr Clin Pharmacol 2009;4:154-58
Tulner LR, van der Jagt-Willlems HC,
Beijnen JH. Intoxication with trazodone caused
by dirty facsimile machine. J Am Geriatr Soc
2009;57:1718-19
Tulner LR, Kuper IM, van Campen JP, Koks CH,
Mac Gillavry MR, Beijnen JH, Brandjes DP.
Treatment of hypertension in an elderly
outpatient population in the Netherlands.
Am J Geriatr Pharmacother 2009;7:204-9
Vainchtein LD, Rosing H, Schellens JH,
Beijnen JH. A new, validated HPLC-MS/MS
method for the simultaneous determination of the
anti-cancer agent capecitabine and its metabolites:
5’-deoxy-5-fluorocytidine, 5’-deoxy-5-fluorouridine,
5-fluorouracil and 5-fluorodihydrouracil, in
human plasma. Biomed Chromatogr 2009
(in press)
Vainchtein LD, Rosing H, Mirejovsky D,
Huynh V, Lenaz L, Schellens JHM, Beijnen JH.
Simultaneous quantitative analysis of EO9
(Apaziquone) and its conversion products EO5a
and EO9-Cl in human and dog urine by highperformance
liquid chromatography coupled with
electrospray tandem mass spectrometry. The Open
Chem Biochem Meth J 2009;2:1-12
Van der Veldt AA, Boven E, Vroling L,
Broxterman HJ, van den Eertwegh AJ,
Haanen JG. Sunitinib-induced hemoglobin
changes are related to the dosing schedule. J Clin
Oncol. 2009;27:1339-40; author reply 1340-2
Vermeulen E, Schmidt MK, Aaronson NK,
Kuenen M, Baas-Vrancken Peeters MJ,
van der Poel H, Horenblas S, Boot H,
Verwaal VJ, Cats A, van Leeuwen FE. A trial of
consent procedures for future research with
clinically-derived biological samples. Br J Cancer.
2009;101:1505-12
Van Vliet M, von Rosenstiel IA, Schindhelm RK,
Brandjes DP, Beijnen JH, Diamant M. Ethnic
differences in cardiometabolic risk profile in an
overweight/obese paediatric cohort in the
Netherlands: a cross-sectional study. Cardiovasc
Diabetol 2009;8:2
Van Vliet M, von Rosenstiel IA, Schindhelm RK,
Brandjes DP, Beijnen JH, Diamant M. The
association of elevated alanine aminotransferase
and the metabolic syndrome in an overweight and
obese pediatric population of multi-ethnic origin.
Eur J Pediatr 2009;168:585-91
Van Vliet M, von Rosenstiel IA, Schindhelm RK,
Brandjes DP, Beijnen JH, Diamant M.
Identifying the Metabolic Syndrome in Obese
Children and Adolescents: Do Age and Definition
Matter? Curr Clin Pharmacol 2009;4:233-38
Vrieling A, Voskuil DW, Bosma1 A, Majoor DM,
van Doorn J, Cats A, Depla ACTM, Timmer R,
Witteman BJM, Wesseling J, Kampman E,
van ’t Veer LJ. Expression of Insulin-like Growth
Factor system components in colorectal tissue and
its relation with serum IGF levels. Growth Horm
IGF Res. 2009;19:126-35
De Vries NA, Beijnen JH, van Tellingen O. Highgrade
glioma mouse models and their applicability
for preclinical testing. Cancer Treat Rev 2009
Vroling L, van der Veldt AA, de Haas RR,
Haanen JB, Schuurhuis GJ, Kuik DJ,
van Cruijsen H, Verheul HM, van den Eertwegh
AJ, Hoekman K, Boven E, van Hinsbergh VW,
Broxterman HJ. Increased numbers of small
circulating endothelial cells in renal cell cancer
patients treated with Sunitinib. Angiogenesis.
2009;12:69-79
Wakelee H, Loo BW Jr, Kernstine KH, Putnam JB
Jr, Edelman MJ, Vokes EE, Schiller JH, Baas P,
Saijo N, Adjei A, Goss G, Choy H, Gandara DR.
Cooperative group research efforts in thoracic
malignancies 2009: a review from the 10th
Annual International Lung Cancer Congress. Clin
Lung Cancer. 2009;10:395-404
Van Waterschoot RA, Rooswinkel RW,
Sparidans RW, van Herwaarden AE, Beijnen JH,
Schinkel AH. Inhibition and stimulation of
intestinal and hepatic CYP3A activity: studies in
humanized CYP3A4 transgenic mice using
triazolam. Drug Metab Dispos 2009
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 AH. Absence of Both Cytochrome P450
3A and P-glycoprotein Dramatically Increases
Docetaxel Oral Bioavailability and Risk of
Intestinal Toxicity. Cancer Res 2009
131
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Van Winden AW, Gast MC, Beijnen JH,
Rutgers EJ, Grobbee DE, Peeters PH,
van Gils CH. Validation of previously
identified serum biomarkers for breast
cancer with SELDI-TOF MS: a case control
study. BMC Med Genomics 2009;2:4
Yang TI, 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. 2009 (in press)
Zandvliet AS, Karlsson MO, Schellens JH,
Copalu W, Beijnen JH, Huitema AD. Twostage
model-based clinical trial design to
optimize phase I development of novel
anticancer agents. Invest New Drugs 2009
(in press)
Zandvliet AS, Schellens JH, Copalu W,
Beijnen JH, Huitema AD. Covariate-based
dose individualization of the cytotoxic drug
indisulam to reduce the risk of severe
myelosuppression. J Pharmacokinet
Pharmacodyn 2009;36:39-62

132
RADIOTHERAPy
Division head, group leader, 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 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
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
Marieke Van Zwienen MD PhD Academic staff
Thelma Witteveen MD PhD Academic staff
Frits Wittkämper PhD Academic staff
Gerben Borst MD PhD Temporary staff
Birgit Hollmann MD Temporary staff
Monique de Jong MD Temporary staff
Bas Kreike MD Temporary staff
Philip Meijnen MD PhD Temporary staff
Stella Mook MD Temporary staff
Marlies Nowee MD PhD Temporary staff
Vera Opperdijk MD Temporary staff
Gabrielle Speijer MD Temporary staff
Roel Steenbakkers MD PhD Temporary staff
Brenda Tomasoa MD Temporary staff
Ben Vanneste MD Temporary staff
Brian Vendel MD Temporary staff
Francine Voncken MD Temporary staff
Moniek Wassenaar MD Temporary staff
DIVISION OF RADIOTHERAPy
In 2009, our department continued to invest in its main research themes: (1)
improving treatment accuracy by optimal dose distribution, geometrical precision
and treatment verification, (2) enhancing radiation response by dose escalation,
chemoradiation and biological response modifiers, and (3) predicting treatment
response and prognosis by genetic profiling, functional imaging and biomarkers.
The use of Intensity Modulated RadioTherapy (IMRT), Image Guided RadioTherapy
(IGRT) and in vivo dosimetry has become indispensible for high dose, high precision
radiation treatment. Further improvement can be achieved by Adaptive RadioTherapy
(ART), a novel strategy that allows a dynamic adaptation of the initial treatment plan
to changes in dosimetry, tumor position, geometry and functional properties during
fractionated radiotherapy. Our department is actively involved in this research area.
Combined modality strategies (chemoradiotherapy/bioradiotherapy) offer yet another
approach to improve treatment outcome. Multiple treatment protocols are active in a
growing number of tumor sites. Functional imaging like FDG-PET and fMRI is
increasingly integrated in our treatment planning protocols and allows a better
prediction of response.
In the 4 th quarter of 2009, we introduced Volumetric Modulated Arc Therapy
(VMAT), a further refinement of existing advanced technologies such as IMRT.
During VMAT the target is continuously irradiated while the source of the beam is
rotated around the patient in single or multiple arcs, while simultaneously
controlling gantry position, gantry speed, MLC leaves, collimator angle and dose rate.
A unique feature developed at our department consists of a continuous dosimetric
verification of VMAT, maximizing the safe delivery of the radiation. We demonstrated
that for early stage lung cancer, prostate, rectal and head and neck cancer VMAT
provides a highly conformal dose distribution delivered in significantly less time than
with current techniques. We are evaluating additional tumor sites for VMAT.
In September 2009, the second scientific meeting of the Descartes Cancer
Consortium (previously ECCCA) was held at Institut Gustave Roussy in Villejuif,
France. 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.
The Radiotherapy department is also an active partner in a consortium that aims at
introducing proton therapy in The Netherlands. In collaboration with Erasmus MC,
Leiden University MC and TU Delft, NKI-AVL aims at establishing a facility for
proton radiotherapy, research and education in the Randstad: the Holland Particle
Therapy Center (HollandPTC).
IMAGE ACQUISITION AND PROCESSING
Tanja Alderliesten, Suzanne Van Beek, Anja Betgen, Johan de Boer, Roman Bohoslavsky, Kristy Brock,
Niel Burnett, Hermine Dees-Ribbers, Andre Dekker2 , Joop Duppen, Rick Haas, Rutger Heddes,
Maarten Hulshof, Rianne de Jong, Vincent Khoo1 , Simon van Kranen, Philippe Lambin2 ,
Joos Lebesque, Corrie Marijnen, Angelo Mencarelli, Cathryn Nagel, Thao-Nguyen Nguyen,
Jasper Nijkamp, Steven Petit2 , Lennert Ploeger, Floris Pos, Coen Rasch, Theo de Reijke3 ,
Peter Remeijer, Simon Rit, Peter de Ruiter, Monique Smitsmans, Sarah Swift, Rajko Topolnjak,
Corine van Vliet-Vroegindeweij, Marnix Witte, Lambert Zijp, Jan-Jakob Sonke, Marcel Van Herk
Intrafraction and interfraction variability of the respiratory motion amplitude
during cone-beam CT acquisition The purpose of this study is to analyze
intrafraction and interfraction variability of the amplitude of the respiratory motion.
From a total of 277 CB scans a respiratory signal was extracted based on the
diaphragm position. The average diaphragm amplitude per patient ranged from
1 The Royal Marsden NHS, London, UK
2 MAASTRO clinic, Maastricht, The Netherlands
3 Free University of Amsterdam, The Netherlands

10.2 mm to 33.2 mm with an average of 17.6 mm. The peak-to-peak amplitude of the
diaphragm motion was found to be slightly more stable inter-fractionally with 2.2 mm
(1 SD) variation than intra-fraction with 3.0 mm variation. The variation reached
3.9 mm when the amplitude was observed regardless of the treatment fraction. This
study fills the gap between studies that had drawn apparently opposite conclusions
on respiratory amplitude acquired on different scanners. Conventional CT scanners
are fast and capture the motion of one or a few cycles only. Observing interfraction
variation of the respiratory amplitude from conventional 4D CT images would therefore
combine both interfraction and intrafraction variations, i.e. 3.9 mm in this study.
In contrast, CBCT scanners are slow and capture the average respiratory cycle over
many cycles, and therefore show only interfraction variation, i.e. 2.2 mm in this study.
Respiration correlated CBCT reduces motion artifacts by using a subset of projection
images for each frame of a 4D dataset at the expense of a longer acquisition time and
reducted image quality. A motion compensation method has been developed, which
is performed during the CBCT reconstruction from all projection images using an a
priori model of the respiratory motion. A model of the patient respiratory motion,
represented by a 4D deformation vector field (DVF) was obtained from the 4D planning
CT with deformable registration. In the new method, the diaphragm position was
used to update the prior 4D deformation vector field based on the observed amplitude
variation. The motion was subsequently compensated in the filtered-backprojection
reconstruction. The method was evaluated on two selected patients: one having
regular breathing with an amplitude similar to the one observed in the planning CT
and one with considerable breathing irregularity. Visual observation of reconstructed
CBCT scans (figure 1) showed that respiratory induced artifacts were considerably
reduced using motion compensation and were further reduced by updating the
model for varying amplitude. We conclude that updating the motion model during
reconstruction is feasible and improves image quality in the presence of irregular
breathing.
Figure 1: Coronal slices of cone-beam CT images for two characteristic patients, one with regular breathing
(top) and one with irregular breathing (bottom). The respiration artifacts (left) are corrected with motioncompensation
assuming a regular breathing (middle) and further reduced by updating the prior model of
the breathing (right).
A practical method for scatter correction in cone beam CT Image quality and
Hounsfield unit accuracy of CBCT are hampered by X-ray scatter. The purpose of this
study was to develop scatter correction algorithm that may be used in an in-line
fashion, i.e., reconstruction should keep up with image acquisition so that volumetric
data is available without delay after scanning. Scatter was modeled as a Gaussian
blurring with an intensity proportional with an attenuation dependent scatter-toprimary
ratio (SPR). To determine SPR and �, various phantom images were acquired
for different beam shapes. Subsequently, SPR and � were fitted minimizing the
difference between the measured and modeled values. During CBCT reconstruction,
the primary x-ray intensity in each projection image is calculated iteratively such that
the sum of a blurred version of the primary intensity (the estimated scatter) plus the
primary intensity itself equals the projection image. To reduce the computational
133
RADIOTHERAPy
Tanja Alderliesten PhD Post-doc
Chun Chen PhD Post-doc
Alessia Gasparini PhD Post-doc
Marnix Maas PhD Post-doc
Anton Mans PhD Post-doc
Leah McDermott 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
Alize Scheenstra PhD Post-doc
Rajko Topolnjak PhD Post-doc
Markus Wendling PhD Post-doc
Marnix Witte PhD Post-doc
Johan De Boer MSc PhD student
Wei Chen PhD Student
Hermine Dees-Ribbers PhD Student
Simon Van Kranen MSc PhD student
Matthijs Kruis PhD Student
Monique Smitsmans MSc PhD student
Jochem Wolthaus MSc PhD student
Jonathan yang MSc PhD student
Hua Zhang PhD Student
Barry Doodeman 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
Jasper Nijkamp MSc 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
Edwin Roosjen Technical staff
Maddalena Rossi MSc Technical staff
René Tielenburg Technical staff
Lambert Zijp MSc Technical staff

134
RADIOTHERAPy
Figure 2: Axial slice without (left) and with
(right) scatter correction
load, scatter is estimated at a relatively low resolution (pixel size: 0.64 cm), making
use of the limited spatial frequency of the scattered irradiation. The intensity
attributed to scatter was locally up to 4.5 times the intensity without scatter, measured
for the largest field size and 30 cm phantom thickness. We found that for practical
application one value for � of 14 cm suffices. The SPR increases with thickness T.
After correction, the primary signal is accurately recovered. Cupping in reconstructed
scans was completely removed (figure 2) and hence the Hounsfield units are more
reliable. The total time needed to perform reconstruction increased only by 14%,
which means that the algorithm is still easily fast enough for in-line reconstruction.
Cone-beam CT guidance for set-up verification in extremity soft tissue
sarcoma patients In 27 patients with lower limb ESTS, volume changes between
the planning CT-scan and CBCT-scans acquired for setup verification were analyzed.
The GTV volume was calculated from each CBCT-scan, and the changes in volumes
were evaluated over time during the five-week treatment interval. In 9 patients an
increase of the GTV up to 26% was seen, 11 patients showed no volume change
during RT and in seven patients a decrease of the GTV up to 58% was observed. All
tumors in the latter group were diagnosed as myxoid liposarcoma (MLS). For the MLS
and tumor boundaries adjacent to bony anatomy, required margins were 0.75 cm in
each direction. For tumor boundaries adjacent to normal tissue or air, required margins
were 1.2 cm in LR and CC direction and 1.4 cm in AP direction. In conclusion,
considerable changes in GTV were seen during the overall treatment time. A reduction
in GTV was only seen for patients with MLS. For these patients a 0.25 cm CTV-to-PTV
margin reduction compared to our clinically prescribed margins of 1 cm is possible,
using online CBCT verification.
Adaptive radiotherapy for rectal cancer patients Substantial shape changes of
the clinical target volume (CTV) of rectal cancer patients have been observed over the
course of radiotherapy (RT). Simple couch corrections cannot reduce such complex
geometric variability. Adaptive RT (ART), on the other hand, has the potential to
minimize the systematic part of this variation by quantifying geometrical uncertainties
over the first few fractions to design a new, more accurate and patient specific
treatment plan for the remaining fractions. The purpose of this study was to find the
optimal ART strategy, and to quantify possible margin reduction. For 55 patients
treated with 5 × 5 Gy pre-operative RT, the mesorectum was delineated as part of the
CTV on the planning CT (pCT) and daily cone-beam CT (CBCT) scans. Three
different ART strategies were tested, ART1) use the delineation of fraction 1 as target
volume for the remainder of the treatment; ART2) generate an average delineation of
fraction 1 and 2 and use that for the remainder of the treatment; ART3) treat fraction
4 and 5 with the average delineation of the first 3 fractions. The required margins for
target deformation were calculated. Without ART systematic errors up to 6 mm SD at
the upper-anterior side adjacent to the bladder/uterus/small bowel were found. No
significant group mean errors in the high variable region were found. Nevertheless,
when the target volume of the first fraction was used for the remainder of the
treatment a decrease of the systematic error to 4 mm SD was found, suggesting a
considerable difference between pCT and the treatment fractions. The optimal
strategy was ART2 with a maximum systematic error of 3 mm SD, while with ART3 a
systematic error comparable with ART1 was found. The gain in margin reduction was
from 15 mm when no ART was used to 10 mm when ART2 was used. This margin
reduction was primarily located at the upper anterior border of the mesorectum,
which is adjacent to organs at risk, making ART clinically interesting. These results
encourage investigation of ART in longer chemo-radiation schedules of 25 fractions,
which is practically more feasible.
Image guided radiotherapy for breast cancer patients: Surgical clips as
surrogate for the breast tumor bed Correct patient positioning is crucial for
proper dose delivery and success of radiotherapy. Breast cancer patients can benefit
from a boost field to the tumor bed. The purpose of this study is to determine the use
of surgical clips as surrogate for locating the tumor bed and to quantify migration of
the clips during the course of RT by using CBCT scans for the breast cancer patients
without seroma. Twenty-one breast cancer patients with surgical clips placed in the

tumor bed were analyzed. CBCT scans were acquired and registered to the planning
CT scans using: 1) grey value registration of the tumor bed region, and 2) matching
of the clips. The migration of the clips was quantified as movement of the center-ofgravity
of the clips according to the center-of-gravity of the tumor bed, and movement
of each separate clip. When the clips are used for setup correction, the position errors
of the tumor bed were small. For the individual clips, however, simultaneous movement
in the direction of center-of-gravity was found. The average distance between the clips
and center-of-gravity was 19 mm (on CT). The average shrinkage towards the centerof-gravity,
observed on the last CBCT scan, was 2 mm with a maximum reduction of
10 mm, showing shrinkage of the tumor bed region. We conclude that clips do not
individually migrate in time, but that they all move towards the center of the tumor
bed due to shrinkage. The clips are a good surrogate for tumor bed, provide small
residual errors and can be used for accurate delivery of boost field to the tumor bed.
Assessment of setup variability for breath-hold radiotherapy for breast cancer
patients by surface imaging Left-sided breast cancer patients treated with
conventional tangential fields frequently have a significant part of heart inside the
irradiation fields causing increased risk for long term heart disease. To decrease the
irradiated heart we use a cone-beam CT guided deep-inspiration breath-hold (DIBH)
treatment technique for these patients. The aim of this study was to quantify the
setup variability during DIBH RT by surface imaging. In each treatment fraction,
three voluntary DIBHs were performed. During the first breath-hold, a CBCT was
acquired for online setup correction. During the second and third breath-hold the
lateral and medial fields were delivered. A 3D surface imaging system (AlignRT,
Vision RT Ltd., London, UK) was used to capture surfaces (5-10 fps) during DIBH RT
of 5 breast cancer patients (5 fractions per patient). For each breath-hold, all captured
surfaces were registered to the planning CT. A large interfraction variability before
correction was found, clearly showing the need for a correction protocol as used.
Interfraction variability after correction and intrafraction variability were of the same
order of magnitude, which is logical as both are mainly caused by intrafraction
breath-hold reproducibility. The intrabeam variability was found to be very small and
likely dominated by small registration errors. In conclusion, patients are well able to
perform a steady voluntarily breath-hold. The variability is acceptable for RT of breast
cancer, provided that setup verification is used.
The influence of the number of implanted fiducial markers on the localization
accuracy of the prostate In our clinic, three fiducial markers are used to localize
the prostate during radiotherapy. Implanting fiducials is uncomfortable for the
patient and the markers introduce imaging artifacts in CT and MRI scans. Therefore,
using fewer markers is preferred. The purpose of this study was to evaluate the
influence of a reduction of the number of markers on localization accuracy of the
prostate. Seven prostate cancer patients were implanted transperineally with three
0.35 × 30 mm or 0.35 × 20 mm gold markers (Visicoil; RadioMed Corp) under
transrectal ultrasound guidance. The markers were aligned to the cranio-caudal axis.
CBCT scans (7-18 per patient) were acquired as part of our correction protocol. CBCT
allows thin, long markers to be used for registration, effectively introducing two
fiducial points per marker, i.e., the endpoints of every marker. Rigid chamfer
matching on any subset of markers was used to register each CBCT to the planning
CT. Using the registration results and the positions of all endpoints, we calculated the
root mean square of the target localization error (s TRE) at the left, right, cranial,
caudal, anterior and posterior side and the center of mass of the prostate. The s TRE
at a given location is the error induced by the localization error of the fiducials and is
a measure of the quality of the registration. For this calculation, rigidity between the
fiducial points was assumed. Seven different configurations of markers were
evaluated: three markers, all combinations of two markers and each marker
separately. To evaluate the configurations, each marker was labeled left or right
according to the lobe it was located in. Configurations with both markers in the same
lobe were grouped, as were configurations with two markers in different lobes and all
configurations with one marker. Compared to a registration on three markers, the
relative s TRE increase over all locations is 19% when registering to two markers in
different lobes. The relative s TRE increase of registering to two markers in the same
Publications
135
RADIOTHERAPy
Ackerstaff AH, Balm AJ, Rasch CR,
de Boer JP, Wiggenraad R, Rietveld DH,
et al. First-year quality of life assessment of
an intra-arterial (RADPLAT) versus
intravenous chemoradiation phase III trial.
Head Neck 2009;31:77-84
Al-Mamgani A, Heemsbergen WD,
Peeters ST, Lebesque JV. Role of intensitymodulated
radiotherapy in reducing toxicity
in dose escalation for localized prostate
cancer. Int J Radiat Oncol Biol Phys
2009;73:685-91
Al-Mamgani A, 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. The Prostate. 2009
(in press)
Al Uwini S, Antonini N, Poortmans PM,
Boersma L, Hurkmans C, Leer JW,
Horiot JC, Struikmans H, Bartelink H.
The influence of the use of CT-planning on
the irradiated boost volume in breast
conserving treatment. Radiother Oncol.
2009;93:87-93
Atsma F, van der Schouw YT, Grobbee DE,
Kors JA, Bartelink ML. Lifetime endogenous
estrogen exposure and electrocardiographic
frontal T axis changes in postmenopausal
women. Maturitas 2009;63:347-51
Aukema TS, Russell NS, Wesseling J,
Rutgers EJ. Extensive soft tissue resection
with autologous tissue closure for locally
recurrent breast cancer: lasting local control
and acceptable morbidity. Eur J Surg Oncol
2009;35:469-74
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. 2009 (in press)
Aukema TS, Valdés Olmos RA, Klomp HM,
Teertstra HJ, Belderbos JS, Vogel WV, et
al. Evaluation of 18F-FDG PET-CT for
Differentiation of Pulmonary Pathology in
an Approach of Outpatient Fast Track
Assessment. J Thorac Oncol 2009

136
RADIOTHERAPy
Publications (continued)
Barcena M, Oostergetel GT, Bartelink W,
Faas FG, Verkleij A, Rottier PJ, et al. Cryoelectron
tomography of mouse hepatitis
virus: Insights into the structure of the
coronavirion. Proc Natl Acad Sci U S A
2009;106:582-7
Bartelink IH, Bredius RG, Belitser SV,
Suttorp MM, Bierings M, Knibbe CA, et
al. Association between busulfan exposure
and outcome in children receiving
intravenous busulfan before hematologic
stem cell transplantation. Biol Blood
Marrow Transplant 2009;15:231-41
Belderbos J, Sonke JJ. State-of-the-art lung
cancer radiation therapy. Expert Rev
Anticancer Ther 2009;9:1353-63
Belderbos JS, Kepka L, Kong FM, Martel MK,
Videtic GM, Jeremic B. Elective nodal
irradiation (ENI) in locally advanced nonsmall-cell
lung cancer (NSCLC): evidence
versus opinion? Int J Radiat Oncol Biol
Phys 2009;74:322-3
Bex A, de Vries R, Pos F, Kerst M,
Horenblas S. Long-term survival after
sequential chemoradiation for limited
disease small cell carcinoma of the bladder.
World J Urol 2009;27:101-6
Bissonnette JP, Franks KN, Purdie TG,
Moseley DJ, Sonke JJ, Jaffray DA, Dawson LA,
Bezjak A. Quantifying interfraction and
intrafraction tumor motion in lung
stereotactic body radiotherapy using
respiration-correlated cone beam computed
tomography. Int J Radiat Oncol Biol Phys.
2009;75:688-95
Bloemen-van Gurp EJ, Haanstra BK,
Murrer LH, van Gils FC, Dekker AL,
Mijnheer BJ, et al. In vivo dosimetry with a
linear MOSFET array to evaluate the
urethra dose during permanent implant
brachytherapy using iodine-125. Int J
Radiat Oncol Biol Phys 2009;75:1266-72
Bloemen-van Gurp EJ, Murrer LH,
Haanstra BK, van Gils FC, Dekker AL,
Mijnheer BJ, Lambin P. In vivo dosimetry
using a linear Mosfet-array dosimeter to
determine the urethra dose in 125I
permanent prostate implants. Int J Radiat
Oncol Biol Phys. 2009;73:314-21
lobe is 75%. This increase is similar to the increase in s TRE (average 73%) when only
one marker is used for the registration. Clearly, the effect of using two markers is lost
when the markers are implanted close together. In conclusion, two instead of three
Visicoil markers can be used to align the prostate, provided that both markers are
implanted at a distance. Using only one marker for registration is possible, but not
recommended with the safety margins in use today.
Improved bladder tumor delineation through registration of planning CT and
cystoscopy images Bladder tumor delineation and localization during treatment are
challenging problems in radiotherapy for bladder cancer. For target volume delineation
and positional verification during treatment we routinely inject lipiodol markers
around the visible tumor during cystoscopy. The purpose of this study is to further
improve tumor delineation by registering cystoscopy images, that more clearly
demarcate the tumor site, with the planning CT. For 10 patients with solitary bladder
tumors, 3-5 small sub-urothelial peri-tumoral deposits of lipiodol (N) were injected
around the tumor during cystoscopy. These deposits were clearly visible on the
planning CT. The registration procedure is outlined in the figure. First, a rough
registration is made to orient the cystoscopy image correctly, using the center of
gravity (CG) of the markers in CT and cystoscopy, the center of the CT bladder, and
one of N markers. Starting from these N orientations, full registrations are
performed taking lens deformation into account. Since a cystoscopy photograph is
2D, each pixel corresponds to a line of sight. The distances between the CT markers
and the lines of sight were minimized using a simplex algorithm. The final cost
function,the RMS distance between corresponding CT dots and lines of sight, was
used to quantify the registration error. The registration with the lowest final function
value was considered to represent the correct orientation. The CT-based delineation
was validated by back-projecting the intersection of the GTV with the bladder wall
onto the cystoscopy image. The average registration error was 4.1 mm, with a SD of
2.7 mm. The main factor determining this relatively large uncertainty is the difficulty
of identifying the center of the marker in CT, since the lipiodol spots are often spread
out. An example registration is shown in the figure. The lower part shows the 3D
reconstruction of the delineated bladder with the cystoscopy projection in ‘throughview’
(left) and the CT based GTV delineation projected on the cystoscopy (right). A
good coverage of the visible tumor can be seen, although the delineation in the upper
part is quite wide. In the 10 patients, however, significant deviations between the
cystoscopy and the CT delineations were observed.In conclusion, registration of
cystoscopic images and planning CT scan is feasible and allows improving tumor
delineation. However, the lipiodol injection protocol needs to be improved to facilitate
identification of markers on both cystoscopy and CT.
The accuracy of deformable registration for adaptive radiotherapy of head and
neck cancer Currently there is a large interest to use deformable image registration
methods for adaptive radiotherapy and dose accumulation. When such tools are used
to support clinical decisions, correct validation of the accuracy is essential. We recently
implemented two different deformable image registration strategies for head and
neck cancer patients. The first is a simple method based on local rigid registration of
multiple regions surrounding bony anatomy, interpolated using thin-plate splines.
This method is close to clinical implementation. The second method is based on
b-splines, and it is more used for research tasks. Both methods employ correlation
ratio as driving cost function. The aim of this paper is to rigorously validate both
methods. Data of 6 patients were analyzed so far. For each patient, the planning CT
(2 mm slices) and 2 CBCT scans (Elekta Synergy, 1mm voxel size) have been analyzed.
Two validation methods are applied. First, two observers and the deformable
registration algorithms identify a set of corresponding anatomical landmarks (30 per
scan pair). The uncertainties in both the human and computer observer are derived
using analysis of variance, taking the inaccuracies of the human observers into
account. Second, small fragments of Visicoil markers (on average 6 per patient) have
been implanted on the border of the tumor. The distance of these markers (which are
ignored by the registration algorithm) after registration was evaluated. Both deformable
registration methods result in visually acceptable registrations. The accuracy of the
multi-region method analyzed from the anatomical landmarks was on the order of

1.4 mm SD for all three axes. B-splines performed better on the landmarks: 1.1. mm
SD. The accuracy derived from the implanted markers was slightly worse, 1.5 mm SD
on average for B-splines. We observed that wound healing processes around the
tumor could result in large marker displacements parallel with anatomical borders.
The landmark data may be considered representative for tissues with visible borders,
i.e., most organs at risk. The implanted markers are representative for tumor borders.
Conclusion: Some head and neck tumor borders show considerable migration during
external beam therapy. The tested deformable image registration methods have an
excellent performance for normal tissues, but are sometimes not capable of capturing
the motion of the tumor borders. Caution should therefore be exercised when using
such algorithms to evaluate accumulated tumor doses.
Tumour control modeling for high risk prostate patients considering
locoregional spread The response to radiotherapy for prostate cancer patients with
a relatively high PSA, Gleason score and/or advanced clinical tumor stage is likely
influenced by the pattern of locoregional cancer spread. Yet, TCP models usually only
consider clonogenic cells inside the prostate (or PTV). We introduced a method to
correlate extraprostatic dose with clinical outcome, and computed a TCP model that
includes tumour burden in locations outside the prostate (e.g. lymph node regions)
that show a strong correlation with outcome. To compare the 3D dose between
patients, maps of the dose surrounding the prostate and surrounding the rectum
were introduced. From patient to patient, these maps identify the dose at a certain
distance (≤ 6 cm) of the organ’s surface, in given directions relative to the center of
mass (prostate) or central axis (rectum). Dose difference maps between patients with
and without failure were computed. Next, a mechanistic TCP model (Webb &
Nahum) was constructed, assuming clonogenic cells were present in the primary
target (prostate an SV), as well as in selected regions outside the prostatic capsule.
The applied regions were selected based on the results of the dose difference maps.
Group III patients without Phoenix failure received on average significantly higher
dose (>7 Gy, p6 Gy, p

138
RADIOTHERAPy
Publications (continued)
de Bruin ML, Dorresteijn LD, van ‘t Veer MB,
Krol AD, van der Pal HJ, Kappelle AC, et
al. Increased risk of stroke and transient
ischemic attack in 5-year survivors of
Hodgkin lymphoma. J Natl Cancer Inst
2009;101:928-37
de Bruin ML, Burgers JA, Baas P,
van ‘t Veer MB, Noordijk EM,
Louwman MW, et al. Malignant
mesothelioma after radiation treatment
for Hodgkin lymphoma. Blood
2009;113:3679-81
Favier O, Heutte N, Stamatoullas-Bastard A,
Carde P, van ‘t Veer MB, Aleman BM,
et al. Survival after Hodgkin lymphoma:
causes of death and excess mortality in
patients treated in 8 consecutive trials.
Cancer 2009;115:1680-91
Fuller CD, Scarbrough TJ, Sonke JJ,
Rasch CR, Choi M, Ting JY, et al. Method
comparison of automated matching
software-assisted cone-beam CT and
stereoscopic kilovoltage x-ray positional
verification image-guided radiation therapy
for head and neck cancer: a prospective
analysis. Phys Med Biol 2009;54:7401-15
Haas RL, Girinsky T, Aleman BM,
Henry-Amar M, de Boer JP, de Jong D.
Low-dose involved-field radiotherapy as
alternative treatment of nodular lymphocyte
predominance Hodgkin’s lymphoma. Int J
Radiat Oncol Biol Phys 2009;74:1199-202
Hamming-Vrieze O, Balm AJ,
Heemsbergen WD, van Hooft HT,
Rasch CR. Regional control of melanoma
neck node metastasis after selective neck
dissection with or without adjuvant
radiotherapy. Arch Otolaryngol Head Neck
Surg 2009;135:795-800
Hartgrink HH, Jansen EP, van Grieken NC,
van de Velde CJ. Gastric cancer. Lancet.
2009;374(9688):477-90
Heemsbergen WD, Al-Mamgani A,
Witte MG, Pos F, 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 2009 (in press)
and validation. For this case, the inter-observer variation for the students prior to the
course for delineation the GTV was 4 mm SD, which reduced significantly to 2 mm
SD for the groups. The validation round resulted in a 3.5 mm SD. Even though the
delineation course has a measurable impact on observer variation, the effect of
delineation in groups is much bigger: a reduction of 50% in inter-observer variation.
This pattern was seen throughout the years and for all four cases. We therefore
conclude that multi-disciplinary discussion plays a major role in improving
delineation consistency.
Application of image guidance to small animal irradiation The image guidance
software developed for human applications has been integrated into a small animal
irradiation platform (figure 3). This work allows irradiation of mouse organs with
sub-mm precision and will enable much more advanced pre-clinical studies on
tumor therapies and normal tissue tolerance.
Figure 3: Screen layout of the software for CBCT guided radiotherapy interfaced to small animal system.
This example shows a complementary color overlay of a pre-treatment reference MRI scan in purple and a
CBCT scan in green. The CBCT scan was registered to the MRI scan by local rigid registration within the
3D rectangular region of interest (ROI) indicated in white. This ROI is selected to be representative of the
target area.
Three orthogonal views of the coronal ( upper-left), sagittal ( upper-right) g pp ), g pp g ) and transverse
(lower-left) allow quick visual inspection of the registration result. Once the user is satisfied, the missposition
of the animal is calculated and automatically corrected to provide the optimal animal setup.
The entire imaging and correction procedure takes about 1-2 minutes.
EPID DOSIMETRy
Pricilla Camargo, Anton Mans, Igor Olaciregui-Ruiz, Raul Pecharromán-Gallego, Jan-Jakob Sonke,
Joep Stroom, Marcel Van Herk, Markus Wendling, Ben Mijnheer
EPID dosimetry is clinically used for verification of all IMRT treatments (~2000/
year) in our department. It is our only form of patient-specific IMRT verification,
used for all curative patient treatments with a few exceptions. In most cases it is
applied in vivo thereby guaranteeing the correct dose delivery to the patient.
In our current procedure, a 2-dimensional (2D) dose distribution is reconstructed
from EPID images in a plane parallel to the EPID intersecting the isocenter by means
of a back-projection algorithm, and compared to the planned dose distribution using
the g-valuation method (3%/3mm criteria). Treatment plans are, however, usually
designed, optimized, and evaluated based on the total 3D dose distribution. We
therefore modified our 2D EPID dosimetry method into a simple 3D method in

which the dose is reconstructed within the patient volume in multiple planes parallel
to the EPID for each gantry angle. By summing the dose grids of all beams, the 3D
dose distribution for the total treatment fraction is obtained. The 3D EPID dosimetry
method was tested for IMRT treatments of prostate, rectum, and head-and-neck
cancer patients. Planned and in vivo-measured dose distributions were within 2% at
the dose prescription point. Within the 50% isodose surface of the prescribed dose, at
least 97% of the points were in agreement. The new method allows accurate in vivo
determination of 3D dose-volume parameters that are common in clinical practice.
Our current back-projection dose-reconstruction algorithm does not account for
tissue inhomogeneities, which gives large deviations for lung cancer treatments.
We therefore developed a new method, in aqua vivo EPID dosimetry, for fast dose
verification in 3D of lung cancer patient treatments. In this method, the dose is
reconstructed in the patient as if the patient would consist entirely of water. For dose
verification, comparison is made against the dose distribution calculated with the
treatment planning system (TPS), where for this specific purpose the inhomogeneity
correction is switched off. 3D dose distributions from the EPID and TPS were
compared using 3D g-evaluation. With this approach we achieved results typical for
in vivo results without large inhomogeneities, such as in head-and-neck cancer
patients. The method is applied clinically now for all our IMRT lung cancer treatments.
Volumetric-modulated arc therapy (VMAT) has recently been introduced in our
department. VMAT is a complex technique in which gantry speed, field shape and
dose rate all are continuously varied during gantry rotation. Consequently, accurate
verification is essential for a safe clinical implementation of VMAT. We therefore
investigated if our back-projection EPID dosimetry method could be used to perform
accurate 3D dosimetric verification of VMAT. For this purpose, several modifications
of our existing approach were implemented including time-resolved data acquisition,
calculation of patient transmission, and 3D dose reconstruction and evaluation.
Planned and EPID reconstructed dose distributions showed good agreement for pretreatment
verification of prostate, stereotactic lung and head-and-neck VMAT plans,
and for in vivo verification of VMAT treatments of prostate (figure 4) and lung cancer
patients. 3D gamma analysis revealed values of mean g-= 0.37, maximum g-= 0.72
and percentage of points with g-=1 = 99%. For in vivo verification, the average
isocentre dose difference was 1.2%. It can be concluded that our portal dosimetry
method has been successfully adapted and can be clinically used for verification of
VMAT treatments, pre-treatment as well as in vivo.
Figure 4: In vivo verification of the first VMAT treatment of prostate cancer. Indicated are the EPID
reconstructed dose distribution (left) and the g-evaluation (right) in three orthogonal planes. The 50%
isodose line is shown in red on the dose distribution and in white on the g-evaluation.
Publications (continued)
139
RADIOTHERAPy
Heideman WH, Russell NS, Gundy C,
Rookus MA, Voskuil DW. The frequency,
magnitude and timing of post-diagnosis
body weight gain in Dutch breast cancer
survivors. Eur J Cancer 2009;45:119-26
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.
2009 (in press)
Jansen EP, Nijkamp J, Gubanski M,
Lind PA, Verheij M. Interobserver Variation
of Clinical Target Volume Delineation in
Gastric Cancer. Int J Radiat Oncol Biol
Phys. 2009 (in press)
Jones HA, Antonini N, Hart AA, Peterse JL,
Horiot JC, Collin F, et al. Impact of
pathological characteristics on local relapse
after breast-conserving therapy: a subgroup
analysis of the EORTC boost versus no
boost trial. J Clin Oncol 2009;27:4939-47
Jurmain R, Bartelink EJ, Leventhal A,
Bellifemine V, Nechayev I, Atwood M,
et al. Paleoepidemiological patterns of
interpersonal aggression in a prehistoric
central California population from CA-
ALA-329. Am J Phys Anthropol
2009;139:462-73
Kappers I, van Sandick JW, Burgers JA,
Belderbos JS, Wouters MW,
van Zandwijk N, et al. Results of combined
modality treatment in patients with nonsmall-cell
lung cancer of the superior sulcus
and the rationale for surgical resection.
Eur J Cardiothorac Surg 2009;36:741-6
Kappers I, van Sandick JW, Burgers SA,
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 2009
Kerckhoffs AP, Akkermans LM,
de Smet MB, Besselink MG, Hietbrink F,
Bartelink IH, et al. Intestinal Permeability
in Irritable Bowel Syndrome Patients:
Effects of NSAIDs. Dig Dis Sci 2009

140
RADIOTHERAPy
Publications (continued)
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 2009 (in press)
Kreike B, Halfwerk H, Armstrong N, Bult P,
Foekens JA, Veltkamp SC, et al. Local
recurrence after breast-conserving therapy
in relation to gene expression patterns in a
large series of patients. Clin Cancer Res
2009;15:4181-90
Kruse JJ, Floot BG, te Poele JA, Russell NS,
Stewart FA. Radiation-induced activation
of TGF-beta signaling pathways in relation
to vascular damage in mouse kidneys.
Radiat Res 2009;171:188-97
Liu G, Bollig-Fischer A, Kreike B,
van de Vijver MJ, Abrams J, Ethier SP,
Yang ZQ. Genomic amplification and
oncogenic properties of the GASC1 histone
demethylase gene in breast cancer.
Oncogene. 2009 (in press)
Mexner V, Wolthaus JW, van Herk M,
Damen EM, Sonke JJ. Effects of
respiration-induced density variations on
dose distributions in radiotherapy of lung
cancer. Int J Radiat Oncol Biol Phys
2009;74:1266-75
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. 2009
(in press)
Nijkamp J, de JR, Sonke JJ, Remeijer P,
van Vliet C, Marijnen C. Target volume
shape variation during hypo-fractionated
preoperative irradiation of rectal cancer
patients. Radiother Oncol 2009;92:202-9
Nijsten SM, van Elmpt WJ, Mijnheer BJ,
Minken AW, Persoon LC, Lambin P, et al.
Prediction of DVH parameter changes due
to setup errors for breast cancer treatment
based on 2D portal dosimetry. Med Phys
2009;36:83-94
Okines A, Verheij M, Allum W,
Cunningham D. ESMO Minimum clinical
recommendations for the diagnosis,
treatment and follow-up of gastric cancer.
Ann Oncol 2009 (in press)
In our model, the EPID reconstructed dose requires the patient transmission, which
is needed to estimate the attenuation between outer contour and reconstruction
plane. The transmission is experimentally determined by a measurement of the ratio
of portal and open image pixel values. In practice this means an extra measurement
of all fields of an IMRT treatment given without a patient. In order to avoid this
additional effort we investigated whether patient transmission could be calculated
using patient CT data. First, the radiological path length d radiol is calculated from the
planning CT scan for every EPID pixel. Next the transmission per pixel is calculated
using an exponential equation: exp(-md radiol + md radiol 2 ) in which the attenuation
coefficient m and the beam hardening coefficient s are experimentally determined by
EPID transmission measurements behind polystyrene slab phantoms of several
thicknesses. EPID reconstruction dose distributions applying either measured or
calculated transmission values are rather similar and are currently compared
quantitatively for a number of treatment sites.
For some treatment techniques EPID dosimetry does not give very accurate results in
the current approach, for instance in case of wedged beams. We therefore modified
our present EPID-based three-dimensional back-projection dose reconstruction
model to be also applicable in wedged beams. For this purpose an energy-dependent
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. Some clinical examples of EPID-based in vivo dosimetry were used to test
the model. For these cases the wedged-beam approach presented significant
improvements compared to using the non-wedged beam model. It can be concluded
that the insertion of a correction factor in the EPID dose-response function provided
an accurate method for pre-treatment and in vivo dosimetry of treatments with
wedged beams.
TREATMENT PLANNING
Corine Van Vliet-Vroegindeweij, Anke Van Mourik, Andrea Holt, Tomas Janssen, Emmy Lamers,
Annemarie Lakeman, Barry Doodeman, Suzanne Den Hollander, Jose Belderbos, Coen Rasch,
Joost Knegjens Jan-Jakob Sonke, Paula Elkhuizen, Tanja Alderliesten, Jonathan Yang, Eugène Damen
New treatment techniques:
Development of Volumetric Modulated Arc Therapy (VMAT) VMAT is a new
form of arc therapy, in which the dose is delivered with continuously variable dose
rate, variable MLC leaf positions and variable gantry speed. In this way a highly
intensity modulated field can be delivered in a relatively short time interval.
For prostate cancer we performed a treatment planning study comparing VMAT with
our current clinical standard, a five-field step-and-shoot IMRT technique. VMAT
parameters (number of arcs, delivery time etc.) and optimization parameters were
varied and the resulting dose distributions were compared to the clinical standard
with respect to tumor coverage and dose to organs at risk. The best VMAT plans
consisted of a single arc of approximately 240�. Target coverage was similar to the
clinical standard, while dose to rectum and anus was reduced considerably. VMAT
resulted in plans that can be delivered faster (6 minutes vs. 90 seconds) and have
less monitor units (650 vs. 300).
For lung SBRT VMAT was compared to the current 15 field IMRT technique. Due to
the high dose that has to be delivered (18 Gy per fraction), two arcs were necessary
with arc lengths varying from patient to patient, depending on tumor position. The
VMAT plans resulted on average in dose distributions that were comparable to the
clinical standard with respect to tumor coverage. Dose to organs at risk was within
tolerance, although dose fall-off in the lung was slightly less steep than in the clinical
plans, resulting in a higher V20 but a lower V5 in lung. The VMAT plans resulted in
a large reduction of treatment delivery time from 25 minutes to 6 minutes.
Both for prostate and SBRT lung VMAT has been introduced successfully in clinical
practice.

Lung
Extending the benefit of hypofractionated SBRT to stage II/III NSCLC:
A feasibility study SBRT for stage I NSCLC, with fractionation schedules up to
3 × 20 Gy, results in very high local control rates of 80-90%. Conventional (chemo-)
radiation for stage II/III disease results in high local recurrence rates, mainly at the
site of the primary tumor. We started a planning study to assess the feasibility of
extending the advantage of SBRT to stage II/III tumors using a novel hybrid
stereotactic/conventional treatment technique: combining SBRT to the peripheral
tumor and conventional fractionated RT to the pathologic lymph nodes.
Nine patients were selected with stage II/III disease and primary tumors < 5 cm in
diameter located > 2 cm from the main bronchial tree. The clinical treatment plan
delivering 24 × 2.75 Gy in 5 weeks to the primary tumor and pathologic lymph nodes
(clinical plan) was compared to a hybrid stereotactic/conventional technique
delivering 3 × 18 Gy to the primary tumor and 24 × 2.75 Gy to pathologic lymph
nodes. PTV margins of the lymph nodes were identical to the clinical plan, while
PTV margins of the primary tumor were reduced because an online setup correction
protocol is used for SBRT. Dose to organs at risk was converted to equivalent dose in
fractions of 2 Gy (EQD2) using the LQ model with a/b of 3 Gy (lung, esophagus,
heart, and Mediastinal Structures (MS)) and 2 Gy (spinal cord), respectively. In this
study we assume that the LQ model is valid for the large fraction sizes used in SBRT.
The treatment plan was judged feasible if the mean lung dose (MLD) ≤ 20 Gy, the
maximum dose (Dmax) to the spinal cord ≤ 50 Gy, the volume of esophagus receiving
35 Gy or more (V35) ≤ 65%, the mean heart dose (MHD) ≤ 46 Gy, and Dmax of the
MS ≤ 94 Gy. In addition, the volume of lung receiving 20 Gy or more (V20) and 5 Gy
or more (V5) was recorded.
Table 1
Average difference ± SD p-value
(hybrid – clinical) (paired t-test)
Lung MLD (Gy) 2.0 ± 3 0.04
V20 (%) –2.6 ± 3 0.02
V5 (%) –5.8 ± 5 0.01
Spinal cord Dmax (Gy) –4.3 ± 12 n.s.
Esophagus V35 (%) –0.9 ± 3 n.s.
Heart MHD (Gy) –1.3 ± 4 n.s.
MS Dmax (Gy) 1.7 ± 6 n.s.
In 8 patients, satisfactory dose coverage of the PTV could be reached while dose to
OARs was within limits. In one patient, the hybrid plan wasn’t feasible due to
violation of the constraints on MLD and maximum dose to the MS. Average changes
in dose values for the organs at risk are listed in the table.
From these results, we concluded that the novel hybrid stereotactic/conventional
technique for stage II/III NSCLC is feasible with possibly a small increased risk to
the lung compared to a conventional treatment plan. We will introduce this hybrid
technique in the clinic in a phase II trial in which the fraction dose to the primary
tumor will be escalated gradually.
Breast
Seroma after breast conserving surgery: A comparison of three radiotherapy
planning techniques In breast cancer patients who develop seroma in the excision
cavity after breast-conserving surgery (BCS), the post-surgical seroma volume is often
used for delineating. However a seroma reduction of 54% during radiotherapy (RT)
has been reported. Since a larger high dose volume is directly associated with fibrosis
and worse cosmetic results, including seroma reduction into RT planning might be
beneficial. In this study, we investigated the difference in breast volume that received
95% of total RT dose (V95), maximum heart dose (maxH) and mean lung dose
(MLD) by comparing 3 breast RT planning techniques.
21 patients who developed seroma after BCS (mean: 63cm 3 (18-218)) were included.
For each patient a pre-treatment CT (CT 1), a CT in the 3 rd (CT 3) and a CT in the fifth
Publications (continued)
141
RADIOTHERAPy
Pengel KE, Loo CE, Teertstra HJ,
Muller SH, Wesseling J, Peterse JL, et al.
The impact of preoperative MRI on breastconserving
surgery of invasive cancer: a
comparative cohort study. Breast Cancer
Res Treat 2009;116:161-9
Poortmans PM, Collette L, Horiot JC,
Van den Bogaert WF, Fourquet A, Kuten A,
et al. Impact of the boost dose of 10 Gy
versus 26 Gy in patients with early stage
breast cancer after a microscopically
incomplete lumpectomy: 10-year results of
the randomised EORTC boost trial.
Radiother Oncol 2009;90:80-5
Pos F, Bex A, Dees-Ribbers HM, Betgen A,
van Herk M, Remeijer P. Lipiodol injection
for target volume delineation and image
guidance during radiotherapy for bladder
cancer. Radiother Oncol 2009;93:364-7
Pos F, Remeijer P. Adaptive Management
of Bladder Cancer Radiotherapy. Semin
Radiat Oncol. 2010 review (in press)
Rijkhorst EJ, Lakeman A, Nijkamp J,
de Bois J, van Herk M, Lebesque JV, et al.
Strategies for online organ motion
correction for intensity-modulated
radiotherapy of prostate cancer: prostate,
rectum, and bladder dose effects. Int J
Radiat Oncol Biol Phys 2009;75:1254-60
Rit S, Wolthaus JW, van Herk M, Sonke JJ.
On-the-fly motion-compensated cone-beam
CT using an a priori model of the
respiratory motion. Med Phys
2009;36:2283-96
Russell NS, Kunkler IH, van Tienhoven G,
Canney PA, Thomas J, Bartlett J, et al.
Postmastectomy radiotherapy: will the
selective use of postmastectomy radiotherapy
study end the debate? J Clin Oncol
2009;27:996-7
Russell NS, Hoving S, Heeneman S,
Hage JJ, Woerdeman LA, de Bree R, et al.
Novel insights into pathological changes in
muscular arteries of radiotherapy patients.
Radiother Oncol 2009;92:477-83
Scharpfenecker M, Floot B, Russell NS,
Ten Dijke P, Stewart FA. Endoglin
haploinsufficiency reduces radiationinduced
fibrosis and telangiectasia
formation in mouse kidneys. Radiother
Oncol 2009;92:484-91

142
RADIOTHERAPy
Publications (continued)
Scharpfenecker M, Kruse JJ, Sprong D,
Russell NS, Ten Dijke P, Stewart FA.
Ionizing radiation shifts the PAI-1/ID-1
balance and activates notch signaling in
endothelial cells. Int J Radiat Oncol Biol
Phys 2009;73:506-13
Schwartz GF, Hughes KS, Lynch HT,
Fabian CJ, Fentiman IS, Robson ME, et al.
Proceedings of the international consensus
conference on breast cancer risk, genetics,
& risk management, April, 2007.
Breast J 2009;15:4-16
Sonke JJ, Rossi M, Wolthaus J, van Herk M,
Damen E, Belderbos J. Frameless
stereotactic body radiotherapy for lung
cancer using four-dimensional cone beam
CT guidance. Int J Radiat Oncol Biol Phys
2009;74:567-74
Straver ME, Rutgers EJ, Russell NS,
Oldenburg HS, Rodenhuis S, Wesseling J,
et al. Towards rational axillary treatment
in relation to neoadjuvant therapy in breast
cancer. Eur J Cancer 2009;45:2284-92
Straver ME, Rutgers EJ, Oldenburg HS,
Wesseling J, Linn SC, Russell NS, et al.
Accurate axillary lymph node dissection is
feasible after neoadjuvant chemotherapy.
Am J Surg 2009;198:46-50
Stroom J, Schlief A, Alderliesten T,
Peterse H, Bartelink H, Gilhuijs K. Using
histopathology breast cancer data to reduce
clinical target volume margins at
radiotherapy. Int J Radiat Oncol Biol Phys
2009;74:898-905
van den Belt-Dusebout AW, Aleman BM,
Besseling G, de Bruin ML, Hauptmann M,
van ‘t Veer MB, et al. Roles of radiation
dose and chemotherapy in the etiology of
stomach cancer as a second malignancy. Int
J Radiat Oncol Biol Phys 2009;75:1420-9
van Blitterswijk WJ, Klarenbeek JB,
van der Luit AH, Alderliesten MC,
van Lummel M, Verheij M. CD95/Fas
downregulation in lymphoma cells through
acquired alkyl-lysophospholipid resistance;
partial role of associated sphingomyelin
deficiency. Biochem J. 2009 (in press)
week (CT 5) of RT were acquired. The seroma (GTV b) was delineated on all CT-scans.
GTV b was expanded by 10 mm. This volume is edited to exclude ribs and muscles
to obtain the clinical target volume (CTV b). The planning target volume (PTV b)
was obtained by an additional 5 mm expansion. Each patient was planned using
3 planning techniques:
1. SIB N: simultaneous inverse optimization of 2 tangential glancing open fields
combined with 2 tangential IMRT fields for the breast (28 × 1.81 Gy) and
2 tangential and 1 orthogonal IMRT fields for PTV b (28 × 0.49 Gy) on CT 1.
2. SIB ART: like SIBN but the first 15 fractions were planned on CT1 and the final 13
on CT 3.
3. SEQ: simultaneous inverse optimization of 2 tangential glancing open fields
combined with 2 tangential IMRT fields for the breast (25 × 2 Gy) on CT 1 and
2 tangential and 1 orthogonal IMRT fields for PTV b (8 × 2 Gy) on CT 5.
Total dose distributions were projected and evaluated on CT5. V95, maxH and MLD
were compared over plans using the Wilcoxon signed rank test. SIB ART resulted in
the lowest V95 in 18 patients (mean=250 cm 3 ) while SEQ resulted in the lowest V95
in 3 patients (mean=293 cm 3 ). Patients who benefited from SEQ had larger seroma
reduction between CT 3 and CT 5 compared to patients who benefited from SIB ART
(mean=42.3 vs. 8.7%). Post-surgical seroma volume on CT 1 did not correlate with
V95 reduction when comparing SIB ART and SEQ (R 2 =0.002). The MLD showed a
minor reduction when comparing SIB ART to SIB N (4.6 vs. 4.7Gy) and when
comparing SEQ to SIB ART (4.2 vs. 4.6Gy). The reduction of maxH differences
between the 3 techniques was also minimal.
From these results we conclude that SIB ART results in the lowest high dose volume
for the majority of patients. Three patients with the largest seroma reduction at the
end of RT benefited from SEQ. Since it is not possible to predict the rate of seroma
reduction at the start of RT, we conclude that SIBART is the optimal planning
technique for breast patients with seroma to reduce excess high dose volume.
Prostate
Probabilistic objectives for IMRT treatment planning using Pinnacle To
develop a set of objective functions in Pinnacle that incorporate the effect of
systematic and random errors during optimization. By replacing the same cost
functions used in the clinic with their probabilistic form, we aim for a system that
resembles current clinical practice and can be easily implemented, allowing clinical
treatment planning without PTV margins in the near future. A customized set of
objectives was created for the research version of the Pinnacle treatment planning
system. Systematic and random error distributions are created for each function at
initialization. On every iteration both error distributions are applied to every function.
Only the ‘best’ 90% of cases are evaluated, i.e., optimization is based on 90%
confidence. We compared 6 clinical treatment plans of prostate cancer patients with
the new plans obtained with our probabilistic objectives. To allow a realistic
comparison between dose distributions, in-house developed software was used to
simulate the actual dose coverage of the target volume (prostate and seminal vesicles)
and the main organ at risk (rectum wall) under the influence of geometrical errors.
Using probabilistic margin-less objective functions we achieved similar dose
distributions when compared to the original margin-based plans, with no noticeable
increase in computation time. The higher degree of freedom obtained with the new
objectives lead to slightly improved treatment plans. A dose increase in the anterior
section of the prostate was observed on new plans, compensated by a shift of the high
dose gradient region away from the rectum wall. With the same or slightly better
target coverage, dosage to the rectum wall was improved, reducing the volume
receiving 70Gy by-2.2±0.9% (from 18.5% to 16.3%). In conclusion, including
geometrical uncertainties during optimization allows plan optimization without
margins, leading effectively to patient-specific anisotropic margins. This ensures
adequate target volume coverage while reducing the rectum wall dosage.
Surprisingly, the treatment planning process is simplified because less structures are
required, and optimization time is not prolonged.

Association between dose exposure in the trigonal bladder neck region and
urinary obstruction after radiotherapy for prostate cancer Side-effects of
radiotherapy for prostate cancer mainly concern the rectum and bladder. A severe
complication can be urinary retention. This symptom is in most cases the result of a
severe outflow obstruction (stricture) in the bladder neck or urethra. We investigated
the relationship between received local dose in the bladder neck region (trigone area)
and the incidence and location of urinary obstruction complaints.
Data of 557 patients of the Dutch escalation study (68 Gy vs 78 Gy) were available, 40
were treated for symptoms of (complete) urinary retention (catheterization, TUR and
/ or dilatation). The local dose was obtained from a dose mapping procedure at about
2 cm above the delineated prostate in the trigone area. From patient files, we learned
that the obstruction was definitely located in or near the bladder neck for 16 patients.
The association with the local dose in the bladder neck was evaluated for both general
obstruction and for identified local bladder neck obstruction. In addition, dose effect
relationships were estimated for early events within 2 years after treatment and for late
events after a lag period of 2 years. We also estimated the effects of other risk factors.
Urinary obstruction within 2 years is associated with urinary problems existing
before RT, acute toxicity, previous TURP and hotspots in the bladder. Events after a
period of 2-7 years are associated with the local dose in the trigone area as well as
with other correlated dose points in this bladder area. The cases with established
bladder neck obstruction showed the strongest correlation with the local dose.
Limiting dose to the bladder neck area is often not an aim in treatment optimization.
Because of the serious nature of urinary obstruction, sparing of the bladder neck area
is however advised in order to prevent the patient from unnecessary risks.
Position variation of the inguinal lymph node region was highly dependent on the
level. Close to the femoral head the variation was small (table 2a), increasing towards
the middle and lower level. The position variation of the femoral artery strongly
correlated with a femur rotation around the AP-axis (left: R2=0.88; right: R2=0.80).
At the low level, an AP-rotation of 6 dg corresponds with a displacement of roughly
1 cm. Since motion of inguinal lymph nodes was highly correlated with the position
of the femora a new study on ten patients with feet fixation was started. With the use
of feet fixation the incidence of lower inguinal lymph nodes more than 1 cm
displaced (vector length) was reduced from 20 to 7%.
Table 2: Systematic errors (S), Random errors (s) and Group Means based on translations
in Left (+)/Right (–), Cranial (+)/Caudal (–) and Anterior (+)/Posterior (–) direction
(in mm).
LR CC AP
Hihg Middle Low Low High Middle Low
a. Anus
Systematic Error 1.1 0.4 0.7 0.0 2.5 0.0 3.1 3.4 2.6
Random Error 1.1 1.0 1.1 0.0 3.5 0.0 3.6 3.4 3.4
Group Mean –0.6 –0.2 –0.4 0.0 –0.7 0.0 –1.8 –2.3 –2.4
Required Margin* 3.4 1.6 2.5 0.0 8.7 0.0 10.3 10.8 8.8
b. Inquinal NL
Systematic Error 0.7 1.8 3.8 – 0.7 1.3 4.2
Random Error 2.1 2.6 4.3 – 0.9 1.9 3.8
Group Mean –0.3 –0.7 –0.6 – –0.4 –0.3 0.5
Required Margin* 3.2 6.3 12.7 – 2.5 4.5 13.1
* The calculated margin, based on the margin recipe of M. van Herk (2.5 • S + 0.7 • s),
is indicative and applicable for position variation only.
Publications (continued)
143
RADIOTHERAPy
van den Broek GB, Wildeman M,
Rasch CR, Armstrong N, Schuuring E,
Begg AC, et al. Molecular markers predict
outcome in squamous cell carcinoma of the
head and neck after concomitant cisplatinbased
chemoradiation. Int J Cancer
2009;124:2643-50
van der Molen L, van Rossum MA,
Ackerstaff AH, Smeele LE, Rasch CR,
Hilgers FJ. Pretreatment organ function in
patients with advanced head and neck
cancer: clinical outcome measures and
patients’ views. BMC Ear Nose Throat
Disord 2009;9:10
Borst GR. Radiotherapy for Lung Cancer,
Amsterdam: University of Amsterdam,
2009
Wolthaus JWH. Four-dimensional imaging
in radiotherapy for lung cancer patient.
Amsterdam: University of Amsterdam,
2009
Figure 5: Kaplan Meier estimates of
established bladder neck obstruction for
3 dose bins.

144
RADIOTHERAPy
Publications (continued)
van der Ploeg IM, Russell NS, Nieweg OE,
Oldenburg HS, Kroon BB, Olmos RA, et
al. Lymphatic drainage patterns in breast
cancer patients who previously underwent
mantle field radiation. Ann Surg Oncol
2009;16:2295-9
van der Putten L, van den Broek GB,
de Bree R, van den Brekel MW, Balm AJ,
Hoebers FJ, et al. Effectiveness of salvage
selective and modified radical neck
dissection for regional pathologic
lymphadenopathy after chemoradiation.
Head Neck 2009;31:593-603
van Kranen S, van Beek S, Rasch C,
van Herk M, Sonke JJ. Setup uncertainties
of anatomical sub-regions in head-and-neck
cancer patients after offline CBCT
guidance.Int J Radiat Oncol Biol Phys.
2009;73:1566-73
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.
2009 (in press)
Verbrugge I, Wissink EH, Rooswinkel RW,
Jongsma J, Beltraminelli N, Dupuis M,
Borst J, Verheij M. Combining
radiotherapy with APO010 in cancer
treatment. Clin Cancer Res. 2009;15:2031-8
Vermeulen IB, Bohte SM, Elkhuizen SG,
Lameris H, Bakker PJ, La Poutré H.
Adaptive resource allocation for efficient
patient scheduling. Artif Intell Med
2009;46:67-80
Weigelt B, Geyer FC, Horlings HM,
Kreike B, Halfwerk H, Reis-Filho JS.
Mucinous and neuroendocrine breast
carcinomas are transcriptionally distinct
from invasive ductal carcinomas of no
special type. Mod Pathol. 2009;22:1401-14
Wendling M, McDermott LN, Mans A,
Sonke JJ, van Herk M, Mijnheer BJ.
A simple backprojection algorithm for 3D in
vivo EPID dosimetry of IMRT treatments.
Med Phys 2009;36:3310-21
CLINICAL APPLICATION OF IMAGE GUIDED RADIOTHERAPy
Anja Betgen, Harry Bartelink, Gerben Borst, Jose Belderbos, Joop Duppen, Eugene Damen,
Wilma HeemsbergenAngelo Mercerelli, Jasper Nijkamp, Floris Pos, Coen Rasch, Danny Minkema,
Peter Remeijer, Maddalena Rossi, Suzanne Van Beek, Marcel Van Herk, Simon Van Kranen,
Corine Van Vliet-Vroegindeweij, Marc Wentink, Jan-Jakob Sonke
Head and Neck
Clinical implementation of a multiple region of interest registration and
correction method in head-and-neck cancer patients Head-and-neck (H&N)
cancer patients frequently exhibit shape and posture changes over the course of
radiotherapy inducing local misalignment of more than 5mm occurs frequently.
Visual validation of a global rigid registration is therefore difficult. We therefore
developed and clinically implemented a multiple region of interest (mROI) registration
and correction method for high-precision radiotherapy of head-and-neck cancer
patients to manage global and local setup errors.
Figure 6: Multiple regions of interest on bony anatomy in head and neck regions used in this study.
Sagittal view of 13 regions of interest (multi-ROI) on bony anatomy in head and neck region.
Adaptive online correction strategies to manage deformations in H&N cancer
patients Considerable deformations and posture changes have been observed over
the course of radiotherapy in H&N cancer patients. The purpose of this study was to
develop a multi-ROI driven correction protocol for optimal couch shifts and adaptive
replanning to manage deformations. Planning-CT to daily cone-beam CT (CBCT)
bony anatomy registrations were performed for: base of skull, vertebrae C2, C4 and
C6, jugular notch, larynx, hyoid, and mandible. We tested two strategies to convert
the multi-ROI registrations into a couch correction: 1) by averaging over the corrections
required to align each region separately (mean correction). 2) by determining the
correction that minimizes the maximum setup error (miniMax correction). Online
corrections were retrospectively simulated for 19 patients (± 30 CBCT scans per
patient). Patients with systematic deformations larger than T mm after mean
corrections over N fractions were identified as candidates for replanning. The mean
correction strategy resulted in accurate online positioning of the vertebrae: systematic
errors were below 1.1 (random: 1.1) mm in all directions, while larynx, hyoid, and
jugular notch exhibited up to two times larger setup errors. The miniMax corrections
reduced maximum errors, mainly at larynx and jugular notch, at the cost of increasing
the vertebrae errors to 1.5(1.4) mm. The effect of the miniMax correction was more
apparent in the reduction of fractions local setup errors (>5mm vector length): from
42% to 20%. Intervention criteria T and N at 4 mm and 8 fractions would lead to a
replanning for 6 patients (32%). We conclude that online couch corrections can be
tailored to minimize overall geometrical uncertainties or maximum errors.

Intervention criteria based on deformations allow objective selection of patients for
adaptive replanning. We are investigating the use of an artificial CT for adaptive
radiotherapy generated from data from the first few CBCT scans.
Twelve to thirteen 3D rectangular-shaped ROI’s were automatically placed around
bony anatomy on the planning CT scans that were individually registered to
subsequent CBCT scan. To simplify the verification of the registration results we
derived a deformation vector field with a thin plate-spline algorithm and overlaid the
deformed CBCT over the original planning CT: any incorrect registration was
immediately identifiable, reducing the visual verification to a single assessment. The
registration and correction method is performed by the RTTs at the treatment machine
and are supervised by specialized imaging RTTs. Only in case of persistent local
residual setup errors that exceed predefined limits, the treating physician is consulted.
The first clinical experience was evaluated on 50 patients.
The preparation and registration time for the mROI method was similar to those of
the single ROI method. Multiple-ROI registration accurately quantifies global and
local setup errors. In 40% of the CBCT scans, one or more ROI-registrations exceeded
predefined limits of 5mm/5°. In 52 times, these local deviations persisted at least
three fractions and a treating physician was consulted to look into the residual errors.
One patient was re-planned based on these observations. In conclusion, the multi-ROI
registration method is easy in use, there is no extra workload, and provides additional
information on local setup errors and helps to select patients for re-planning.
Development of an application to gather information on delivered treatment
The path from new patient to the end of IMRT delivery consists of a series of steps.
After acquisition of a planning CT, a treatment plan is calculated and transferred to a
treatment scheduling system and finally the beamlets are delivered to the patient in
several fractions. During treatment delivery, patient setup and plan can be recorded
and corrected to account for systematic changes in the patient. In all these steps
several different applications are used and often no complete overview of what has
been delivered is available. This hampers quality control and (retrospective) analysis
of clinical data. The purpose of this study was to develop an application which
automatically gathers the information and is able to rapidly produce reports for
individuals and groups of patients on actual delivered treatment. We combined
therefore the following data in one database: 1) Treatment plan dose per beamlet and
contours from the planning system; 2) Actual treatment delivery data as recorded by
the treatment scheduling system; 3) Patient setup data from either cone-beam CT or
EPID. Report tools were developed to produce dose volume histograms, DVH’s, on
1) the treatment as planned in the planning system; and 2) the treatment corrected
for measured setup errors. The currently stored data contains the 7000 treatments
since 2006. The tool we have developed can analyse the treatment to a group of
patients within a few hours whereas manually this would have cost us days.
Effect of setup errors on delivered maximum spinal cord dose for head &
neck patients The spinal cord is a critical serial organ-at-risk in radiotherapy for
Head & Neck patients. The maximum dose should not exceed 50 Gy to prevent
myelopathy and paraplegia. We used our developed database for recalculation of
treatment plans to evaluate the impact of setup errors on the delivered maximum
dose to the spinal cord and compare it to the planned dose. We analysed data of
35 patients planned and treated with 35 fractions of 2 Gy (IMRT) in the period
January - July 2008. Setup verification was performed with an offline bony anatomy
protocol. Data on translational errors was available for part of the treatment (median
of 9 days). For days without setup information, the data of the nearest fraction were
copied. The dose was converted to equivalent dose in 2 Gy/fraction (EQD2) for the
spinal cord. Maximum planned spinal cord dose was critical (between 45-50 Gy) in
8/35 patients. Systematic standard deviations for setup were 1.2 mm, 0.8 mm and 1.0
mm (mean -0.1 mm) for the left-right, craniocaudal and anterior-posterior direction,
respectively. Deviations in the delivered maximum dose were roughly between -6 %
and +6 % with one outlier of +11 % where the CTV was very close to the spinal cord.
One oropharynx patient exceeded the level of 50 Gy: the estimated delivered dose was
3 % higher (plan 49.3 Gy, delivered 50.8 Gy). We concluded that setup errors have a
modest effect on delivered maximum dose to the spinal cord (deviations of about
Publications (continued)
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RADIOTHERAPy
Wertheim GB, Yang TW, Pan TC, Ramne A,
Liu Z, Gardner HP, Dugan KD, Kristel P,
Kreike B, van de Vijver MJ, Cardiff RD,
Reynolds C, Chodosh LA. The Snf1-related
kinase, Hunk, is essential for mammary
tumor metastasis. Proc Natl Acad Sci U S
A. 2009;106:15855-60
Wildeman MA, Gibcus JH, Hauptmann M,
Begg AC, van Velthuysen ML, Hoebers FJ,
et al. Radiotherapy in laryngeal carcinoma:
can a panel of 13 markers predict response?
Laryngoscope 2009;119:316-22
Yang TI, 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. 2009 (in press)
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 Multi-institutional Analysis. Int
J Radiat Oncol Biol Phys 2009
Zerp SF, Stoter R, Kuipers G, Yang D,
Lippman ME, van Blitterswijk WJ, et al.
AT-101, a small molecule inhibitor of antiapoptotic
Bcl-2 family members, activates
the SAPK/JNK pathway and enhances
radiation-induced apoptosis. Radiat Oncol
2009;4:47
Zuur CL, Simis YJ, Lamers EA, Hart AA,
Dreschler WA, Balm AJ, et al. Risk factors
for hearing loss in patients treated with
intensity-modulated radiotherapy for headand-neck
tumors. Int J Radiat Oncol Biol
Phys 2009;74:490-6

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RADIOTHERAPy
1-3 Gy), and large deviations (> 5 Gy) are not to be expected. However, we did not
study other effects that could have influenced the delivered treatment dose, like
changes in the position of the spinal cord (bending) or significant weight loss.
Clinical implementation of a CBCT guided correction protocol for differential
motion between tumor and organs at risk in stereotactic body radiotherapy
Four dimensional CBCT allows accurate positioning of the target during
radiotherapy. Baseline shifts (differential motion between tumor and organs at risk
(OARs)) can be substantial during treatment and compromise dose to the OARs.
This is especially problematic in Stereotactic Body Radiotherapy (SBRT) due to the
high dose per fraction (3x18Gy). We therefore clinically implemented a protocol to
safely align the target while respecting the dose limits to the OARs.
During treatment planning, OARs such as spinal cord, oesophagus and heart are
expanded by 1cm. If dose limits to these expanded OARs can be met, a baseline shift
of the tumor up to 1cm in any direction can be safely corrected during treatment. In
some cases, expansions were reduced in order to remain within dose constraints.
Expansions were then converted into non-isotropic limits on residual misalignments
of OARs. A fixed isotropic limit of 4mm is used for the target. Limits were entered
into the CBCT software. Two regions of interest (ROIs) are registered (dual
registration): a 3D rectangular shaped ROI on the bony anatomy of the vertebrae
(surrogate for OARs) and a shaped region of interest around the tumor. Couch
corrections are initially based on the tumor registration, but if the residual OAR
misalignment exceeds the limits, a warning message appears. The software then
allows manual adjustments to find a correction that remains within the limits of
OARs and tumor.
Of 136 NSCLC patients treated for SBRT, 30 patients had limits smaller than 1cm due
to violation of the dose to the OARs. In 15 of these 136 patients, limits of 1cm or
smaller were effectuated in one or more fractions. In conclusion, the implementation
of the IGRT protocol evaluating both tumor and OARs position allows accurate dose
delivery to the tumor without the risk of overdosing the OARs.
Bladder
Online CBCT guided corrections for bladder cancer Variations in bladder filling
are the predominant cause of geometrical uncertainties in radiotherapy of bladder
cancer. Online corrections can be a solution, but need to be reliable and fast, with
regard to urinary inflow and efficiency. We therefore developed and clinically
implemented a simple correction method based on CBCT guidance using lipiodol as
a surrogate for the tumor. Lipiodol is a fatty radio-opaque substance, which can be
inserted with relative ease during cystoscopy. Since the correction needs to be done
online, only translations are corrected in the proposed protocol. Residual errors due
delineation uncertainties, matching inaccuracy, rotation and deformation, and
changes caused by urinary inflow are accounted for by the PTV margin.
Shape analysis based on 21 patients showed that tumor deformations, nontranslational
and delineation errors range between 2-3mm (SD), depending slightly
on the location of the tumor in the bladder. The lipiodol fiducials were well visible in
both CT and CBCT and did not exhibit significant washout, even weeks or months
after injection. Analysis of match accuracy for 10 patients showed that chamfer
matching of lipiodol was more accurate than grey value registration (0.8, 0.9, 1.7mm
SD, compared to 1.1, 1.8, 2.6mm SD along the three Cartesian axes) or manual
registration (1.8, 1.7, 2.1mm SD). The average error caused by urinary inflow over a
period of 10 minutes (typical for a bladder cancer treatment) was 3.5mm in the
cranial-anterior direction for cranially located tumors. The inter- and intra-patient
(i.e. systematic and random) SDs for this time interval were 2.4mm and 3.7mm,
respectively. Urinary inflow variability still presents an important uncertainty in the
treatment of bladder cancer. If we compensate for the population averaged inflow, a
margin of 13mm will be possible. With a near perfect estimate of the urinary inflow,
the margin will be limited to 10mm, which is then governed by the non-translational
errors, shape variability and delineation uncertainties. In conclusion, the proposed
CBCT guided online correction strategy provides an efficient way to significantly
reduce the treatment margins required for radiotherapy of bladder cancer by 50-75%.

Breast
Clinical implementation of image guided deep inspiration breath hold breast
irradiation An increased risk of cardiovascular related mortality is observed after leftsided
breast or thoracic wall irradiation applied to breast cancer patients by many
studies. We therefore developed a voluntary deep inspiration breath-hold (DIBH)
treatment protocol using CBCT to correct the patient setup error and monitored
breath-hold depth and stability by 2D kV and MV fluoroscopy. The feasibility, cardiac
dose reduction and the influence of the setup error on the delivered dose for this
protocol was evaluated on nineteen patients treated according to the DIBH protocol
and compared to the Free Breathing (FB) protocol. All patients completed the DIBH
treatment successfully. The largest setup variability was observed in the direction
perpendicular to the RT field (μ=-0.8mm, S=2.9mm, s=2.0mm). The setup
variability did not result in a worse dose delivery to the breast or thoracic wall. The
mean (Dmean) and maximum (Dmax) doses of the DIBH treatment plan was
significantly lower compared to the FB treatment plan for the heart (34% and 25%,
p

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RADIOTHERAPy
In conclusion, shape variation of the mesorectal part of the target volume in rectal
cancer patients is substantial, heterogeneous and different between male and female
patient. Differences between prone and supine orientation are, however, small.
Treatment margins to account for these variations should be increased from 10 to
about 20 mm and differentiated in position along the cranio-caudal axis, in anteriorposterior
direction and for gender. To further investigate how to estimate and take the
shape variation uncertainties into account during treatment a repeat CT study has
been started. In this study not only treatment with 5x5 Gy will be investigated, but
also the 25x2 Gy chemo-radiation scheme. For both patient groups 20 male and 20
female patients will be included. significant weight loss.
BREAST CANCER
Aline Van Giersbergen, Sandra Vreeswijk, Suzanne Den Hollander, Marc Van De Vijver1 ,
Jelle Wesseling, Emiel Rutgers, Hester Oldenburg, Claudette Loo, Adrian Begg, Wouter Vogel,
Corine Van Vliet, Harry Bartelink, Paula Elkhuizen
Image guided Preoperative Accelerated partial Breast Irradiation (PAPBI):
defining radiotherapy sensitivity 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 defined 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 ab 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 profile 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 (1-3cm) pN0 M0 breast cancer; sentinel node procedure before irradiation.
Patients will be treated by a preoperative APBI (PAPBI) consisting in delivering 10 ×
4 Gy over 12 days. Six weeks after PAPBI, a wide local excision will be performed. As
the tumor remains ‘in situ’ during irradiation, accurate tumor delineation and control
of accurate radiation dose delivery to the tumor becomes possible by treating these
patients with a cone beam CT linear accelerator. To identify a subgroup of breast
cancer radiosensitivity, biological studies planned are gene expression profiling from
RNA and DNA isolated from biopsies and fine needle aspiration taken of the tumor
before, during radiotherapy and at time of operation. The mRNA gene expression
profiles, the miRNA expression profiles and the DNA copy number changes will be
correlated with response to radiotherapy, defined as pathologic response at the time
of the lumpectomy (i.e. 6 weeks after the completion of the PAPBI).
This trial will accrue 120 patients over a period of years. First, 60 patients will be
studied as a test set to identify predictive profiles 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-profiling and (iii) to
evaluate the early functional imaging modifications. The Institut Gustave Roussy
(France) and the Karolinska Institut (Sweden) will participate in this study (Descartes
Cancer Consortium).
1 AMC, Department of Pathology, Amsterdam

COMBINATION OF RADIOTHERAPy AND CHEMOTHERAPy
Berthe Aleman, Ibrahím Al-Mamgani 1 , Harry Bartelink, José Belderbos, Henk Boot,
Aannemieke Cats, Ewout Courrech Staal, Otilia Dalesio, Luc Dewit, Johan Dikken,
Wilma Heemsbergen, Michel Van Den Heuvel, Frans Hilgers, Edwin Jansen, Rianne De Jong,
Corrie Marijnen, L. Van De Molen, Maurits Swellengrebel, Renato Valdes Olmos, Karijn Verschueren,
Coen Rasch, Marcel Verheij
Head and Neck - Dysphagia and decreased mouth opening after radiotherapy
for head & neck cancer: Dose-effect relationships Head and Neck patients
treated with Intensity Modulated Radiotherapy are at risk for swallowing problems
and decreased maximum mouth opening (MMO). We assessed the relationship
between functional loss and dose received by structures involved in swallowing,
chewing and opening the mouth in 48 patients treated with concurrent
chemoradiotherapy. At 10 weeks post-treatment, swallowing problems were determined
by videofluoroscopy and the MMO was measured with a ruler. Subjective complaints
were scored on a questionnaire. We found no significant correlations between mean
dose to swallowing structures and objective dysphagia whereas subjective dysphagia
was correlated with several dose parameters. Decreased MMO was, as expected,
associated with dose to the muscles involved in opening the mouth except for the
temporalis muscle. An example of an estimated dose-effect relationship is shown in
the figure 7.
Figure 7: Dose-effect relation for mouth opening in chemoradiated head and neck cancer patients
Head and neck - Chemoradiotherapy Following the favorable results of neoadjuvant
TPF (Docetaxel, Cisplatinum and 5-Fluoro-uracil) chemotherapy, from
September 2008 onwards a collaborative trial with the University of Nijmegen was
opened for accrual at the NKI. The aim of the trial is to determine the optimal
chemoradiation regimen after neo-adjuvant chemotherapy. The trial concerns
patients below 60 years with stage III/IV head and neck cancer. Patients are first to
receive 3-4 courses of TPF chemotherapy followed by a randomization between two
regimens of chemoradiation. Fourteen of 70 patients have entered the trial so far.
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
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 efficacy of three
different regimens of concurrent chemoradiation (CRT) regimens 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-fluorouracil
1 Erasmus Medical Center, Rotterdam, The Netherlands
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(group A, n=65), radiotherapy to 50.4 Gy in 28 fractions with concurrent carboplatin
and paclitaxel (group B, n=16) or radiotherapy to 66 Gy in 33 fractions with low-dose
cisplatin (group C, n=13). Patient-tailored treatment and frequent evaluation all
regimens of chemoradiotherapy for esophageal cancer resulted in acceptable rates of
toxicity (Grade 3/4 hematological toxicity in 19% of patients and grade 3 nonhematological
toxicity in 9% of patients) and efficacy (pathological complete response
27% a 3-year survival of 41% for all patients). In addition, a systematic review was
performed including recent literature (2000-2008) on neoadjuvant CRT for
oesophageal cancer.
Gastroenterology – Gastric cancer Combined radiotherapy and chemotherapy
(CRT) has improved treatment outcome and organ preservation in a growing number
of solid tumors. In the US Intergroup 0116 study, a significant survival benefit in
postoperative CRT was reported in gastric cancer. Based on these results we finished
three adjuvant chemoradiotherapy phase I-II trials in gastric cancer. In the first trial,
postoperative radiotherapy was combined with escalating doses of daily cisplatin and
capecitabine. The maximal tolerated dose (MTD) was established as 5 mg/m 2 for
cisplatin and 650 mg/m 2 bid for capecitabine. Very recently we analyzed cisplatin in
a weekly schedule in this combination. The MTD was cisplatin 20 mg/m 2 and
capecitabine 575 mg/m 2 bid. In the third trial radiotherapy was combined with
escalating doses of capecitabine only. In this study, with a dose of 1000 mg/mv the
MTD was not reached.
Until now, we have treated over 200 gastric cancer patients with CRT. Together with
the Leiden University medical center we compared 116 patients from our phase I-II
studies with 694 patients that had veen treated in the randomized D1(limited lymph
node dissection) vs. D2 (extended lymph node dissection) surgical trial. We found
that CRT significantly decreased local recurrence rates (17 vs. 5%); especially after a
D1 resection. Furthermore, with long-term follow up using serial renography to
monitor kidney function, a 27% and 52% decrease in left renal function was observed
at 18 and 24 months, respectively. With ongoing follow up this functional
deterioration further progresses. This has prompted us to apply more sophisticated
radiotherapy strategies (IMRT; IGRT) to minimize normal tissue toxicity. We have
started accrual in a large international phase III study (CRITICS) in which patients
with operable gastric cancer will receive preoperative chemotherapy, surgery and then
are being randomized between continuation of chemotherapy or postoperative CRT
in a schedule based on the above-mentioned phase I-II studies. At this moment over
200 patients have been randomized. In addition, Sweden has joined the trial. We
were able to demonstrate large inter-observer variation in delineation of the clinical
target volume (CTV). To minimize this in the multicenter study, we developed a CTV
contouring atlas. Furthermore, we have designed 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 (NARCIS).
Gastroenterology – Rectal cancer The Dutch TME trial demonstrated that shortterm
preoperative radiotherapy is effective for the prevention of local recurrences, but
not in patients with a positive resection margin, which occurs more often in the more
advanced tumors (T3/T4). For these tumors, downstaging needs to be achieved to
facilitate a curative resection. Downstaging is dependent on the total radiotherapy
dose and the interval between the end of radiotherapy and surgery, at least 4 weeks.
Chemotherapeutic agents serve as radiosensitizers to improve the therapeutic
efficacy of radiotherapy, and the preferred schedule should be identified.
We have started a multicenter feasibility study (RAX trial), in which patients at risk
for a positive resection margin will receive additional bevacizumab to the established
schedule of preoperative radiotherapy combined with daily capecitabine. The main
endpoints are tolerability and, toxicity. Until now 32 patients have been included.
Furthermore we participate in the rectal cancer M1 study. In this phase II trial
patients with resectable primary metastasized rectal cancer are included to evaluate
the efficacy of a new treatment regimen. In case of primary stage IV rectal cancer,
resection of the primary tumor and metastatic disease may result in an overall
survival of 30%. The majority of these primary tumors are T3 or N+ lesions,
requiring long course preoperative radiotherapy for down staging. Metastatic disease

may respond to chemotherapy (up to 40%), neoadjuvant chemotherapy may increase
the fraction of patients with resectable disease, or may allow for smaller resections.
To overcome the logistical problem of combining long course radiotherapy to the
pelvis with adequate neoadjuvant chemotherapy for systemic disease, the proposed
regimen consists of a short course preoperative radiotherapy (5x5 Gy) followed by
combination chemotherapy (bevacizumab, capecitabine and oxaliplatin). The main
objective is to evaluate the efficacy of the proposed regimen. Primary endpoint is the
fraction of patients who undergo an R0 resection of all tumor sites. Accrual of
patients within this trial is almost complete, as 49 of the planned 50 patients have
been included.
Gastroenterology – Anal cancer To analyze the interfraction variation of the anus
and the inguinal lymph node region during radiation for anal cancer,the target
volumes were delineated on the planning CT and on weekly cone-beam CT (CBCT)
in 11 patients. For the anus, systematic (S) and random (s) position variation was
found in AP and CC direction in the order of 3 mm (table 2b, see page 143). In the
LR direction the variation was negligible. The diameter of the anus was on average
stable for each level, but considerable variation was observed (systematic SD 2-3 mm
for LR and 3-6 mm for AP).
Lung - NSCLC Combining concurrent chemotherapy with radiotherapy (CRT) is the
standard treatment of locally advanced NSCLC. The EGFR monoclonal antibody
Cetuximab has been shown to be active in NSCLC. The addition of Cetuximab to our
concurrent CRT was well tolerated in a phase I trial in 12 patients treated between
April and July 2008, with dermatitis and radiation esophagitis being the most
common side effects. We are currently continuing the feasibility of combining
Cetuximab with concurrent CRT in the RADITUX trial (M07CCL). In this multicenter
randomized phase II trial patients with inoperable locally advanced NSCLC
are allocated to our CRT regimen (66 Gy in 24 fractions and daily dose Cisplatin
6 mg/m 2 ) with or without the addition of Cetuximab. Between February and
November 2009, 37 patients were treated within this trial (32 patients from NKI-
AVL). We expect to finish this trial in 2010. Figure 8.
Figure 8: Partial Metabolic response in an 53 year old female with cT3N0 NSCLC shortly after
chemoradiiation with cetuximmab (M07CCL)
Lung - SCLC For limited stage small cell lung cancer (LS-SCLC), the combination of
chemotherapy and thoracic radiotherapy is the standard treatment. Indeed two metaanalyses
have shown that thoracic radiotherapy given concurrently with chemotherapy
improves both survival and local control. Although thoracic radiotherapy is now
integrated in the routine treatment of LS-SCLC, several important questions remain
unanswered including the optimal total radiation dose and radiation fractionation.
To establish a standard chemo-radiotherapy regimen for LS-SCLC, an EORTC
international, multicentre randomised phase III trial started in 2009 comparing
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twice daily with high dose radiation delivered once daily (CONVERT trial).
Chemotherapy is the cornerstone in the treatment of extensive stage small cell lung
cancer (ES-SCLC. Intrathoracic tumor control is a major problem in ES-SCLC. 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 will be
evaluated in a recently opened 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. At the NKI-AVL,
patient accrual to this trial started in September 2009.
BRACHyTHERAPy
Berthe Aleman, Marcel Steggerda, Baukelien van Triest, Thelma Witteveen en Simon Horenblas,
Luc Moonen, Floris Pos,
Bladder preservation with brachytherapy for bladder cancer Conservative
treatment is standard of care for numerous malignancies. For patients with muscle
invasive bladder cancer, however, radical cystectomy with a pelvic lymph node
dissection is considered standard treatment and organ preservation is only rarely
considered. Brachytherapy for bladder cancer is a bladder preserving alternative to
cystectomy for patients with solitary T1-T2N0M0 bladder cancer < 5 cm. This
procedure has been performed in our hospital for over more than 40 years. In recent
years, the brachytherapy procedures have been brought up to date. Furthermore, we
have analyzed the results and compared the outcome with contemporary cystecomy
data in our own hospital. We have shown that brachytherapy most likely does not
compromise survival and offers a high probability of bladder preservation. The next
step will be a joint analysis of all bladder cancer patients treated with brachytherapy
in the Netherlands. The results of this analysis are expected in 2010.
MECHANISMS, MODULATION AND PREDICTION OF
RADIATION-INDUCED CELL DEATH
Maaike Alderliesten, Tjeerd Aukema, Wim Van Blitterswijk, Jannie Borst, Sjaak Burgers,
Albert Van Hell, Gerben Koning1 , Rogier Rooswinkel, Renato Valdes Olmos, Baukelien Van Triest,
Inge Verbrugge, Jonathan Yang, Shuraila Zerp, Marcel Verheij
The research performed within our group extends on the interface between
laboratory and clinic. It focuses on (1) the identification 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
efficacy 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
identified 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,
death receptor ligands (TRAIL, CD95L/FasL), and small molecule inhibitors of Bcl-2
(Gossypol/AT-101, ABT737). To monitor tumor response and predict treatment
outcome, we explore new functional imaging modalities including in vivo imaging of
apoptosis by annexin V scintigraphy and MIBI SPECT-CT.
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 such as the orally available Perifosine represent a
first 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,
1 Erasmus MC, Rotterdam the Netherlands

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. Besides these radiosensitizing properties, APLs show potent
anti-angiogenic effects in vitro. In an ongoing separate project (in collaboration with
W Van Blitterswijk, Division XX) we study mechanisms of cellular APL uptake and
transport, and address the functional implications of an altered membrane lipid
composition on cellular drug uptake, signal transduction and radiosensitivity. We found
that the uptake of APLs via lipid rafts is essential for their cytotoxic effect, because
disruption of these membrane structures by pharmacological (SM degradation/
cholesterol depletion) or genetic (SM synthase knock down) approaches, results in
resistance to APL. Interestingly, these cells that lack SM and thus have defective rafts,
show cross-resistance to a variety of other, unrelated apoptotic stimuli, including
death receptor ligands and DNA-damaging regimens. This ‘multi-stress resistance’ is
subject of ongoing research and is associated with downregulation of the enzymes
SMS1 and SHIP1.
Following in vitro analyses, in vivo proof-of-concept experiments and a clinical phase
I/pharmacokinetic study, we performed an international multicenter placebocontrolled
phase II study in locally advanced NSCLC, randomizing 168 patients
between radiation + placebo and radiation + daily Perifosine. In the third quarter of
2009 disclosure of the results is scheduled. Other promising APL derivatives with
potentially better bioavailability and radiosensitizing properties, like the i.v.
compound ErPC, have become available and are studied in vitro and in vivo.
Other radiosensitizers in preclinical development In two separate projects with
0rst (Division IV) 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) using a
Bcl-2-overexpressing T-leukemic cell line (Jurkat), a colon carcinoma cell line
(HCT116) and a mesothelioma cell line. While APO010 and radiation had a clear
combined cytotoxic effect on tumor cells in vitro, a combined therapeutic effect on the
same cells subcutaneously grafted in mice was not achieved at APO010 doses
approximating the maximally tolerable level. Our current research focuses on the
molecular mechanism underlying the synergy between death receptor ligands and
DNA damage. We demonstrated that c-FLIP downregulation is an important
common mechanism by which various stress factors, including radiation, sensitize
cells to FasL-mediated apoptosis. Finally, we study the impact of TRAIL death
receptor trafficking on pro-apoptotic signaling and found that internalization of
TRAIL death receptors is not required for TRAIL-induced apoptosis.
In collaboration with the University of Michigan and the division of Radiobiology at
the Free University of Amsterdam, we have initiated preclinical efficacy testing of
small-molecule inhibitors of anti-apoptotic members of the Bcl-2 family (AT-101 and
ABT-737). AT-101 was shown to induce apoptosis and enhance radiation-induced cell
death in human leukemic cells. Activation of the JNK/SAPK pathway appeared to
critically contribute to AT-101-induced apoptosis. 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. ABT-737 has been chemically designed to
specifically 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 using inducible
overexpression of 6 different anti-apoptotic proteins in Jurkat T cells.
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RADIOTHERAPy

154
RADIOTHERAPy
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 efficacy 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 reflect 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 evaluated two non-invasive
cell death imaging techniques in collaboration with the department of nuclear
medicine (Valdés Olmos, Division XIII): 99mTc-Annexin V scintigraphy (TAVS) and
99mTc-MIBI single photon emission computed tomography (MIBI SPECT-CT).
TAVS was evaluated in a variety of tumor types, including NHL, NSCLC, SCLC,
H&NSCC and sarcomas treated by radiotherapy and/or chemotherapy. A statistically
significant correlation was found between the increase in TAV tumor uptake on the
post-treatment scan and tumor response in 65 patients. These results indicate that
TAVS might be useful as a non-invasive predictive test for treatment outcome,
ultimately allowing an early switch to more effective regimens. More recently, we
examined a group of 11 patients with advanced NSCLC (20 lesions) who received
MIBI SPECT-CT before their first course of chemotherapy and analyzed the
relationship between uptake and response to treatment. A statistically highly
significant relationship was found between 99mTc-MIBI uptake and change in
tumor size (r 2 =0.53, p

DIVISION OF SURGICAL ONCOLOGY
GYNAECOLOGY
Gemma Kenter, Marc van Beurden, Willemien van Driel, Petra Biewenga, Jan Lange,
Lotti Lubsen-Brandsma
The department focuses on innovative treatment for ovarian cancer, on interventions
to improve quality of life for premature (iatrogenic) menopausal symptoms and
immunotherapy for (premalignant) genital neoplasms.
Ovary A randomized multicenter phase III clinical trial for stage III ovarian carcinoma
has started investigating the effect of secondary debulking surgery with or without
hyperthermic intraperitoneal cisplatinum. Inclusion started March 2007 and
currently 5 centers in The Netherlands are participating. 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 hyperthermic condition during surgery. Primary endpoint of this
study is progression free survival (KWF CKTO2006-16).
Participation in a randomized, multicenter, phase III study of erlotinib versus
observation in patients with no evidence of progression after first line, platinumbased
chemotherapy for high-risk stage I and stage II-IV ovarian epithelial, primary
peritoneal, or fallopian tube cancer. Results are expected in 2010 (EORTC 55041).
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
patients experiencing treatment-induced menopause is ongoing in 10 hospitals
(KWF project: NKI 2006-3470).
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 sentunal 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 efficacy 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 efficacy study). The benefit of wearing therapeutic
elastic stockings in order to prevent lymphedema for patients treated with inguinal
lymph node dissection is conducted in a non-blinded, randomized controlled trial
(M06PRO ‘Kousen’ study). Inclusion was finalized and results will be available in
2010. HPV 16 E6/E7 DNA prime and E6/E7 long peptide boost vaccination for
multifocal VIN will be tested in a randomized study. (KWF project: NKI 2007-3943).
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 efficacy 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 confirmed by normalization of p16INK4a expression and in
normalization of immunocompetent cells in the dermis which is strongly correlated
with histologic regression. Preparations for a phase 1 trial with DNA HPV 16 E7
vaccination in patients with cervical carcinoma are being made.
155
SURGICAL ONCOLOGY
Division head Theo Ruers
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
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
Bas Polle MD Fellow
Nikola Kimmings MD PhD Fellow
Pieter Tanis MD Fellow
Ewout Courrech Staal MD Research physician
Ingrid Kappers MD Research physician
Iris Van der Ploeg Research physician
Jeffrey Schaap Graduate student
Lotte Nieuwenhuis Graduate student
Marieke Straver Research assistant
Rachel Numan Graduate student
Ronald de Vreeze Research physician
Sjoerd Bruin Research physician
Danny Evers Research physician
Head and Neck Oncology and Surgery
Michiel Van den Brekel MD PhD Head
Gwijde Adriaensen MD Resident
Annemieke Ackerstaff PhD Academic staff
Alfons Balm MD PhD FRCS FACS Academic staff
Cindy van den Boer MD Research physician
Tom Geurts MD Research physician
Fleur Hansen MD Resident
Frans Hilgers MD PhD Academic staff
Irene Jacobi PhD Phoniatrician
Luuk Janssen MD Fellow
Baris Karakullukcu MD Fellow
Martin Klop MD Fellow
Menno Krap DDS Academic staff
Annemarijn Kreeft MD Research physician
Anne-Marije van Kuijen MD Resident
Wouter Lodder MD Research physician

156
SURGICAL ONCOLOGY
Peter Lohuis MD PhD Academic staff
Lisette Van der Molen Bsc Research fellow
Hester van Monsjou MD Research physician
Heike Nyst MD Research physician
Jimmy Pramana MD Research physician
Renske Scheenstra MD Research physician
Ludi Smeele MD DDS PhD Academic staff
Bing Tan MD PhD Academic staff
Adriaan Timmers DDS Academic staff
Corina Van As-Brooks PhD Academic staff
Caroline Verhagen MD Research physician
Melchert Wildeman 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
Joost Leijte Research physician
Igor Cordia MD Fellow
Chantal Nunnink Assistant
Niels Graafland Research physician
Gynaecology
Gemma Kenter MD PhD Academic staff
Marc van Beurden MD PhD Academic staff
Willemien van Driel MD PhD Academic staff
Jan Lange MD Academic staff
Lottie Lubsen–Brandsma MD Academic staff
Plastic and Reconstructive Surgery
Joris Hage MD PhD Head
Arnoud van Turnhout MD PhD 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
May Ronday MD Academic staff
Michael Srámek MD PhD Academic staff
Julia ten Cate MD Academic staff
Ingeborg Vergouwe MD Academic staff
Lenie Hulshoff 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
BREAST CANCER
Emiel Rutgers, Hester Oldenburg, Marie-Jeanne Vrancken Peeters, Jos van der Hage,
Marieke Straver, Tjeerd Aukema, Iris van de Ploeg, Lenka Vermeeren, Saskia Duijts, Mila Donker
In a study of 167 patients we showed that the 70-gene profile predicts the outcome of
neo adjuvant chemotherapy: 20% CR in High Risk tumours, versus 0% in low risk.
Further in an IHC study we found that the increase in breast conserving surgery and
the complete remission of the tumour, is related to different histological and receptorbased
subtypes of breast cancer. Early response monitoring provides the opportunity
to adjust the therapy to the response of the primary tumour; the first steps towards
patient-tailored treatment. Currently we are analyzing the use of PET/CT beside MRI
for this purpose. The additional aim is to accurately select those patients that can be
safely treated with breast and axilla conserving surgery. We analyzed the potential of
MRI to guide selection of surgery after neo adjuvant chemotherapy. Currently we are
investigating the combination of axillary response monitoring with FDG-PET/CT and
marking lymph nodes with radioactive Iodine-125 seeds in order to selectively remove
them after neo adjuvant chemotherapy.
PET-CT scanning: currently we are evaluating the role of PET-CT scanning in T2 and
T3 cancers and those patients who are scheduled for neo adjuvant chemotherapy with
respect to the loco regional extend and distant metastasis. Further the role of PET-CT
scanning in patients with loco regional recurrent breast cancer is evaluated.
Prediction and prognosis. Our group is instrumental in the development and the
implementation of the international MINDACT study, where the role of the
Mammaprint – 70 gene array – is evaluated in patients with early breast cancer.
This year a multicenter study of sentinel node biopsy in multi centric breast cancer is
started (MULTISENT).
The EVA project: In cooperation with the PSOE department a multicenter
randomized trial is evaluating the effectiveness of cognitive behavioral therapy,
physical exercise and the combination of both interventions in alleviating climacteric
symptoms in breast cancer patients experiencing treatment-induced menopause.
Inclusion is completed this year; first results will follow next year.
GASTRO-INTESTINAL CANCER
Arend Aalbers, Frits van Coevorden, Theo Ruers, Johanna van Sandick, Vic Verwaal,
Marie-Jeanne Vrancken-Peeters, Ewout Courrech Staal, Danny Evers
Oesophageal cancer In 2009, Ewout Courrech Staal continued to work as a fulltime
research physician on his thesis-project Multimodality treatment for oesophageal
cancer. Outcome of low-volume esophageal cancer surgery in the NKI-AVL has been
analyzed and compared with published data from high-surgical-volume hospitals.
Results indicated that more relevant factors other than hospital volume alone should
be taken into account to improve outcome after oesophageal cancer surgery (Ann
Surg Oncol 2009;16:3219-26). In a study on concurrent chemo radiotherapy (CRT)
as neo-adjuvant or definitive treatment for locally advanced oesophageal cancer, we
found that with individual treatment planning, different regimens of CRT resulted in
acceptable rates of toxicity and efficacy (e.g., 86% of patients completed the chemo
radiation as planned, and pathological complete response rate was 27%) (accepted by
Am J Clin Oncol). We performed a systematic review of the literature on neoadjuvant
CRT for esophageal cancer. Thirty-eight papers (comprising 3910 patients)
met the predefined inclusion criteria. CRT regimens varied widely. Besides
traditional outcome parameters (e.g., survival), other parameters should be analyzed
(e.g., toxicity, quality of life) to assess whether the risks of CRT are sufficiently
compensated for by the benefits (accepted by Br J Surg).

In a study on long-term quality of life, it was found that patients who survive one year
or more after potentially curative treatment for esophageal cancer can lead
satisfactory lives (manuscript in preparation).
Several studies to investigate whether biological markers can improve individual
treatment planning in patients with non-metastatic oesophageal cancer are ongoing.
E.g., the tumour-stroma ratio has been identified as a practicable prognostic tumour
characteristic that can discriminate resected oesophageal cancer patients with a poor
outcome from those with a better outcome (accepted in Eur J Cancer).
Hipec The results from the randomized trial, finished in 2003, were recently
updated. At the time of this update, the median follow-up was almost 8 years (range
72–115 months). In the standard arm, 4 patients were still alive, 2 with and 2 without
disease; in the “HIPEC” arm, 5 patients were still alive, 2 with and 3 without disease.
The median progression-free survival was 7.7 months in the control arm and 12.6
months in the HIPEC arm (P = 0.020). The median disease-specific survival was
12.6 months in the control arm and 22.2 months in the HIPEC arm (P = 0.028).
The 5-year survival was 45% for those patients in whom a R1 resection was achieved.
With 90% of all events having taken place up to this time, this randomized trial
shows that cytoreduction followed by HIPEC does significantly add survival time to
patients affected by peritoneal carcinomatosis of colorectal origin. For a selected
group, there is a possibility of long-term survival.
Photonics and Image Guided Treatment In close collaboration with the University
of Twente a project has started on the application of nanoparticles for margin
detection and treatment in cancer. The project investigates the use of gold nanoshells
in combination with NIR light for tumor detection and tumor destruction of positive
tumor resection margin during surgery. On the project a post doc is employed on a
grant of the UTwente. In collaboration with partners from industry we started to
investigate the use of spectroscopy for tumor diagnosis using minimal invasive
techniques. A PhD student has started in the last half of 2009. In collaboration with
UMCU, TU Eindhoven, UMCN and Philips a CTMM grant was awarded in 2009 for
the development of HIFU in patients with colorectal liver metastases. The project fits
into an established research line on local treatment of colorectal liver metastases
(KWF grant, EORTC study)
THORACIC CANCER SURGERY
Houke Klomp, Michel Wouters, Johanna van Sandick, Eva Schaake, Tjeerd Aukema
Preoperative EGFR tyrosinekinase inhibitor treatment in patients with NSCLC
proved to be feasible. FDG PET/CT was considerably more informative than
diagnostic CT (RECIST) in assessing response to neo-adjuvant Erlotinib therapy.
Evaluation of the outpatient fast track diagnostic facility for lung nodules showed that
availability of such a facilities including PET/CT can contribute to appropriate and
timely evaluation of lung malignancies. In locally advanced NSCLC, combined
modality treatment has been continuously evaluated. High pCR rate (62%) was
found in resection specimens of patients locally advanced NSCLC of the superior
sulcus following concurrent chemo radiotherapy, consisting of daily low dose
Cisplatin 6 mg/m² and accelerated high-dose radiotherapy (RT) of 66 Gy in
24 fractions. Complete pathological response was associated with better survival;
however pCR cannot be sufficiently recognized preoperatively. In locally advanced
stage IIIA-N2 NSCLC, evaluation of induction chemotherapy followed by either
surgical resection or radiotherapy, showed that outcome was favorable after
lobectomy, but not after pneumonectomy (5-yr survival 43% versus 16%, 5-yr survival
after radiotherapy 16%). Newer induction protocols are ongoing; the M07CCL
(Raditux) study using cetuximab in combination with concurrent chemoradiotherapy,
and the N08CPA study for mesothelioma, using chemotherapy
(Cisplatin and Pemetrexed) with Axitinib.
Publications
157
SURGICAL ONCOLOGY
Aukema TS, Russell NS, Wesseling J,
Rutgers EJ. Extensive soft tissue resection
with autologous tissue closure for locally
recurrent breast cancer: lasting local control
and acceptable morbidity. Eur J Surg
Oncol. 2009;35:469-74
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. 2009
Aukema TS, Valdés Olmos RA, Klomp HM,
Teertstra HJ, Belderbos JS, Vogel WV,
Baas P, Burgers SA, van den Heuvel MM.
Evaluation of 18F-FDG PET-CT for
Differentiation of Pulmonary Pathology in
an Approach of Outpatient Fast Track
Assessment. J Thorac Oncol. 2009
Ackerstaff AH, Balm AJM, Rasch CRN,
De Boer JP, Wiggenraad R, Rietveld DHF,
Gregor RT, Kröger R, Hilgers FJM. Firstyear
quality of life assessment of an intraarterial
(RADPLAT) versus intravenous
chemoradiation phase III trial. Head Neck
2009;31:77-84
Ackerstaff AH, Hilgers FJM. Pulmonary
rehabilitation after total laryngectomy. In:
Voice restoration after total laryngectomy;
Current science and future perspectives.
Edited by Umanath K. Nayak, Aand
Rehan Kazi. With Forewords by Eric D.
Blom and Frans J.M. Hilgers. Publisher:
Byword Books Private Limited, Delhi,
India; 2009:89-100
Ackerstaff AH, Hilgers FJM. Quality-of-life
issues after total laryngectomy. In: Voice
restoration after total laryngectomy; Current
science and future perspectives. Edited by
Umanath K. Nayak, Aand Rehan Kazi.
With Forewords by Eric D. Blom and Frans
J.M. Hilgers. Publisher: Byword Books
Private Limited, Delhi, India; 2009:101-112
Balch CM, MortonDL, Gershenwald J,
McMasters K, Nieweg OE, Powell B,
Ross M, Sondak VK, Thompson JF.
Sentinel node bopsy and standard of care
for melanoma. J Am Acad Dermatol
2009;60:872-5

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Publications (continued)
Bex A, de Vries R, Pos F, Kerst M,
Horenblas S. Long-term survival after
sequential chemoradiation for limited
disease small cell carcinoma of the bladder.
World J Urol. 2009;27:101-6
Bex A, van der Veldt AA, Blank C,
van den Eertwegh AJ, Boven E,
Horenblas S, Haanen J. Neoadjuvant
sunitinib for surgically complex advanced
renal cell cancer of doubtful resectability:
initial experience with downsizing to
reconsider cytoreductive surgery.
World J Urol. 2009;27:533-9
Bex A. Editorial Comment on: Hybrid
Renal Cell Carcinomas Containing
Histopathologic Features of Chromophobe
Renal Cell Carcinomas and Oncocytomas
Have Excellent Oncologic Outcomes. Eur
Urol. 2009
Blank LE, Koedooder K, Pieters BR,
Van der Grient HNB, Van de Kar M,
Buwalda J, Balm AJM, Merks JHM,
Strackee SD, Freling NJ, Koning CC. The
AMORE protocol for advanced-stage and
recurrent nonorbital rhabdomyosarcoma in
the head-and-neck region of children:
A radiation oncology view. Int J Radiat
Oncol Biol Phys 2009;74:1555-1562
Bueno-de-Mesquita JM, Linn SC, Keijzer R,
Wesseling J, Nuyten DS, van Krimpen C,
Meijers C, de Graaf PW, Bos MM, Hart AA,
Rutgers EJ, Peterse JL, Halfwerk H,
de Groot R, Pronk A, Floore AN, Glas AM,
Van ‘t Veer LJ, van de Vijver MJ. Validation
of 70-gene prognosis signature in nodenegative
breast cancer. Breast Cancer Res
Treat. 2009;117:483-95
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, Trifiró 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. Eur
J Nucl Med Mol Imaging 2009;36:1713-42
Corten EML, Hage JJ, Schellekens PP,
Kreulen M, Kon M. Clinical application and
outcome of the segmental pectoralis major
free flap in five head and neck patients.
Plast Reconstr Surg 2009;123: 1462-1467
LIPOSARCOMA STUDY GROUP
Frits van Coevorden, Daphne de Jong, Petra Nederlof, Rick Haas, Harm van Tinteren,
Ronald de Vreeze
Morphological and molecular genetic differences can nowadays help to differentiate
lipomatous tumour from other sarcomas and classify them as specific subtype of
lipomatous tumour. As the natural history and clinical behaviour of lipomatous
tumours can vary enormously and their response to therapy also varies, we have put a
lot of effort in analyzing our liposarcoma database of 325 patients between 1977 and
2007 and subjecting the available material to additional tests for improved
classification and grading. By adding information on the choice of therapy, therapy
outcome and follow up of these cases an interesting set of data came available for
further study and analysis. Our studies generated the following publications:
Ann Surg Oncol.: The additional value of molecular biological analysis for diagnosing
and clinical management of liposarcoma patients; a 30 year single institution’s
experience. In this study we have compared the results of the pathology conclusions
within the 30 years of this study period, comparing primary reports, revision reports
without and with addition of molecular biological and genetic tests. By adding the
clinical history of these patients an interesting added value is shown for certain
groups, suggesting the additional use of molecular and genetic tests in specific
situations, where treatment choices can be questioned.
Mod Pathol.: Primary retroperitoneal myxoid-round cell liposarcoma is a non existing
disease. An immunohistochemical and molecular biological analysis.
In this study we showed by additional molecular biological tests that all primary
myxoid sarcomas in the retroperitoneum were variants of well or dedifferentiated
liposarcoma and that molecular proven (true) myxoid liposarcomas found in the
retroperitoneum were metastases of primary MLS outside the retroperitoneum.
Journal of Molecular Diagnostics: Multifocal myxoid liposarcoma; metastasis or second
primary? A molecular biological analysis.
In this study with similar techniques as in the 1 st study we have analysed those
patients in our database where metachrounous appearance of myxoid liposarcomas
in other sites than the lungs, could have generated the discussion whether these
secondary tumours are of metastatic nature or second primaries. Our study shows a
high correlation between the several tumors occurring within one patient. And in
none of the cases in this study a true second primary nature could be established.
These findings can have impact on the strategic approach in these clinical situations.
MELANOMA
Jos van der Hage, Bin Kroon, Omgo Nieweg, Iris van der Ploeg, Michel Wouters
The research activities of the surgical melanoma group concern 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 and aspects of
regional isolation perfusion. Melanoma surgeons are struggling with the question
whether every patient with a tumour-positive sentinel node needs a completion node
dissection and if so, to what extent. They are attempting to create criteria to select
patients in whom the positive sentinel node is likely to be the only involved node and
who may be spared the morbidity of a complete lymph node dissection. Minimal
tumour load in the sentinel node may be such a criterion and this was the subject of
three studies. In one of these, three micromorphometric parameters of sentinel node
metastases were compared: invasion depth from the capsule (Starz-classification),
maximum diameter and location within the node. Sub capsular metastases with an
invasion depth under 0.3 mm and metastases with a diameter less than 0.1 mm
irrespective of their location were not associated with additional involved nodes.
Other nodes were involved in 2.6% of the patients with sub capsular metastases,
8.5% in case of both subcapsular and parenchymal metastases, and 33% when
multifocal or extensive disease was present. The criterion that best determined both

the risk of additional metastases and survival was 0.4 mm invasion depth. The issue
of the extent of a completion groin lymph node dissection in case of a positive sentinel
node was investigated through the tumour status of second-tier nodes. This study
concerned 42 patients and indicated that, in case of a tumour-positive sentinel node,
the strategy to determine the extent of the groin dissection based on the location of
the second-tier nodes appears to be valid.
HEAD AND NECK ONCOLOGY AND SURGERY
Alfons Balm, Michiel van den Brekel, Frans Hilgers, Martin Klop, Peter Lohuis, Ludi Smeele,
I Bing Tan, Luuk Janssen, M Baris Karakullukcu, Govert Salverda, Charlotte Zuur,
Annemieke Ackerstaff, Jan Paul de Boer, Olga Hamming-Vrieze, Frank Hoebers, Saar Muller,
Coen Rasch, Margot Tesselaar, Arash Navran, Louise van Velthuysen, Adrian Begg, Conchita Vens
The department of head and neck surgery and oncology is a national referral centre
and one of the larger clinical departments in this field 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. There is a close
clinical and research cooperation between the department of head and neck surgery
and the departments of radiotherapy, medical oncology and experimental therapy.
Furthermore, at a clinical level there is close cooperation with the department of
surgery (melanoma, thyroid cancer), dermatology, pulmonology (research on
squamous cancer and second primaries) as well as pediatric oncology at the AMC
(surgery + brachytherapy).
In 2009 the head and neck department was involved in several translational projects.
Together with Adrian Begg and Conchita Vens we studied gene expression in
laryngeal cancer and found that CD44 is a key protein in predicting radiosensitivity.
After testing gene expression using Illumina Beat Arrays in a matched series of small
laryngeal cancers, the clinical significance of overexpression of this gene was
validated using immunohistochemistry on a tissue array. Further validation studies
will follow. Also studies on the mechanisms related to this gene will be initialized.
Possibly it is a marker for stem cells. In another study on gene expression in patients
with large tumors treated with chemoradiation we found that gene expression
signatures for HPV as well as the “chung” signature are predictors independent of
clinical factors for patients treated with chemoradiation. The most important clinical
factors were tumor volume, T stage and oral site. In A project together with Marcel
Verheij the preclinical work on the mechanisms of BCL2 inhibitors such as gossypol
have been studied. A clinical phase two study will soon start using gossypol together
with cisplatin. In a project focusing on fanconi pathway defects in HNSCC clinical
preparations have been done to start mass sequencing. In cooperation with the
university of Maastricht and Nijmegen genetic markers predicting progression from
dysplasia to cancer are studied. 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.
We cooperate in a national validation study using the “Roepman”expression profile
predicting lymph node metastases in oral cancer.
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. Also, the value of PDT in recurrences after
radiotherapy of nasopharyngeal cancer is studied. In a cooperative study with
Yogyakarta in Indonesia this technique has shown promising results. 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.
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SURGICAL ONCOLOGY
Publications (continued)
Corten EML, Schellekens PP, Hage JJ,
Kon M. Clinical outcome after a pedicled
segmental pectoralis major island flap for
head and neck reconstruction. Ann Plast
Surg 2009
Courrech Staal EFW, van Coevorden F,
Cats A, Aleman BMP,
van Velthuysen MLF, Boot H,
Vrancken Peeters MTFD, van Sandick JW.
Outcome of low-volume surgery for
esophageal cancer in a high–volume referral
center. Ann Surg Oncol. 2009;16:3219-26
De Bock GH, Putter H, Bonnema J,
van der Hage JA, Bartelink H,
van de Velde. CJ. The impact of locoregional
recurrences on metastatic
progression in early-stage breast cancer: a
multistate model. Breast Cancer Res Treat.
2009;117:401-8
De Boer M, van Deurzen CH, van Dijck JA,
Borm GF, van Diest PJ, Adang EM,
Nortier JW, Rutgers EJ, Seynaeve C,
Menke-Pluymers MB, Bult P,
Tjan-Heijnen VC. Micrometastases or
isolated tumor cells and the outcome of
breast cancer. N Engl J Med. 2009;
36:653-63
De Snoo F, Bender R, Glas A, Rutgers E.
Gene expression profiling: decoding breast
cancer. Surg Oncol. 2009;18:366-78
(review)
De Vos van Steenwijk P, Piersma SJ,
Welters MJP, van der Hulst JM, Fleuren G,
Hellebrekers BWJ, Kenter GG,
Van der Burg SH. Surgery followed by
persistence of high-grade squamous
intraepithelial lesions is associated with the
induction of a dysfunctional HPV16-specific
T-cell response. Clin Cancer Res
2008;14:7188-95
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. 2009

160
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Publications (continued)
De Vries RR, Nieuwenhuijzen JA,
Meinhardt W, Bais EM, Horenblas S.
Long-term survival after combined modality
treatment in metastatic bladder cancer
patients presenting with supra-regional
tumor positive lymph nodes only. Eur J
Surg Oncol. 2009;35:352-5
De Vries RR, Nieuwenhuijzen JA,
Meinhardt W, Bais EM, Horenblas S.
Langdurige overleving na combinatie
therapie voor gemetastaseerde blaaskanker.
Ned Tijdschr Urol 2009;17:75-7
De Vries RR, Nieuwenhuijzen JA,
van Tinteren H, Oddens JR, Visser O,
van der Poel HG, Bex A, Meinhardt W,
Horenblas S. Prostate-sparing cystectomy:
long-term oncological results. BJU Int
2009;104:1239-43.
Duijts SFA, Oldenburg HSA,
Van Beurden M, Aaronson NK. Cognitive
behavioural therapy and physical exercise
for climacteric symptoms in breast cancer
patients experiencing treatment-induced
menopause: design of a multicenter trial.
BMC Women’s Health 2009;9:15
Duller P, Van der Veen JPW, Evers A et al.
Dermatitis artefacta: aanbevelingen voor de
diagnostiek en behandeling. Ned. Tijdschr.
Dermatol. Venereol. 2009;19:398-402
Fongers A, Wolkerstorfer A,
Nieuweboer-Krobotova L, Krawczyk P,
Tóth GG, van der Veen JP. Long-term
results of 2-mm punch grafting in patients
with vitiligo vulgaris and segmental vitiligo:
effect of disease activity. Br J Dermatol.
2009
Gast MC, Van Gils CH, Wessels LF,
Harris N, Bonfrer JM, Rutgers EJ,
Schellens JH, Beijnen JH. Serum protein
profiling for diagnosis of breast cancer
using SELDI-TOF MS. Oncol Rep.
2009;22:205-13
Gast MC, van Gils CH, Wessels LF,
Harris N, Bonfrer JM, Rutgers EJ,
Schellens JH, Beijnen JH. Influence of
sample storage duration on serum protein
profiles assessed by surface-enhanced laser
desorption/ionisation time-of-flight mass
spectrometry (SELDI-TOF MS). Clin
Chem Lab Med. 2009;47:694-705
Rehabilitation research focuses on several topics. Prevention of trismus, swallowing
and speech problems in patients treated with chemo-radiation for advanced head and
neck cancer is one field of research. An evidence based rehabilitation program is
being set up. Furthermore, the fields of radiotherapy are adjusted to diminish these
side effects. In a project on pulmonary rehabilitation after laryngectomy, new heatand-moist-exchangers
are being developed and tested using an artificial airway
system, but also in patients. Also together with Atos Medical, a new voice prosthesis
has been developed, the Provox ® Vega that is introduced in 2009. The valve system
has been internalized, airflow resistance decreased and the insertion system
optimized (Smart-inserter). Together with the University of Amsterdam Institute of
Phonetic Sciences, an automatic speech analyzer is being developed for pathologic
voices, such as after laryngectomy or chemoradiation. This tool can be used in
assessment of different rehabilitation programs.
Clinical research is quite diverse. A focus is on sentinel node detection and imaging.
The use of SPECT-CT and intraoperative imaging is assessed and has been found
very useful both in oral cancer and melanoma. The patterns of spread of melanoma
has been studied, and over 15% of the patients have tumor draining lymph nodes
outside the expected levels. Assessment of tumor thickness was studied in oral
cancer and high frequency ultrasound has been found to be the optimal technique
for this. This is important in selecting patients for PDT but also to estimate the risk
of occult metastases and adapt the policy for the neck accordingly.
In a project on (in)operability, definitions of inoperability and how to predict it is
studied clinically and in model systems. It has been shown that in maxillectomy, over
half of the patients have tumor positive resection margins, This is an argument to
look for other treatment strategies in a subgroup of these patients. This is done in
cooperation with the Technical University Twente and VUmc.
In a national study, the efficacy of hyperbaric oxygen in radiation induced
osteoradionecrosis is studied in a randomized multicenter trial led by our institute.
Together with Nijmegen University a phase 2 trial on induction TPF with either
conventional chemoradiation or accelerated chemoradiation is being conducted.
UROLOGIC ONCOLOGY
Simon Horenblas, Wim Meinhardt, Axel Bex, Henk van der Poel, Igor Cordia, Chantal Nunnik
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 treatment results.
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 findings were observed. The sentinel node strategy together
with the Sentinella were 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 fluorescence camera ICG is seen after i.v. injection. In testicular
cancer also no false negative findings 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 last year, with the aim of elucidating the
lymphatic pathways of renal cancer and the immunological effect of renal cancer in
the sentinel nodes.

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 coordinated by Axel
Bex, followed the experiences of the phase II study with neo-adjuvant 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 is anticipated at beginning of 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 benefit for
reconstruction of the median fibrous raphe in men after RALP.
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 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 findings 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 Griffioen at the Free University medical centre and Eric
Jonasch, MD Anderson Cancer Center, Houston, kidney cancer tissue is analysed,
specifically 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).
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%).
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Publications (continued)
Gemma G. Kenter, Marij J.P. Welters,
Peggy J. Vos van Steenwijk,
Margriet J.G. Lowik, Dorien M.A.
Berends-van der Meer, Farah Essahsah,
Linda F.M. Stynenbosch, Annelies P.G.
Vloon, Tamara H. Ramwadhdoebe, Sytse J.
Piersma, Jeanette M. van der Hulst,
A. Rob P.M. Valentijn, Lorraine
M. Fathers, Jan Wouter Drijfhout,
Kees L.M.C. Franken, Jaap Oostendorp,
Gert Jan Fleuren, Sjoerd H. van der Burg
and Cornelis J.M. Melief. Effective
Treatment of Vulvar Intraepithelial
Neoplasia by Vaccination against the
Oncoproteins E6 and E7 of Human
Papillomavirus Type 16. Accepted NEJM,
2009
Geurts TW, Balm AJM, Van Velthuysen ML,
Van Tinteren H, Burgers JA,
Van Zandwijk N, Klomp HM. Survival
after surgical resection of pulmonary
metastases and second primary squamous
cell lung carcinomas in head and neck
cancer. Head Neck 2009;31:220-226
Geurts TW, van Velthuysen ML,
Broekman F, van Huysduynen TH,
van den Brekel MW, van Zandwijk N,
van Tinteren H, Nederlof P, Balm AJ,
Brakenhoff RH. Differential diagnosis of
pulmonary carcinoma following head and
neck cancer by genetic analysis. Clin Cancer
Res. 2009;15:980-5
Goldhirsch A, Ingle JN, Gelber RD,
Coates AS, Thürlimann B, Senn HJ;
Panel members. Thresholds for therapies:
highlights of the St Gallen International
Expert Consensus on the primary therapy of
early breast cancer 2009. Ann Oncol.
2009;20:1319-29
Graafland NM, Leijte JA, Valdés Olmos RA,
Hoefnagel CA, Teertstra HJ, Horenblas S.
Scanning with 18F-FDG-PET/CT for
detection of pelvic nodal involvement in
inguinal node-positive penile carcinoma.
Eur Urol. 2009;56:339-45
Graafland NM, Leijte JA, Valdés Olmos RA,
van Boven HH, Nieweg OE, Horenblas S.
Repeat dynamic sentinel node biopsy in
locally recurrent penile carcinoma.
BJU Int. 2009
Hage JJ, van der Steen LPE. Locking,
jamming, and ratchet mechanisms of
sliding knots topologically revisited. World J
Surg 2009;33:751-757

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Publications (continued)
Hage JJ. Ease of tying arthroscopic knots
(letter) J Shoulder Elbow Surg 2009;18:e50
Hage JJ. Flipping and recruitment of
instrumentally tied knots as a means to
save suture material. Plast Reconstr Surg
2009
Hage JJ. Invited discussion of: Evaluation
of surgical procedures for sex reassignment:
a systematical review JPRAS 2009;
62:307-308
Hage JJ. Plastic surgery of the genital area
(Editorial). JPRAS 2009;62:292-293
Hage JJ. Simple, safe, and satisfactory
secondary penile enhancement after neartotal
oncologic amputation. Ann Plast Surg
2009;62:685-689
Hamming-Vrieze O, Balm AJM,
Heemsbergen WD, Hooft van Huysduynen T,
Rasch CRN. Regional control of melanoma
neck node metastasis after selective neck
dissection with or without adjuvant
radiotherapy. Arch Otolaryngol Head Neck
Surg 2009;135:795-800
Hilgers FJM. Primary postlaryngectomy
vocal, pulmonary and olfactory
rehabilitation: an update on the present
global state of the art. J Jap
Bronchoesophagol Soc 2009;60:142-150
Hughes BE, Leijte JA, Kroon BK, Shabbir MA,
Swallow TW, Heenan SD, Corbishley CM,
van Boven HH, Perry MJ, Watkin NA,
Horenblas S. Lymph Node Metastasis in
Intermediate-Risk Penile Squamous Cell
Cancer: A Two-Centre Experience. Eur
Urol. 2009
Jacobi I, Ackerstaff AH, Van Rossum,
Hilgers FJM. Acoustic evaluation and
speaker-subjective perception of
tracheoesophageal voicing. Proceedings of
the AVFA 3rd Advanced Voice Function
International Workshop, Madrid, Spain,
May 18-20, 2009;77-80
Johannsen M, Flörcken A, Bex A, Roigas J,
Cosentino M, Ficarra V, Kloeters C,
Rief M, Rogalla P, Miller K, Grünwald V.
Can tyrosine kinase inhibitors be
discontinued in patients with metastatic
renal cell carcinoma and a complete
response to treatment? A multicentre,
retrospective analysis. Eur Urol.
2009;55:1430-8
ANESTHESIA AND INTENSIVE CARE
Peter Schutte, Dirk Buitelaar, Tina Efthymiou, Christoph Hahn, Lenie Hulshoff, May Ronday,
Michael Srámek, Julia ten Cate, Ingeborg Vergouwe
In 2009 the emphasis of the activities of the Department of Anesthesia and Intensive
Care Medicine had to be on providing daily anesthesia and intensive care services due
to lack of personnel because of the departure of two staff members and illness of a
third. Our present research focuses on the role of peripheral nerve blockade with
long acting local anesthetic solutions as an adjuvant to general anesthesia in
oncologic surgery. The purpose is the reduction of both opioid consumption and
postoperative pain in oncologic surgical patients in order to prevent the development
of chronic pain and to shorten the hospital stay. Collaboration with the departments
of surgical oncology, head and neck oncology and urologic oncology innovations in
this respect are introduced. E.g. superficial cervical plexus anesthesia in patients
undergoing neck dissections and parotidectomies, field block in breast surgery and
transversus abdominis plane block (TAP-block) in robot assisted laparoscopic
prostatectomies. The trial N07TAP which evaluates the role of TAP-block in the
prevention of painful urine bladder spasms after robot assisted laparoscopic
prostatectomy is closed and the data are presently evaluated. The results will be
submitted in 2010. Future focus is on the role of regional and general anesthesia in
the prognosis of surgically treated oncologic patients.
PLASTIC SURGERY AND RECONSTRUCTIVE SURGERY
Joris Hage, Marieke van den Berg, Martine van Huizum, Arnoud van Turnhout, Leonie Woerdeman,
Brigitte Drost
Up until the year 2000, plastic surgical support in the NKI-AVL was seconded from
the Department of Plastic, Reconstructive and Handsurgery of the Academic Medical
Center in Amsterdam. Then, the Board of Directors and Surgical Oncologic Disciplines
of the NKI-AVL were committed to set up a full-fledged in-house department to
better facilitate the other surgical disciplines and further improve the quality of care.
Since, the academic staff of the department has increased from 0.8 FTE to 5.2 FTE,
including 1.0 FTE resident-in-training and 0.4 medical dermatographist. The
number of patients treated by this staff has, likewise, increased 600%.
After the initial period with emphasis on quantity and clinical quality of care, the
activities of the Department of Plastic and Reconstructive Surgery have concentrated
on reconstructive procedures following oncological resections performed by general
surgeons, urologists, and gynecologists. This reflects on the research items that are
focused predominately on primary and secondary breast reconstruction, and the
vascular and functional anatomy of tissue flaps. Parts of the research are conducted
in collaboration with the Departments of Radiology and Radiotherapy of the NKI-
AVL, with the Departments of Plastic and Reconstructive Surgery at the Academic
Medical Center in Amsterdam and the University Medical Center in Utrecht, and
with the Faculty of Movement Sciences at the Vrije Universiteit in Amsterdam.
(Inter)national collaborations
Joined training program for plastic surgical residents with the Departments of
Plastic, Reconstructive and Handsurgery at the Academic Medical Center,
Amsterdam, and the Red Cross Hospital, Beverwijk, The Netherlands.
Joined research program for PhD students with the Department of Plastic,
Reconstructive and Handsurgery at the University Medical Center Utrecht, Utrecht,
The Netherlands.
Joined research program for MSc students with the Faculty of Movement Sciences at
the Vrije Universiteit in Amsterdam, The Netherlands.

Research lines
Primary and secondary breast reconstruction
This part of research includes the surgical development, clinical implementation and
follow-up surveillance of innovative techniques for breast reconstruction. Functional
trade-offs are studied in collaboration with H.J. Veeger, MSc, PhD of the Faculty of
Movement Sciences at the Vrije Universiteit in Amsterdam, The Netherlands.
Vascular and functional anatomy of flaps
This part of research includes the development, implementation and surveillance of
pedicled and free transplantation of tissue flaps. Part of this study is done in
collaboration with prof. M. Kon, MD, PhD, Chief of the Department of Plastic,
Reconstructive and Handsurgery at the University Medical Center Utrecht, Utrecht,
The Netherlands.
DERMATOLOGY-MELANOMA
Esther Tjin, Henk Mallo, Wietze Van der Veen, Omgo Nieweg, Bin Kroon, John Haanen,
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, asignificant 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. To discriminate between these processes, we have
developed an ex vivo tumor explant model in a patient-specific setting. To determine
the presence and localisation of anti-melanoma immunity in patients, we are
analysing the presence of melanoma antigen-specific T cells in the melanoma tissue,
as compared to T cells in the adjacent skin tissue and in the peripheral blood.
Moreover, we are analysing the expression in melanoma of molecules that inhibit
T cell function, such as PD-1, PD-L1 and galectin-3.
Immunotherapy by the synergism of Monobenzone, Imiquimod and CpG for
metastatic melanoma patients
Hansje-Eva Teulings, Jasper van den Boorn, Debby Konijnenberg, Esther Tjin, Daisy Picavet,
Nico Meeuwenoord, Dmitri Filippov, Wietze van der Veen, Jan Bos, Cornelis Melief, John Haanen,
Rosalie Luiten
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 adjuvants imiquimod and cytosine-guanine
oligodeoxynucleotides (CpG) (MIC therapy). MIC treatment induced strong
melanoma-reactive immunity, which effectively eradicated established melanoma in
mice. MIC treatment also induced a protective memory response that conferred longterm
tumor-free survival, and protected against secondary tumor growth.
We will start a phase I clinical trial of MIC therapy in melanoma patients at the NKI-
AVL in 2010. The MIC 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-specific in vitro cultures nor non-myeloablative lymphodepletion, reducing
patient treatment burden. The MIC 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 toxicity of MIC therapy in melanoma patients as well as the
induction of melanocyte/melanoma-specific immunity.
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Publications (continued)
Jordanova ES, Gorter A, Ayachi O, Prins F,
Durrant LG, Kenter GG, van der Burg SH,
Fleuren GJ. Human Leukocyte Antigen
Class I, MHC Class I Chain-Related
Molecule A, and CD8+/Regulatory T-Cell
Ratio: Which Variable Determines Survival
of Cervical Cancer Patients?’ Clin Cancer
Res. 2008;14:2028-2035
Kappers I, van Sandick JW, Burgers SA,
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. 2009
Kappers I, van Sandick JW, Burgers JA,
Belderbos JS, Wouters MW,
van Zandwijk N, Klomp HM. Results of
combined modality treatment in patients
with non-small-cell lung cancer of the
superior sulcus and the rationale for
surgical resection. Eur J Cardiothorac Surg.
2009;36:741-6
Keijzer Ch, Buitelaar DR, Efthymiou KM,
Šrámek M, ten Cate J, Ronday M, Stoppa T,
Huitink JM, Schutte P. A Comparision of
Postoperative Throat and Neck Complaints
after the Use of the i-gel ® and the La
Premiere ® Disposable Laryngeal Mask: A
Double-Blinded, Randomized, Controlled
Trial. Anesth Analg 2009;109:1092-1094
Kenter GG. Therapeutische vaccinaties bij
HPV gerelateerde afwijkingen.
Reproduktieve geneeskunde, obstetrie en
gynaecologie anno 2009. Proceedings
Doelencongres 2009, Rotterdam
Kenter GG. Vaccinatie tegen HPV16 als
preventie of behandeling bij
cervixcarcinoom. Modern Medicine, 2009;1
Klomp HM, Kappers I. EGFR-TKI
treatment and surgical resection for
oligometastatic NSCLC? Onkologie.
2009;32:627-8
Klomp HM, Kappers I. Is pneumonectomy
justified in patients with locally advanced
NSCLC and persistent N2 disease after
induction chemotherapy? Ann Thorac
Surg. 2009;87:990-1
Klomp HM, Steyerberg EW, Wittens CH,
van Urk H, Habbema JD. A prognostic
model for amputation in critical lower limb
ischemia. Vasc Med. 2009;14:109-15

164
SURGICAL ONCOLOGY
Publications (continued)
Klomp HM, Steyerberg EW, Habbema JD,
van Urk H; ESES study group. What is the
evidence on efficacy of spinal cord
stimulation in (subgroups of) patients with
critical limb ischemia? Ann Vasc Surg.
2009;23:355-63 (review)
Kloth JN, Gorter A, Fleuren GJ, Oosting J,
Uljee S, ter Haar N, Dreef EJ, Kenter GG,
Jordanova ES Elevated expression of Serpin
A1 and SerpinA3 in HLA-positive cervical
carcinoma J Pathol. 2008;215:222-230
Kloth JN, Kenter GG, Spijker HS, Uljee S,
Corver WE, Jordanova ES, Fleuren GJ,
Gorter A. Expression of Smad2 and Smad4
in cervical cancer: absent nuclear Smad4
expression correlates with poor survival.
Mod Pathol. 2008;21:866-875
Kluijt I, de Jong D, Teertstra HJ,
Axwijk PH, Gille JJ, Bell K, van Rens A,
van der Velden AW, Middelton L,
Horenblas S. Early onset of renal cancer in
a family with Birt-Hogg-Dubé syndrome.
Clin Genet. 2009;75:537-43
Korse CM, Bonfrer JM, Beurden M van,
Verheijen RH, Rookus MA. Estradiol and
testosterone levels are lower after
oophorectomy than after natural
menopause. Tumour Biol 2009;30:37-42
Kreeft A, Lohuis PJ, Smeele LE. Stent
repair of an anastomotic pseudoaneurysm
of the carotid artery after free fibula
transplantation. Br J Oral Maxillofac Surg
2009;47:225-227
Kreeft A, Tan IB, van den Brekel MW,
Hilgers FJ, Balm AJ. The surgical dilemma
of ‘functional inoperability’ in oral and
oropharyngeal cancer: current consensus on
operability with regard to functional results.
Clin Otolaryngol. 2009;34:140-6
Kreeft A, Van der Molen L, Hilgers FJM,
Balm AJM. Speech and swallowing after
surgical treatment of advanced oral and
oropharyngeal carcinoma: a systemic review
of the literature. Eur Arch Otorhinolaryngol
2009;266:1687-1696
Lans TE, van der Pol C, Wouters MW,
Schmitz PI, van Geel AN. Complications
in wound healing after chest wall resection
in cancer patients; a multivariate analysis
of 220 patients. J Thorac Oncol.
2009;4:639-43
Langenhoff BS, Krabbe PF, Ruers TJ.
Efficacy of follow-up after surgical
treatment of colorectal liver metastases.
Eur J Surg Oncol. 2009;35:180-6
Leijte JA, Graafland NM, Valdés Olmos RA,
van Boven HH, Hoefnagel CA,
Horenblas S. Prospective evaluation of
hybrid 18F-fluorodeoxyglucose positron
emission tomography/computed
tomography in staging clinically nodenegative
patients with penile carcinoma.
BJU Int. 2009;104:640-4
Leijte JA, Hughes B, Graafland NM,
Kroon BK, Olmos RA, Nieweg OE,
Corbishley C, Heenan S, Watkin N,
Horenblas S. Two-center evaluation of
dynamic sentinel node biopsy for
squamous cell carcinoma of the penis.
J Clin Oncol. 2009;27:3325-9
Leijte JA, van der Ploeg IM,
Valdés Olmos RA, Nieweg OE,
Horenblas S. Visualization of tumor
blockage and rerouting of lymphatic
drainage in penile cancer patients by use
of SPECT/CT. J Nucl Med.
2009;50:364-7
Linthorst Homan MW, Spuls PI,
de Korte J, Bos JD, Sprangers MA,
van der Veen JP. The burden of vitiligo:
patient characteristics associated with
quality of life. J Am Acad Dermatol.
2009;61:411-20
Lohuis PJ, Tan ML, Bonte K,
van den Brekel MW, Balm AJ,
Vermeersch HB. Superficial
parotidectomy via facelift incision. Ann
Otol Rhinol Laryngol. 2009;118:276-80
Metha AM, Jordanova ES, Corver WE,
van Wezel T, Uh HW, Kenter GG,
Jan Fleuren G. Single nucleotide
polymorphisms in antigen processing
machinery component ERAP 1
significantly associate with clinical
outcome in cervical carcinoma.. Genes
Chromosomes Cancer. 2009;48:410-8
Mook S, Bonnefoi H, Pruneri G,
Larsimont D, Jaskiewicz J, Sabadell MD,
Macgrogan G, Van ‘t Veer LJ, Cardoso F,
Rutgers EJ. Daily clinical practice of
fresh tumour tissue freezing and gene
expression profiling; logistics pilot study
preceding the MINDACT trial. Eur J
Cancer. 2009;45:1201-8
Mook S, Schmidt MK, Rutgers EJ,
van de Velde AO, Visser O, Rutgers SM,
Armstrong N, Van ‘t Veer LJ, Ravdin PM.
Calibration and discriminatory accuracy
of prognosis calculation for breast cancer
with the online Adjuvant! program: a
hospital-based retrospective cohort study.
Lancet Oncol. 2009;10:1070-6
Mook S, Schmidt MK, Viale G, Pruneri G,
Eekhout I, Floore A, Glas AM, Bogaerts J,
Cardoso F, Piccart-Gebhart MJ, Rutgers ET,
Van ‘t Veer LJ; TRANSBIG Consortium.
The 70-gene prognosis-signature predicts
disease outcome in breast cancer patients
with 1-3 positive lymph nodes in an
independent validation study. Breast
Cancer Res Treat. 2009;116:295-302
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. 2009
Nieweg OE. False-negative sentinel
node biopsy. Ann Surg Oncol.
2009;16:2089-91
Nieweg OE. Local treatment for primary
melanoma. In: Atlas of procedures in
surgical oncology with critical, evidencebased
commentary notes. Audisio RA,
redacteur. World Scientific Publishing Co
Pte Ltd, Singapore, 2009:219-22
Nyst HJ, Tan IB, Stewart FA, Balm AJM.
Is photodynamic therapy a good
alternative to surgery and radiotherapy
in the treatment of head and neck
cancer? Photodiagnosis Photodyn Ther
2009;1:3-11
Ortonne JP, Arellano I, Berneburg M,
Cestari T, Chan H, Grimes P, Hexsel D,
Im S, Lim J, Lui H, Pandya A, Picardo M,
Rendon M, Taylor S, Van Der Veen JP,
Westerhof W. A global survey of the role
of ultraviolet radiation and hormonal
influences in the development of
melasma. J Eur Acad Dermatol Venereol.
2009

Pengel KE, Loo CE, Teertstra HJ, Muller SH,
Wesseling J, Peterse JL, Bartelink H,
Rutgers EJ, Gilhuijs KG. The impact of
preoperative MRI on breast-conserving
surgery of invasive cancer: a comparative
cohort study. Breast Cancer Res Treat.
2009;116:161-9
Pettaway CA, Horenblas S. Penile
cancer: incremental insights into etiology,
diagnosis, staging, and management.
World J Urol. 2009;27:139-40
Piersma SJ, Welters MJ, van der Hulst JM,
Kloth JN, Kwappenberg KM, Trimbos BJ,
Melief CJ, Hellebrekers BW, Fleuren GJ,
Kenter GG, Offringa R, van der Burg SH.
Human papilloma virus specific T cells
infiltrating cervical cancer and draining
lymph nodes show remarkably frequent
use of HLA-DQ and -DP as a restriction
element. Int J Cancer. 2008;122:486-94
Protzel C, Alcaraz A, Horenblas S,
Pizzocaro G, Zlotta A, Hakenberg OW.
Lymphadenectomy in the surgical
management of penile cancer. Eur Urol.
2009;55:1075-88
Rezaul Karim ,Ekaterina Jordanova,
Sytse Piersma, Gemma Kenter,
Lieping Chen, Judith Boer,
Cornelis Melief, Sjoerd van der Burg.
B7-H1 (PD-L1) expressed on cancer cells
confers survival benefit to patients with
cervical carcinoma highly infiltrated by
regulatory T-cells. Accepted CCR 2009
Rickelt J, Hoekstra H, van Coevorden F,
de Vreeze R, Verhoef C, van Geel AN.
Forequarter amputation for malignancy.
Br J Surg. 2009
Ruers TJ, Wiering B, van der Sijp JR,
Roumen RM, de Jong KP, Comans EF,
Pruim J, Dekker HM, Krabbe PF, Oyen WJ.
Improved Selection of Patients for
Hepatic Surgery of Colorectal Liver
Metastases with 18F-FDG PET: A
Randomized Study. Nucl Med.
2009;50:1036-1041
Russell NS, Hoving S, Heeneman S,
Hage JJ, Woerdeman LA, de Bree R,
Lohuis PJ, Smeele LE, Cleutjens J,
Valenkamp A, Dorresteijn LD, Dalesio O,
Daemen MJ, Stewart FA. Novel insights
into pathological changes in muscular
arteries of radiotherapy patients.
Radiother Oncol 2009;92:477-483
Scheenstra RJ, Muller SH, Vincent A,
Sinaasappel M, Zuur JK, Hilgers FJM.
Endotracheal temperature and humidity
measurements in laryngectomized
patients: intra- and inter-patient
variability. Med Biol Eng Comput
2009;47:773-782
Stollman TH, Ruers TJ, Oyen WJ,
Boerman OC. New targeted probes for
radioimaging of angiogenesis.Methods.
2009;48:188-92
Stollman TH, Scheer MG, Franssen GM,
Verrijp KN, Oyen WJ, Ruers TJ,
Leenders WP, Boerman OC. Tumor
accumulation of radiolabeled
bevacizumab due to targeting of celland
matrix-associated VEGF-A isoforms.
Cancer Biother Radiopharm.
2009;24:195-200
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. 2009
Straver ME, Rutgers EJ, Oldenburg HS,
Wesseling J, Linn SC, Russell NS,
Vrancken Peeters MJ. Accurate axillary
lymph node dissection is feasible after
neoadjuvant chemotherapy. Am J Surg.
2009;198:46-50
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;45:2284-92
Swellengrebel HA, Zoetmulder FA,
Smeenk RM, Antonini N, Verwaal VJ.
Quantitative intra-operative assessment
of peritoneal carcinomatosis - a
comparison of three prognostic tools.
Eur J Surg Oncol. 2009;35:1078-84
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. 2009
165
SURGICAL ONCOLOGY
Terlou A, Hage JJ, Beurden M van.
Skinning clitorectomy and skin replacement
in women with vulvar intraepithelial
neoplasia. J Plast Reconstr Aesthet Surg
2009;62:341-5
Te Velde EA, Wouters MJWM, Nieweg OE,
Haanen JBAG, Verwaal VJ. Partiële
dunnedarmresectie voor metastasen
van melanoom. Ned Tijdschr Oncol
2009;6:165-70
Te Velde E.A, Veerman T, Subramaniam V,
Ruers T. The Use of Fluorescent Dyes and
Probes in Surgical Oncology. Eur J Surg
Oncol. 2009
Upile T, Jerjes W, Sterenborg HJ,
El-Naggar AK, Sandison A, Witjes MJ,
Biel MA, Bigio I, Wong BJ, Gillenwater A,
Macrobert AJ, Robinson DJ, Betz CS,
Stepp H, Bolotine L, McKenzie G,
Mosse CA, Barr H, Chen Z, Berg K,
D’Cruz AK, Stone N, Kendall C, Fisher S,
Leunig A, Olivo M, Richards-Kortum R,
Soo KC, Bagnato V, Choo-Smith LP,
Svanberg K, Tan IB, Wilson BC,
Wolfsen H, Yodh AG, Hopper C. Head
and neck optical diagnostics: vision of the
future of surgery. Head Neck Oncol
2009;1:25
Valdés Olmos RA, Vidal-Sicart
S, Nieweg OE. SPECT-CT and real-time
intraoperative imaging: new tools for
sentinel node localization and radioguided
surgery? Eur J Nucl Med Mol Imaging
2009;36:1-5
Van den Boorn JG, Konijnenberg D,
Douwenga W, Van Swieten P,
Drijfhout J-W, Van der Veen JPW, Bos JD,
Melief CJM, Luiten RM. Chemically
induced vitiligo sheds new light on
melanoma immunotherapy. Pigment Cell &
Melanoma Research 2008;21-327
Van den Boorn J.G, Konijnenberg D,
Dellemijn T.A, Van der Veen J.P.W,
Bos J.D, Melief C.J, Vyth-Dreese F.A,
Luiten R.M. Autoimmune destruction of
Skin Melanocytes by Perilesional T Cells
from Vitiligo Patients. J Invest Dermatol.
2009;129:2220-32

166
SURGICAL ONCOLOGY
Publications (continued)
Van den Broek GB, Wildeman M, Rasch CR,
Armstrong N, Schuuring E, Begg AC,
Looijenga LH, Scheper R, van der Wal JE,
Menkema L, van Diest PJ, Balm AJ,
van Velthuysen ML, van den Brekel MW.
Molecular markers predict outcome in
squamous cell carcinoma of the head and
neck after concomitant cisplatin-based
chemoradiation. Int J Cancer.
2009;124:2643-50
Van den Hende M, van Poelgeest MI,
van der Hulst JM, de Jong J, Drijfhout JW,
Fleuren GJ, Valentijn AR, Wafelman AR,
Slappendel GM, Melief CJ, Offringa R,
van der Burg SH, Kenter GG. Skin
reactions to human papillomavirus (HPV)
16 specific antigens intradermally injected
in healthy subjects and patients with
cervical neoplasia. Int J Cancer.
2008;123:146-52
Van der Molen L, Van Rossum MA,
Ackerstaff AH, Smeele LE, Rasch CRN,
Hilgers FJM. Pretreatment organ function
in patients with advanced head and neck
cancer: clinical outcome measures and
patients’ views. BMC Ear Nose Throat
Disord. 2009;9:10
Van der Molen L, Van Rossum MA,
Burkhead LM, Smeele LE, Hilgers FJM.
Functional outcomes and rehabilitation
strategies in patients, treated with chemoradiation
for advanced head and neck
cancer: A systematic review. Eur Arch
Otorhinolaryngol 2009;266:889-900
Van den Tillaart SA, Kenter GG, Peters AA,
Dekker FW, Gaarenstroom KN, Fleuren GJ,
Trimbos JB. Nerve-sparing radical
hysterectomy: local recurrence rate,
feasibility, and safety in cervical cancer
patients stage IA to IIA Int J Gynecol
Cancer. 2009;19:39-45
Van der Ploeg IM, Kroon BB, Antonini N,
Valdés Olmos RA, Nieweg OE.
Comparison of three micromorphometric
pathology classifications of melanoma
metastases in the sentinel node. Ann Surg.
2009;250:301-4
Van der Ploeg IMC, Kroon BBR, Antonini N,
Valdés Olmos RA, Nieweg OE. Is
completion lymph node dissection needed in
case of minimal melanoma metastasis in
the sentinel node? Ann Surg
2009;249:003-7
Van der Ploeg IM, Kroon BB,
Valdés Olmos RA, Nieweg OE.
Evaluation of Lymphatic Drainage
Patterns to the Groin and Implications
for the Extent of Groin Dissection in
Melanoma Patients. Ann Surg Oncol.
2009
Van der Ploeg IM, Nieweg OE, Kroon BB,
Rutgers EJ, Baas-Vrancken Peeters MJ,
Vogel WV, Hoefnagel CA, Olmos RA.
The yield of SPECT/CT for anatomical
lymphatic mapping in patients with
breast cancer. Eur J Nucl Med Mol
Imaging. 2009;36:903-9
Van der Ploeg IM, Oldenburg HS,
Rutgers EJ, Baas-Vrancken Peeters MJ,
Kroon BB, Valdés Olmos RA, Nieweg OE.
Lymphatic Drainage Patterns from the
Treated Breast. Ann Surg Oncol. 2009
Van der Ploeg IM, Russell NS, Nieweg OE,
Oldenburg HS, Kroon BB, Olmos RA,
Rutgers EJ. Lymphatic drainage patterns
in breast cancer patients who previously
underwent mantle field radiation. Ann
Surg Oncol. 2009;16:2295-9
Van der Ploeg IM, Valdes Olmos RA,
Kroon BB, Rutgers EJ, Nieweg OE. The
hidden sentinel node and SPECT/CT in
breast cancer patients. Eur J Nucl Med
Mol Imaging. 2009;36:6-11
Van der Ploeg IM, Valdés Olmos RA,
Kroon BB, Wouters MW,
van den Brekel MW, Vogel WV,
Hoefnagel CA, Nieweg OE. The yield of
SPECT/CT for anatomical lymphatic
mapping in patients with melanoma.
Ann Surg Oncol. 2009;16:1537-42
Van der Ploeg IMC, Valdés Olmos RA,
Kroon BBR, Nieweg OE. Evaluation of
lymphatic drainage patterns to the groin
and implications for the extent of groin
dissection in melanoma patients. Ann
Surg Oncol, 2009;16:2994-9
Van der Poel HG, W. de Blok. Role of
the extent of fascia preservation and
erectile function after robot-assisted
laparoscopic prostatectomy. Urology.
2009;73:816-21
Van der Poel HG, W. de Blok, N. Joshi,
E. van Muilekom. Urine continence after
robot-assisted laparoscopic prostatectomy
improves by preservation of the lateral
prostate fascia Eur Urol 2009
Van der Poel HG. Chemical castration
and survival in low risk prostate cancer.
Eur Urol 2009
Van der Poel HG. Editorial Comment
on: A Randomized Phase 1 Study of
Testosterone Replacement for Patients
with Low-Risk Castration-Resistant
Prostate Cancer. Eur Urol. 2009
Van der Poorten VLM, Hart A,
Vauterin T, Jeunen G, Schoenaers J,
Hamoir M, Balm AJM, Stennert E,
Guntinas-Lichius O, Delaere P.
Prognostic index for patients with parotid
carcinoma: international external
validation in a Belgian-German
database. Cancer 2009;115:540-550
Van der Putten L, van den Broek GB,
de Bree R, van den Brekel MW, Balm AJ,
Hoebers FJ, Doornaert P, Leemans CR,
Rasch CR. Effectiveness of salvage
selective and modified radical neck
dissection for regional pathologic
lymphadenopathy after chemoradiation.
Head Neck. 2009;31:593-603
Van der Veen JPW. Nieuwe
ontwikkelingen bij vitiligo. Ned. Tijdschr.
Dermatol 2009;19:97
Van Gijn W, Wouters MW, Peeters KC,
van de Velde CJ. Nationwide outcome
registrations to improve quality of care in
rectal surgery. An initiative of the
European Society of Surgical Oncology.
J Surg Oncol. 2009;99:491-6
Van Riel E, Wárlám-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 2009
Van Rossum MA, Van As-Brooks CJ,
Hilgers FJM, Roozen M. Quality of
‘glottal’ stops in tracheoesophageal
speakers. Clin Linguist Phon 2009:1:1-14

Van Rossum MA, Jongmans P,
Van As-Brooks CJ, Hilgers FJM.
Spraakverstaanbaarheid bij
tracheoesophageal sprekers; een
therapieprogramma voor het verbeteren
van spraakverstaanbaarheid. Logopedie
en Foniatrie 2009;5:172-176
Van Winden AW, Gast MC, Beijnen JH,
Rutgers EJ, Grobbee DE, Peeters PH,
van Gils CH. Validation of previously
identified serum biomarkers for breast
cancer with SELDI-TOF MS: a case
control study. BMC Med Genomics.
2009;2:4
Veenstra HJ, Van der Ploeg IMC,
Wouters MWJM, Kroon BBR, Nieweg OE.
Re-evaluation of the rate of local-regional
recurrence in melanoma patients with a
positive sentinel node compared to
patients with palpable nodal
involvement. Ann Surg Oncol. 2009
Vermeeren L, Valdes Olmos RA,
Meinhardt W, Bex A, van der Poel HG,
Vogel WV, Sivro F, Hoefnagel CA,
Horenblas S. Value of SPECT/CT for
detection and anatomic localization of
sentinel lymph node before laparoscopic
sentinel node lymphadenectomy in
prostate carcinoma. J Nucl Med.
2009;50:865-70
Vermeeren L, Valdés Olmos RA,
Meinhardt W, Bex A, van der Poel HG,
Vogel WV, Sivro F, Hoefnagel CA,
Horenblas S. Intraoperative
radioguidance with a portable gamma
camera: a novel technique for
laparoscopic sentinel node localisation in
urological malignancies. Eur J Nucl Med
Mol Imaging. 2009;36:1029-36
Vermeeren L, Valdés Olmos RA,
Meinhardt W, Bex A, van der Poel HG,
Vogel WV, Sivro F, Hoefnagel CA,
Horenblas S. Value of SPECT/CT for
detection and anatomic localization of
sentinel lymph nodes before laparoscopic
sentinel node lymphadenectomy in
prostate carcinoma. J Nucl Med.
2009;50:865-70
Vermeeren L, van der Ploeg IM, Olmos RA,
Meinhardt W, Klop WM, Kroon BB,
Nieweg OE. SPECT/CT for preoperative
sentinel node localization. J Surg Oncol.
2009
Verwaal VJ. Long-term results of
cytoreduction and HIPEC followed by
systemic chemotherapy. Cancer J.
2009;15:212-5
Wildeman MA, Gibcus JH,
Hauptmann M, Begg AC,
van Velthuysen ML, Hoebers FJ,
Mastik MF, Schuuring E, van der Wal JE,
van den Brekel MW. Radiotherapy in
laryngeal carcinoma: can a panel of
13 markers predict response?
Laryngoscope. 2009;119:316-22
Wouters MW, Krijnen P, Le Cessie S,
Gooiker GA, Guicherit OR, Marinelli AW,
Kievit J, Tollenaar RA. Volume- or
outcome-based referral to improve quality
of care for esophageal cancer surgery in
The Netherlands. J Surg Oncol.
2009;99:481-7
Wouters MW, Gooiker GA,
van der Hoeven JJM, Klinkenbijl JHG,
Koning CCE en Tollenaar RAEM.
Ketenzorg wordt geketende zorg:
overregulering oncologie dreigt door
bureaucratisch beleid Inspectie. Medisch
Contact 2009;64:1167-1170
Wouters MW, Karim-Kos HE, le Cessie S,
Wijnhoven BP, Stassen LP, Steup WH,
Tilanus HW, Tollenaar RA.
Centralization of esophageal cancer
surgery: does it improve clinical outcome?
Ann Surg Oncol. 2009;16:1789-98
Wreesmann VB, Smeele LM,
Hilgers FJM, Lohuis PJFM. Closure of
tracheoesophageal fistula with prefabricated
revascularized bilaminar radial forearm free
flap. Head Neck 2009;31:838-842
Zijlmans HJ, Fleuren GJ, Hazelbag S,
Sier CF, Dreef EJ, Kenter GG, Gorter A.
Expression of endoglin (CD105) in
cervical cancer. Br J Cancer. 2009
Zuur CL, Simis YJ, Lamers EA, Hart AA,
Dreschler WA, Balm AJM, Rasch CR.
Risk factors for hearing loss in patients
treated with intensity-modulated
radiotherapy for head-and-neck tumors.
Int J Radiat Oncol Biol Phys
2009;74:490-496
167
SURGICAL ONCOLOGY
Zuur JK, Muller SH, Vincent A,
Sinaasappel M, De Jongh FHC,
Hilgers FJM. The influence of a heat and
moisture exchanger on tracheal climate in a
cold environment. Medical Engineering and
Physics 2009;31:852-857
Book chapters
Ackerstaff AH, Hilgers FJM. Pulmonary
rehabilitation after total laryngectomy. In:
Voice restoration after total laryngectomy;
Current science and future perspectives.
Edited by Umanath K. Nayak, and Rehan
Kazi. With Forewords by Eric D. Blom and
Frans J.M. Hilgers. Publisher: Byword
Books Private Limited, Delhi, India; 2009,
pp 89-100
Ackerstaff AH, Hilgers FJM. Quality-of-life
issues after total laryngectomy. In: Voice
restoration after total laryngectomy; Current
science and future perspectives. Edited by
Umanath K. Nayak, and Rehan Kazi.
With Forewords by Eric D. Blom and Frans
J.M. Hilgers. Publisher: Byword Books
Private Limited, Delhi, India; 2009,
pp 101-112
Hilgers FJM, Tan IB. Comprehensive
(prosthetic) voice, pulmonary and olfaction
rehabilitation after total laryngectomy.
Book chapter (chapter 96) in: ‘Head and
Neck Surgery’; editor in chief Chris de
Souza; Jaypee Brothers Medical Publishers
(P) LTD, New Delhi, India, 2009;
pp 1390-1408
Jacobi I, Ackerstaff AH, Van Rossum MA,
Hilgers FJM. Acoustic evaluation and
speaker-subjective perception of
tracheoesophageal voicing. Proceedings of
the AVFA 3rd Advanced Voice Function
International Workshop, Madrid, Spain,
2009; pp. 77-80
Menke HE, Van der Veen JPW. Vitiligo.
hoofdstuk 15, p.153-160. In: Samenvatting
richtlijnen Dermatologie 2009, Red Van
Everdingen JJE, Borgonjen RJ. DCHG,
medische uitgeverij, Haarlem 2009

168
SURGICAL ONCOLOGY
In press
Bień S, Okła S, Van As-Brooks CJ,
Ackerstaff AH. The effect of a Heat and
Moisture Exchanger (Provox((R))
HME) on pulmonary protection after
total laryngectomy: a randomized
controlled study. Eur Arch
Otorhinolaryngol.
Dirven R, Kooijman PGC, Maal TJJ,
Hilgers FJM, Bergé SJ, Marres HAM.
An external neck brace to support the
peristomal fixation of an automatic
stoma valve (ASV): 3D
sterophotogrammetrical assessment. Acta
Otolaryngol.
Hilgers FJM, Ackerstaff AH,
Van Rossum MA, Jacobi I, Balm AJM,
Tan IB, Van den Brekel MWM. Clinical
phase I / feasibility study of the next
generation indwelling Provox voice
prosthesis (Provox Vega). Acta
Otolaryngol.
Hilgers FJM, Balm AJM,
van den Brekel MWM, Tan IB. Chapter
13. Surgery for larynx and
hypopharyngeal cancer; e. Voice
restoration. In: Surgery of Larynx and
Trachea. Editors: Remacle and Eckel.
Springer Verlag, Stuttgart
Hilgers FJM, Van den Brekel MWM.
Chapter 113: Vocal and Speech
Rehabilitation Following Laryngectomy.
In: Cummings Otolaryngology: Head
and Neck Surgery. 5 th Edition. Editors:
Flint, Haughey, Richardson, Robbins,
Thomas, Niparko, and Lund. Elsevier,
Philadelphia
Jongmans P, Wempe AG,
Van Tinteren H, Hilgers FJM, Pols LCW,
Van As-Brooks C. The acoustic analysis
of the voiced-voiceless distinction in
tracheoesophageal speech. J Speech Lang
Hear Res.
Keijzer C, Buitelaar D, Efthymiou K,
Šrámek M, ten Cate J, Ronday M,
Stoppa T, Huitink J, Schutte P. A
Comparison of Postoperative Throat and
Neck Complaints After the Use of the
i-gel ® and the La Premiere ® Disposable
Laryngeal Mask: A Double-Blinded,
Randomized, Controlled Trial. Anesth
Analg 2009
Rasch CRN, Hauptmann CRN,
Schornagel JH, Wijers O, Buter J,
Gregor T, Wiggenraad R, De Boer JP,
Ackerstaff AH, Kroger R, Hoebers FJP,
Hilgers FJP, Balm AJM. Intra-arterial
versus intravenous chemoradiation for
advanced head and neck cancer: results of
a randomized phase III trial. Cancer
Scheenstra RJ, Muller SH, Vincent A,
Ackerstaff AH, Jacobi I, Hilgers FJM.
Short-term endotracheal climate changes
and clinical effects of a heat and moisture
exchanger with an integrated electrostatic
virus and bacterial filter developed for
laryngectomized individuals. Acta
Otolaryngol.
Scheenstra RJ, Muller SH, Vincent A,
Sinaasappel M, Hilgers FJM. The
influence of breathing resistance of heat
and moisture exchangers on tracheal
climate and breathing pattern in
laryngectomized individuals. Head Neck.
Van der Poel HG, Zevenhoven J,
Bergman A. Pim1 regulates androgendependent
survival signaling in prostate
cancer cells. Urol Int
Vermeeren L, Van der Ploeg IM,
Olmos RA, Meinhardt W, Klop WMC,
Kroon BBR, Nieweg OE. SPECT/CT for
preoperative sentinel node localization. J
Surg Oncol.
Vermeeren L, Tanis PJ, Nieweg OE,
Valdés Olmos RA. Lymphoscintigraphy
of a breast tumor showing focal tracer
accumulation along the falciform
ligament of the liver. Clin Nucl Med.

BIOMETRICS DEPARTMENT
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 notifications 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).
Figure 1: TENALEA logo
TENALEA (figure 1) is a service that provides these online tools to support the data
management process for clinical trials. The service is currently co-funded by the
eTEN programme of the European Union. The TENALEA-ID project – TENALEA
Initial Deployment – 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, and will continue until the end of
2010. We are the initiators and coordinators of the project. As initiators of the project
we stimulate different (European) Data Coordinating Centers to adopt our online
tools to support the process of clinical trial data capture and data management. As
coordinator of the project, the Biometrics department is responsible for the technical,
administrative and financial management of the project. The focus of the project is
deployment of a randomization service and an electronic Case Report Forms (eCRF)
service (figure 2). 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.
Figure 2: Example eCRF screen
169
BIOMETRICS
Department head, group leader 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
Danny Baars Technical staff
Frauwkje Bessels Technical staff
Audi Boucher Technical staff
Monique Carreno Technical staff
Jolien Claassen Technical staff
Saskia Cooke Technical staff
Gerda de Jong Technical staff
Marjolijn De Waal MSc Technical staff
Wilma Deurloo Technical staff
Brigitte Dufournij Technical staff
Ernesto Fernandez Salcedo MSc Technical staff
Christiane Hagenaars Technical staff
Song-Hieng Hau MSc Technical staff
Annelies Hiemstra Technical staff
Paula Hoekstra MSc 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
Carla Modder Technical staff
Pietje Muller Technical staff
Cecile Paulus van Pauwvliet Technical staff
Loes Pronk MSc Technical staff
Harriet Rehorst Technical staff
Anneke Reinders Technical staff
Jolanda Remmelzwaal Technical staff
Daniel Roberts Technical staff
Marielle Roskam Technical staff
Dea Storm Technical staff
Beata Sznajder Technical staff
Ria Tilgenkamp Technical staff
Alex Torres Acosta Technical staff
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
Anneke Wals Technical staff
Lidwina Wever Technical staff
Els Willemse Technical staff
Jeroen Zandbergen MSc Technical staff
Lonny Ziblat Technical staff

170
BIOMETRICS
Publications
Al Uwini S, Antonini N, Poortmans PM,
Boersma L, Hurkmans C, Leer JW,
Horiot JC, Struikmans H, Bartelink H.
The influence of the use of CT-planning on
the irradiated boost volume in breast
conserving treatment. Radiother Oncol
2009;93:87-93
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 2009
Borgemeester MC, van den Brekel MW,
van Tinteren H, Smeele LE, Pameijer FA,
van Velthuysen ML, Balm AJ. Ultrasoundguided
aspiration cytology for the
assessment of the clinically N0 neck: factors
influencing its accuracy. Head Neck
2008;30:1505-13
Bueno-de-Mesquita JM, Nuyten DS,
Wesseling J, van Tinteren H, Linn SC,
van de Vijver MJ. The impact of interobserver
variation in pathological
assessment of node-negative breast cancer
on clinical risk assessment and patient
selection for adjuvant systemic treatment.
Ann Oncol 2009
de Vries RR, Nieuwenhuijzen JA,
van Tinteren H, Oddens JR, Visser O,
van der Poel HG, Bex A, Meinhardt W,
Horenblas S. Prostate-sparing cystectomy:
long-term oncological results. BJU Int
2009;104:1239-43
Gast MC, van Tinteren H, Bontenbal M,
van Hoesel RQ, Nooij MA, Rodenhuis S,
Span PN, Tjan-Heijnen VC, de Vries EG,
Harris N, Twisk JW, Schellens JH,
Beijnen JH. Haptoglobin phenotype is not
a predictor of recurrence free survival in
high-risk primary breast cancer patients.
BMC Cancer 2008;8:389
Geurts TW, van Velthuysen ML,
Broekman F, van Huysduynen TH,
van den Brekel MW, van Zandwijk N,
van Tinteren H, Nederlof P, Balm AJ,
Brakenhoff RH. Differential diagnosis of
pulmonary carcinoma following head and
neck cancer by genetic analysis. Clin
Cancer Res 2009;15:980-5
The TENALEA Initial Deployment project completed its third Milestone term with an
Audit Certificate in November 2009. The Audit Certificate, which was issued by
IT&GCP Consulting SPRL, marks the completion of a long period of software
enhancement, documentation improvement and QA Standard Operating Procedures
development required to bring the TENALEA Randomization service and the
TENALEA eCRF service into a validated state. The Audit Certificate provides a
confirmation by external auditors of TENALEA compliance to regulatory
requirements, and is essential for customers of the service requiring a validated
system, such as pharmaceutical industry and the large Academic clinical trial
coordination centers.
During 2009, TENALEA extended its service portfolio with three new services, each
based on caBIG software modules. The cancer Biomedical Informatics Grid, or
caBIG (figure 3) is an initiative of the National Cancer Institute, part of the National
Institutes of Health in the USA. caBIG is a voluntary virtual informatics infrastructure
intended to connect data, research tools, scientists, and organizations to leverage
their combined strengths and expertise in an open federated environment with
widely accepted standards and shared tools. TENALEA is currently piloting caTissue,
caAERS and caCORE EVS. caTissue is caBIG’s bio-repository tool for bio-specimen
inventory management, tracking, and annotation. This tool permits users to enter
and retrieve data concerning the collection, storage, quality assurance, and
distribution of bio-specimens. The Cancer Adverse Event Reporting System (caAERS)
is an open source software tool that is used to collect, process, and report adverse
events that occur during clinical trials. This tool supports regulatory and protocol
compliance for adverse event report and allows local collection, management, and
querying of adverse event data, whether routine or serious. This tool also supports
service based integration of data from other clinical trials management systems.
The Enterprise Vocabulary Services or EVS encompass terminology development,
licensing and publication, software development and licensing, and operations
support to address the broad spectrum of terminology needs in Cancer Clinical
Research. The adoption of EVS in TENALEA Randomization and eCRF will allow
better standardization of Clinical Trial forms, and adherence to International
standards. The caTissue and EVS services are intended to be operated with the
TENALEA eCRF Service for the AirForce project of the CTMM early 2010.
Figure 3: caBIG logo
The growth in number of trials and clinical trial coordination centers using
TENALEA Services has continued over 2009 as can be seen in figure 4. Indeed,
250 clinical trials running all over the world are using the TENALEA Randomization
service, while the TENALEA eCRF service – which was only recently introduced – is
operated for 8 clinical trials in UK, France, Poland, Indonesia and the Netherlands.
The difference in number of trials using TENALEA Randomization and the number
of trials using TENALEA eCRF is also related to the higher impact the introduction of
eCRF has on the operations of a clinical trial. With the validated state of the services
being achieved, TENALEA Randomization and TENALEA eCRF are now ready to
target larger trial centers.

Figure 4: Progress of the project measured as number of studies using ALEA and eCRFs.
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 randomization services, quality assurance, data
handling 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.
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 first to study
a reduction of therapy in Wilms’ tumors. Over the years the number of participating
centres 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 is
planned to finish inclusion by the end of 2009. The SIOP 2001 study introduced also
several new treatment strategies for patient not eligible for randomization compared
to previous practice. The SIOP 2001 study will remain open for patient inclusion to
strengthen descriptive exploratory analyses of other subgroups. The coming two
years also extra effort will be put in the collection of tumor specimen for the studies
on genotyping.
The SIOP 9301 and 2001 register now includes data on more than 6000
nephroblastomas and other renal malignancies (figure 5), 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%.
NVALT cancer studies We also function as Data Centre for the cancer studies of the
Dutch Chest Physician Association (NVALT) and we collaborate in planning,
designing and running their clinical trials in lung cancer and mesothelioma. More
than 75 hospitals in the Netherlands participate in their clinical trials. The Dutch
Cancer Society has given support for the management of data of 5 studies of the
12 trials that have or are being run by the group.
Publications (continued)
171
BIOMETRICS
Geurts TW, Balm AJ, van Velthuysen ML,
van Tinteren H, Burgers JA,
van Zandwijk N, Klomp HM. Survival
after surgical resection of pulmonary
metastases and second primary squamous
cell lung carcinomas in head and neck
cancer. Head Neck 2009;31:220-6
Jones HA, Antonini N, Hart AA, Peterse JL,
Horiot JC, Collin F, Poortmans PM,
Oei SB, Collette L, Struikmans H,
Van den Bogaert WF, Fourquet A, Jager JJ,
Schinagl DA, Warlam-Rodenhuis CC,
Bartelink H. Impact of pathological
characteristics on local relapse after breastconserving
therapy: a subgroup analysis
of the EORTC boost versus no boost trial.
J Clin Oncol 2009;27:4939-47
Koopman M, Venderbosch S,
van Tinteren H, Ligtenberg MJ,
Nagtegaal I, van Krieken JH, Punt CJ.
Predictive and prognostic markers for the
outcome of chemotherapy in advanced
colorectal cancer, a retrospective analysis of
the phase III randomised CAIRO study.
Eur J Cancer 2009;45:1999-2006
Maaskant JM, De Boer JP, Dalesio O,
Holtkamp MJ, Lucas C. The effectiveness of
chlorhexidine-silver sulfadiazine
impregnated central venous catheters in
patients receiving high-dose chemotherapy
followed by peripheral stem cell
transplantation. Eur J Cancer Care.
2009;18:477-82
Pantarotto JR, Piet AH, Vincent A,
van Sornsen de Koste JR, Senan S.
Motion analysis of 100 mediastinal lymph
nodes: potential pitfalls in treatment
planning and adaptive strategies. Int J
Radiat Oncol Biol Phys 2009;74:1092-9
Russell NS, Hoving S, Heeneman S,
Hage JJ, Woerdeman LA, de Bree R,
Lohuis PJ, Smeele L, Cleutjens J,
Valenkamp A, Dorresteijn LD, Dalesio O,
Daemen MJ, Stewart FA. Novel insights
into pathological changes in muscular
arteries of radiotherapy patients. Radiother
Oncol. 2009;92:477-83
Scheenstra RJ, Muller SH, Vincent A,
Sinaasappel M, Zuur JK, Hilgers FJ.
Endotracheal temperature and humidity
measurements in laryngectomized patients:
intra- and inter-patient variability. Med
Biol Eng Comput 2009;47:773-82

172
BIOMETRICS
Publications (continued)
Smit EF, Burgers SA, Biesma B, Smit HJ,
Eppinga P, Dingemans AM, Joerger M,
Schellens JH, Vincent A, van Zandwijk N,
Groen HJ. Randomized phase II and
pharmacogenetic study of pemetrexed
compared with pemetrexed plus carboplatin
in pretreated patients with advanced nonsmall-cell
lung cancer. J Clin Oncol
2009;27:2038-45
Spoelstra FO, van Sornsen de Koste JR,
Vincent A, Cuijpers JP, Slotman BJ,
Senan S. An evaluation of two internal
surrogates for determining the threedimensional
position of peripheral lung
tumors. Int J Radiat Oncol Biol Phys
2009;74:623-9
Straver ME, van Adrichem JC, Rutgers EJ,
Rodenhuis S, Linn SC, Loo CE, Gilhuijs KG,
Oldenburg HS, Wesseling J, Russell NS,
Antonini N, Vrancken Peeters MT.
Neoadjuvant systemic therapy in patients
with operable primary breast cancer: more
benefits than breast-conserving therapy. Ned
Tijdschr Geneeskd 2008;152:2519-25
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;45:2284-92
Swellengrebel HA, Zoetmulder FA,
Smeenk RM, Antonini N, Verwaal VJ.
Quantitative intra-operative assessment of
peritoneal carcinomatosis - a comparison of
three prognostic tools. Eur J Surg Oncol
2009;35:1078-84
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
2009
Tol J, Koopman M, Cats A, Rodenburg CJ,
Creemers GJ, Schrama JG, Erdkamp FL,
Vos AH, van Groeningen CJ, Sinnige HA,
Richel DJ, Voest EE, Dijkstra JR,
Vink-Borger ME, Antonini NF, Mol L,
van Krieken JH, Dalesio O, Punt CJ.
Chemotherapy, bevacizumab, and
cetuximab in metastatic colorectal cancer.
N Engl J Med 2009;360:563-72
Figure 5: Age distribution by sex for 6300 renal tumors in children aged 0 -10 years
In 2009 the results of the study on the role of platinum compounds as part of second
line therapies in advanced NSCLC (NVALT7) were published in the Journal of Clinical
Oncology. This was a randomized trial comparing pemetrexed (P) and pemetrexedcarboplatin
(PC) in patients relapsing after platinum-based chemotherapy. The study
showed that PC as second line treatment for relapsed NSCLC resulted in a statistically
significant 33% reduction of hazard of progression as compared to P alone. The
NVALT 10 is the follower of this study and compares erlotinib vs erlotinib and
chemotherapy combination in pretreated patients with advanced NSCLC. The study
was been opened to entry this year and already 41 patients have been randomized.
561 patients were randomized in the NVALT4 trial. The statistical report on this
randomized placebo-controlled study of docetaxel/carboplatin with celecoxib or
placebo in patients with locally advanced or metastatic non-small cell lung cancer,
was prepared in 2009. However, it became apparent that further data on the
characteristics of the tumor needed to be collected. Specimens and pathology slides
are currently being collected and analyzed.
The NVALT 5 is a phase III trial of the antiangiogenic agent Thalidomide in patients
with malignant pleural mesothelioma after first line chemotherapy. Centers in
Australia have also started participating and it is expected that the required number
of patients will be completed in 2010.
The accrual to 2 studies in resectable NSCLC low and high risk patients as
determined by preoperative PET SUVmax (NVALT8 A and B), has been
disappointing, with 52 patients entered in total. The organization of the study is
rather complex and involves a multidisciplinary team including surgeons, lung
physicians, nuclear medicine experts and pathologists, and requiring central analysis
of PET values. A study similar to the B study is being carried out in France and the
NVALT group is currently discussing cooperation with them to complete the research.
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).
To date a total of 27 have been included in this study.

DCCG studies Collaboration with the Dutch Colorectal Cancer Group (DCCG) has
resulted in several large phase III randomized studies in patients with colorectal
carcinoma for which we are the Statistical Center.
CAIRO 1 and CAIRO 2 studies in advanced colorectal cancer previously untreated
patients were rapidly carried out and in 2009 results of the CAIRO 2 were published
in the New England Journal of Medicine. Multiple ancillary questions based on the
patient data collected in the CAIRO studies are been performed and many have
already resulted in publications as can be seen from the publication list.
The CAIRO 3 is the third randomized phase III trial of the DCCG in patients with
advanced colorectal carcinoma. The study compares maintenance treatment with
capecitabine and bevacizumab versus observation after induction treatment with
capecitabine, oxaliplatin, and bevacizumab as first-line treatment. For this study, we
have developed an e-CRFs application. 339 patients have already been randomized by
61 centers. We will perform a confidential interim analysis mid 2010, about half way
in the study accrual.
In addition, the design of a study after radical resection of liver metastases of
colorectal cancer comparing bevacizumab in combination with capecitabine and
eloxatin vs eloxatin alone as adjuvant treatment (the HEPATICA study) has been
modified allowing patients receiving 3 courses of capecitabine and eloxatin preoperatively
and also allowing radiofrequency ablation of the metastses, in an attempt
to increase the accrual rate. This amendment has recently been approved by the
central Ethics Committee. To date 58 patients have been randomized in the study.
Other studies We are also involved in the coordination of the Rosel study, which is
another study of the DLCRG. This is a randomized clinical trial of radio-surgery
(stereotactic radiotherapy) or surgery in patients with stage A non-small cell lung
cancer who are fit to undergo primary resection. This study is partially supported by
the ZonW. Accrual is slow and a revision of the design, endpoints and sample size is
currently taking place.
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, 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 first confidential interim analysis report to be discussed by the independent data
monitoring committee. The second is a randomized double blind study of treatment
of hot flashes in breast cancer patients in menopause following chemotherapy for
their breast cancer. It is expected that accrual will be completed beginning 2010
allowing for the final analysis next year.
We coordinate with the radiotherapy department a large randomized study in young
women with early breast cancer. Almost 1700 patients have been entered by
participating centers in the Netherlands, France (288 patients) and Germany
(21 patients). Tumor material is being collected for translational research analyses.
Cosmetics results are being evaluated with pictures taken in series.
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.
Publications (continued)
173
BIOMETRICS
van den Berg JH, Nujien B, Beijnen JH,
Vincent A, van Tinteren H, Kluge J,
Woerdeman LA, Hennink WE, Storm G,
Schumacher TN, Haanen JB.
Optimization of intradermal vaccination
by DNA tattooing in human skin. Hum
Gene Ther 2009;20:181-9
van der Ploeg IM, Kroon BB, Antonini N,
Valdés Olmos RA, Nieweg OE.
Comparison of three micromorphometric
pathology classifications of melanoma
metastases in the sentinel node. Ann Surg
2009;250:301-4
van der Ploeg IM, Kroon BB, Antonini N,
Valdés Olmos RA, Nieweg OE. Is
completion lymph node dissection needed in
case of minimal melanoma metastasis in
the sentinel node? Ann Surg
2009;249:1003-7
van der Sande JJ, van Tinteren H,
Brandsma D, Jobsis GJ, Boogerd W. Brain
metastases in patients with pelvic or
abdominal malignancy do not prevail in the
posterior fossa: a retrospective study. J
Neurol 2009;256:1485-7
Vujanic GM, Harms D, Bohoslavsky R,
Leuschner I, de Kraker J, Sandstedt B.
Nonviable tumor tissue should not upstage
Wilms’ tumor from stage I to stage II: a
report from the SIOP 93-01 nephroblastoma
trial and study. Pediatr Dev Pathol
2009;12:111-5
Yang TI, 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 2009
Zuur JK, Muller SH, Vincent A,
Sinaasappel M, de Jongh FH, Hilgers FJ.
The influence of a heat and moisture
exchanger on tracheal climate in a cold
environment. Med Eng Phys 2009;31:852-7

174
CLINICAL TrIALS
CLINICAL TrIALS IN The
NeTherLANdS CANCer INSTITuTe
Type of Title Study Phase Activated No pts.
cancer study coordinator (closed) in NKI-
(nick name) in NKI-AVL AVL per
15/12/
ALL SITeS
2009
M05HOP An open label phase I dose escalation study of JHM Schellens I 28/06/2005 41
(HOPE) E7080 (01/08/2009)
M05RTB A phase III international randomized trial of M Verheij III 10/01/2006 4
single versus multiple fractions for re-irradiation
of painful bone metastases
M06OGP Phase I dose escalation study of oral JHM Schellens I 29/08/2006 38
(OGP) gemcitabine prodrug (LY2334737) alone and in (27/08/2009)
combination with erlotinib (Tarceva) in patients
with advanced solid tumors
M07CUP Molecular profiling as a strategy for the M Kerst other 27/03/2008 17
(CUP-PRINT) identification of primary solid tumour in (01/07/2009)
metastatic cancer patients
M07ERI An open label parallel group study to explore the JHM Schellens other 11/03/2008 10
(108) pharmacokinetics of eribulin mesylate (E7389)
in patients with advanced solid tumors and
normal or reduced hepatic function according to
the Child-Pugh system
M07HHK Cost effectiveness of scalp cooling in the JW Baars other 20/05/2008 *
prevention of chemotherapy-induced hair loss (19/11/2009)
M07KUC A phase I, open-label, study to assess the safety JHM Schellens I 25/06/2007 60
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 tumours
M07MKC A phase I dose escalating study evaluating MK-1775 JHM Schellens I 21/02/2008 42
in both monotherapy and in combination
with Gemcitabine, Cisplatin or Carboplatin in
adult patients with advanced solid tumors
M07OTS A phase I study of oral topotecan in subjects with JHM Schellens I 18/02/2008 5
cancer and impaired renal function
M07PFU Pharmacogenomic and pharmacokinetic safety JHM Schellens other 16/05/2007 13
and cost-saving analysis in patients treated with
fluoropyrimidines
* = not registered at trialbureau

175
CLINICAL TrIALS
Type of Title Study Phase Activated No pts.
cancer study coordinator (closed) in NKI-
(nick name) in NKI-AVL AVL per
15/12/
2009
M07POB The relationship between cost-effectiveness of WJ Nooijen other 10/01/2007 *
blood safety measures and the distribution of (23/11/2009)
patients receiving blood
M08CIP Chemotherapy induced peripheral neuropathy W Boogerd other 11/03/2009 *
(CI-PERINOMS) outcome measures standardisation study
M08EVC A phase I/II, non-randomized, multicenter, dose- JHM Schellens I/II 26/08/2008 1
(mTOR) escalating, two-stage efficacy and feasablity (14/12/2009)
study of the combination of everolimus and
capecitabine in patients with advanced malignancies
M08HYT A phase I open-label study of the safety, tolerability JHM Schellens I 15/09/2008 9
and pharmacokinetics of two schedules of oral
topotecan in combination with pazopanib in subjects
with advanced solid tumors
M08MEK Open-label, multicenter, dose-escalation phase I JHM Schellens I 04/02/2009 7
study with extension to evaluate safety,
pharmacokinetics and activitiy of RO4987655, a MEK
inhibitor, administered orally as monotherapy in
patients with advanced tumors
M09BGJ A phase I open-label, multicenter, dose escalation JHM Schellens I 10/12/2009 0
study of oral BGJ398, a pan FGF-R kinase inhibitor
in adult patients with advanced solid malignancies
M09DAZ A phase I open label multicenter study to assess JHM Schellens I 14/10/2009 3
the safety and tolerability, pharmacokinetics,
preliminary anti-tumor activity of ascending doses
of AZD4547 in patients with advanced solid malignancies
M09GDC A phase Ib open label, dose-escalation study of the JHM Schellens I 12/08/2009 4
safety and pharmacology of GDC-0941 in combination
with erlotinib with advanced solid tumours
M09LAP An open label phase Ib continuation study of lapatinib JHM Schellens I 26/11/2009 0
(E111767) monotherapy or lapatinib in combination with other
anti-cancer treatment in patients with solid tumors
M09NIB The NIB-Cohort study, therapeutic drug JHM Schellens other 09/06/2009 26
monitoring of tyrosine kinase inhibitors
M09OCP A phase I clinical study of oral CP-4126 in JHM Schellens I 02/04/2009 4
patients with advanced solid tumour
M09RIF An open-label phase I study to assess the effect of JHM Schellens I 01/12/2009 0
rifampicin on the pharmacokinetics of eribulin mesylate
(E7389) in subjects with advanced solid tumors
N06PFB Pleural fluid bank MM van den Heuvel other 27/09/2006 *
N07DOW Weekly administration of oral Docetaxel in JHM Schellens I 14/11/2007 53
combinaton with Ritonavir
N07NEX Phase I study of gemcitabine and carboplatin JHM Schellens I 29/01/2008 16
plus sorafenib in patients with advanced solid tumors
* = not registered at trialbureau

176
CLINICAL TrIALS
Type of Title Study Phase Activated No pts.
cancer study coordinator (closed) in NKI-
(nick name) in NKI-AVL AVL per
15/12/
2009
N08BEN An open label study to investigate the pharmacokinetics JHM Schellens I 09/01/2009 6
(distribution, metabolism and excertion) of bendamustine
hydrochloride following intravenous infusion of
[14C]bendamustine hydrochloride in patients with
relapsed or refractory malignancy (hematologic or
non-hematologic)
N08BPM 24 hour ambulatory blood pressure monitoring in patients JHM Schellens other 02/09/2008 5
with malignancies enrolled in phase I oncological studies
N08CER An open-label, non-randomized, single-center study JHM Schellens I 02/02/2009 6
(BOLD 103) to determine 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 JHM Schellens other 31/07/2008 47
techniques in patients with advanced solid tumors
N08EDO Phase I interaction study of docetaxel with JHM Schellens I 03/02/2009 10
supplementation of St. John’s wort or Echinacea
N08KER An open-label phase I study to evaluate the JHM Schellens I 02/02/2009 12
(BOLD 109) pharmacokinetics and tolerance of co-administration
of oral multiple dose of ketoconazole and an IV (bolus)
infusion of Eribulin in patients with advanced solid tumors
N08LRE An open-label non randomized single center JHM Schellens I 11/05/2009 6
study to determine the metabolism and (30/09/2009)
elimination of 14C-E7080 in patients with
advanced solid tumours of lymphoma who are
unsuitable for or have failed existing therapies
N08NPM NVALT Palliative care Protocol on malignant MM van den Heuvel other 13/03/2008 83
pleural effusion
BrAIN / CNS
N05THB Totale hersenbestraling met een eenmalige boost bij LGH Dewit other 07/04/2005 41
patiënten met solitaire hersenmetastase van een
epitheliale tumor - een registratiestudie
BreAST
E10031 A phase III trial evaluating the role of ovarian function GS Sonke III 20/07/2005 4
(SOFT) suppression 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
E10041 Micro-array in node-negative disease may Avoid EJTh Rutgers III 02/01/2007 266
(MINDACT) Chemotherapy: 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

177
CLINICAL TrIALS
Type of Title Study Phase Activated No pts.
cancer study coordinator (closed) in NKI-
(nick name) in NKI-AVL AVL per
15/12/
2009
E10981 After Mapping of the Axilla: Radiotherapy Or EJTh Rutgers III 21/12/2000 470
(AMAROS) Surgery?
E22051 SUPREMO, an MRC phase III randomised trial to NS Russell III 27/02/2007 23
(SUPREMO) assess the role of adjuvant chest wall irradiation
in ‘intermediate risk’ operable breast cancer
following mastectomy
M03RBC Radiation dose intensity study in breast cancer in GMM Bartelink III 29/03/2004 131
(YOUNG young women: a randomized phase III trial of
BOOST) additional dose to the tumor bed (‘young boost’)
M04MAT Microarray Analysis in breast cancer to Tailor SC Linn III 26/04/2004 65
(MATADOR) Adjuvant Drugs Or Regimens (MATADOR)
M05BRI Long term risk of breast cancer following NS Russell other 05/01/2006 92
(BRIGHT) treatment of Hodgkin’s disease
M05HIR Hormonal substitution after prophylactic adnectomy M van Beurden other 31/05/2005 *
(HIRISE) in women with an increased risk for breast- and ovarian
cancer due to a genetic predisposition: HIRISE
(High-Risk women and hormonal Substitution Exposure)
M06DAT A prospective randomised open multicentre phase III SC Linn III 01/05/2007 47
(DATA) study to assess different Durations of Anastrozol therapy (29/07/2009)
after 2 to 3 years Tamoxifen as Adjuvant therapy in
postmenopausal women with breast cancer
M06HER Prospective randomized pharmacological intervention JHM Schellens III 18/06/2007 18
(CANDY) study; evaluating the effect of angiotensin II-receptor
(AT1) blocker candesartan versus placebo in prevention
of trastuzumab-associated cardiotoxicity in patients with
primary breast cancer treated with trastuzumab
M06TM2 A. Multicentre, prospective phase II trial investigating SC Linn III 26/09/2006 45
(TEAM II) the efficacy of neoadjuvant hormonal therapy with
exemestane for six months B. Randomised, multicentre,
prospective, phase III trial investigating the efficacy and
safety of the addition of ibandronate to adjuvant
hormonal therapy in postmenopausal women with
M07ATX Phase II randomized trial of combination therapy SC Linn II 12/12/2007 14
(ATX) of paclitaxel and bevacizumab versus paclitaxel,
capecitabine and bevacizumab as first-line treatment
for locally recurrent or metastatic breast cancer patients
with HER2/neu negative tumor
M07CBE Late effects of chemotherapy on brain functioning SSB Schagen other 01/07/2008 *
in the elderly
M07FEP A randomized double-blind phase 2 trial of Fulvestrant GS Sonke II 12/09/2007 8
plus Enzastaurin versus Fulvestrant plus placebo in
aromatase inhibitor-resistant metastatic breast cancer
* = not registered at trialbureau

178
CLINICAL TrIALS
Type of Title Study Phase Activated No pts.
cancer study coordinator (closed) in NKI-
(nick name) in NKI-AVL AVL per
15/12/
2009
M07LET IDEAL: Investigation on the duration of extended EJTh Rutgers III 16/11/2007 36
(IDEAL) adjuvant 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
M07LTB A randomised, multi-centre, open-label, phase III GS Sonke III 27/05/2008 0
(ALTTO) study of adjuvant lapatinib, trastuzumab, their sequence (24/02/2009)
and their combination in patients with HER2/ErbB2
positive primary breast cancer; ALTTO study
(Adjuvant Lapatinib and/or Trastuzumab Treatment
Optimisation) study
M08BCP Prospective and Retrospective register study of SC Linn other 27/03/2008 32
(BOOG 2003-04) the German Adjuvant Cancer Study Group (GABG) for
diagnosis and treatment of breast cancer in pregnancy
M08CPE A two-arm randomized open label phase 2 study SC Linn II 27/01/2009 0
of CP-751,871 in combination with exemestane
versus exemestane alone as first line treatment
for postmenopausal patients with hormone
receptor positive advanced breast cancer
M08HAT A randomized phase II study of concomitant SC Linn II 20/04/2009 3
trastuzumab, bevacizumab with paclitaxel versus
trastuzumab and bevacizumab followed by the
combination of trastuzumab, bevacizumab and
paclitaxel at progression as first-line treatment of
patients with metastatic breast cancer with
Her2-neu overexpression
M08MAM An exploratory clinical study for initial validation RA Valdés Olmos other 29/04/2009 *
(MARI) of a novel small ring device for positron emission
mammotomography
M08MUL Multicenter feasibility study of the sentinel node HSA Oldenburg other 16/04/2009 *
(MULTISENT) procedure in patients with multiple breast tumors
(MULTISENT)
M08PBI Image guided Preoperative Accelerated partial PH Elkhuizen other 01/10/2009 0
(PAPBI) Breast Irradiation (PAPBI): defining radiotherapy
sensitivity
M08PTP A phase Ib open-label, two arm study of i.v. and JHM Schellens I 11/12/2008 1
oral panobinostat(LBH589) in combination with (30/09/2009)
i.v. trastuzumab (Herceptin) and i.v. paclitaxel as
treatment for adult female patients with HER2
overexpressing metastatic breast cancer (MBC)
M08TRA Trastuzumab in a neoadjuvant regimen for GS Sonke II 18/09/2008 24
(TRAIN) Her2+ breast cancer - the TRAIN study
M09MLA An open label study to examine the effects of JHM Schellens I 26/11/2009 0
(EGF111582) low-fat and high-fat meals on the pharmacokinetics
of orally administered lapatinib in metastatic Erb2
positive breast cancer patients
* = not registered at trialbureau

179
CLINICAL TrIALS
Type of Title Study Phase Activated No pts.
cancer study coordinator (closed) in NKI-
(nick name) in NKI-AVL AVL per
15/12/
2009
M09SRB Sentinal Node and recurrent breastcancer; EJTh Rutgers other 27/10/2009 *
(SNARB) regional staging and registration (SNARB)
N04POM Tailored preoperative chemotherapy in stage II S Rodenhuis II 21/03/2005 89
or III breast cancer with either a primary tumor
over 3 cm in size or a clinically tumor-positive axilla
N04RTB Effecten van bestraling op bloedvaten NS Russell other 05/10/2004 70
N05ECP Prospectief, dubbelblind, gerandomiseerde JHM Schellens III 10/10/2005 94
placebo gecontroleerde farmacologisch (28/08/2009)
interventieonderzoek: evaluatie van de frequentie,
duur en intensiteit van opvliegers na interventie
met Venlafaxine (Efexor ® )) en Clonidine versus
placebo bij vrouwen met vervroegd postmenopauzale
klachten tgv adjuvant chemotherapie en/of hormonale
N05MIB 99mTc-methoxyisobutylisonitrile (MIBI) for imaging RA Valdés Olmos other 07/06/2005 *
of apoptosis and prediction of tumour respons to
chemotherapy and/or radiotherapy in cancer patients
N05TOM A clinical trial to compare the efficacy of the HJ Teertstra other 07/02/2006 *
lorad tomosynthesis mammography system to (25/11/2009)
conventional mammography
N06GLB Phase I study of gemcitabine plus lapatinib JHM Schellens I 16/05/2007 15
(GW572016) in women with advanced breast cancer
N06IAA Randomized phase II/III study of intensified S Rodenhuis II/II 08/01/2007 12
alkylating agent chemotherapy with peripheral
blood progenitor cell support in the preoperative
chemotherapy of breast tumors that are deficient
for homologous recombination
N06TRZ The effect of Trastuzumab treatment on B-cell JHM Schellens other 11/12/2006 23
kinase activities assessed by microarray derived
phosphorylation profiles
N06VNI Meting van de functie van de arm en de bijdrage JJ Hage other 26/09/2006 *
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
N07BOS Genetic determinants of survival and second EJTh Rutgers other 12/12/2007 *
(BOSOM) breast cancer development in premenopausal
breast cancer patients
N07MAN Randomized phase II/III study of second-line S Rodenhuis II/II 03/04/2008 7
(Mandjes studie) endrocrine treatment followed by capecitabine
versus capecitabine followed by endrocrine
treatment in patients with metastatic ER
positive breast cancer
* = not registered at trialbureau

180
CLINICAL TrIALS
Type of Title Study Phase Activated No pts.
cancer study coordinator (closed) in NKI-
(nick name) in NKI-AVL AVL per
15/12/
2009
N08AFT A randomized prospective trial of 2-6 weeks SC Linn II 04/08/2008 13
(AFTER) pre-operative hormonal treatment for hormone
receptor positive breast cancer: Anastrozole +/-
fulvestrant or tamoxifen exposure - response in
molecular profile (AFTER-study)
N08RMB Tumorresponse monitoring in patients with breast MTFD Vrancken other 23/09/2008 37
cancer treated with primary systemic therapy: Peeters
towards predicting response in both the primary
tumor and in axillary lymph nodes
N09TOL Long-term safety of trastuzumab in patients with JHM Schellens 22/10/2009 51
HER2-positive breast cancer
GASTro INTeSTINAL
M05RAX Pre-operative chemoradiotherapy regimen with A Cats II 20/03/2006 8
(RAX) capecitabine and bevacuzimab in locally
advanced rectal cancer. A feasability study (RAX)
M06CRI A multicenter randomized phase III trial of neo- M Verheij III 11/01/2007 27
(CRITICS) adjuvant 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 22/02/2007 2
(M1 study) neoadjuvant bevacizumab, capecitabine and
oxaliplatin and radical resection of primary tumor
and metastases in primary stage IV rectal cancer
M06SCR A multicenter phase III randomised trial comparing A Cats III 09/05/2006 7
(SCRIPT) total 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 A Cats III 04/09/2007 7
(CAIRO3) bevacuzimab versus observation after induction
treatment with capecitabine, oxaliplatin and
bevacuzimab as first-line treatment in patients with
advanced colorectal carcinoma, a randomised
phase III study (CAIRO3)
M07DDO Development of a docetaxel, oxaliplatin, capecitabine A Cats I 23/07/2007 22
(D-DOCS) combination schedule in patients with advanced
cancer of the stomach or the gastro-esophageal
junction, a phase I study
M07HBT Feasibility study of external beam radiation therapy B van Triest I/II 25/07/2007 10
(HerBerT) followed by high-dose rate endorectal brachytherapy
(HDBRT) in inoperable rectal cancer patients

181
CLINICAL TrIALS
Type of Title Study Phase Activated No pts.
cancer study coordinator (closed) in NKI-
(nick name) in NKI-AVL AVL per
15/12/
2009
M07HEP A randomized two arm phase III study in patients A Cats III 28/04/2008 5
(HEPATICA) post radical 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
M07NAR Neo-Aduvant Radiotherapy-Chemotherapy In Stomach E Jansen I/II 28/01/2008 1
(NARCIS) Cancer. Induction therapy with carboplatin, paclitaxel
and radiotherapy in patients with locally advanced
gastric cancer. The ‘NARCIS’ study
M07RBV Clinical pilot study: radiolabeled bevacizumab TJM Ruers I 12/02/2008 *
as a tracer of VEGF expression in patients with
colorectal liver metastases
M07RCM Simultaneous integrated boost radiation therapy A Cats I 31/01/2008 18
with concomitant capecitabine and mitomycin-C
chemotherapy for locally advanced anal carcinoma
M08ACL Accelerated growth of synchronous colorectal TJM Ruers II 21/02/2008 9
(SILENT) liver metastases: effects of neo-adjuvant therapy
M08DCS Tumor destruction and DC activation in situ: TJM Ruers Pilot 18/09/2008 4
towards in vivo loaded DC vaccines
M08GPO Genetic and protein profiling in patients with JW van Sandick other 01/09/2008 11
(PROFOC) oesophageal cancer
N02ECC A single institution phase II study of ECC H Boot II 29/08/2002 133
(Epirubicin, Cisplatin, Capecitabin) in locally
advanced or metastatic gastric cancer and
adenocarcinoma of the oesophago-gastric
junction and distal oesophagus
N05GEA Saving the gastro-epiploic artery at omentectomy VJ Verwaal III 21/03/2006 42
and the effect on post-surgical recovering gastric function (03/08/2009)
N05MCT A phase II study of treatment with the combination ME Smits other 05/12/2005 3
of unlabelled (‘cold’) and radioactive (‘hot’)
meta-iodobenzylguanidin (MIBG) in metastatic
carcinoid tumours
N05STP Serum and tissue protein profiling and tumour genetic ME Smits other 19/01/2006 140
analysis in patients with potential premalignant
conditions or colorectal cancer
N06DCM Feasibility of the analysis of platinum-DNA adducts JHM Schellens other 12/12/2006 4
in tumour tissue in cisplatinum treated with (15/04/2009)
advanced gastric cancer
N08ICG Pilot study on the use of fluorecence imaging of TJM Ruers Pilot 30/12/2008 *
lymph nodes during colorectal lymphadenectomy
using indocyanine green
* = not registered at trialbureau

182
CLINICAL TrIALS
Type of Title Study Phase Activated No pts.
cancer study coordinator (closed) in NKI-
(nick name) in NKI-AVL AVL per
15/12/
2009
N08RCT Deformation of target volume during radiotherapy M Verheij other 14/01/2009 *
for rectal cancer, a repeat CT study
GyNAeCoLoGICAL
M05HIR Hormonal substitution after prophylactic M van Beurden other 31/05/2005 *
(HIRISE) adnectomy in women 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 WJ van Driel other 12/01/2006 *
cancer patients
M05SNV GROningen International study on sentinel WJ van Driel other 26/03/2007 22
(GROINSS-V II) nodes in vulvar cancer (GROINSS-V) II
M06HRT The effect of hormonal replacement therapy on CM Korse other 25/09/2006 61
(NOVARIA) menopausal complaints related to biochemical
changes in surgically and naturally postmenopausal
women. A prospective observational comparative study
M06OVH Phase III randomised clinical trial for stage III ovarian WJ van Driel III 04/01/2006 26
(OVHIPEC) carcinoma randomising between secondary debulking
surgery with or without hyperthermic intraperitoneal
chemotherapy (OVHIPEC-1)
M06RTE Randomised phase III trial comparing concurrent B van Triest III 28/03/2007 4
(PORTEC-3) chemoradiation and adjuvant chemotherapy with
pelvic radiation alone in high risk and advanced
stage endometrial carcinoma: PORTEC-3
M07OVE A phase II, double blind placebo controlled S Rodenhuis II 01/07/2008 9
(OVERT-1) multicenter randomised study of AZD0530 in (28/02/2009)
patients with ovarian cancer sensitive to platinum
based chemotherapy (OVERT-1)
M07RCV Phase II study of definitive radiochemotherapy WJ van Driel II 26/06/2007 0
for locally advanced squamous cell cancer of the
vulva: an efficacy study
M09ALC Double-blind, randomized phase II study to evaluate JHM Schellens II 13/07/2009 4
(REASON) the safety and efficacy of Acetyl-L-Carnitine in the (14/10/2009)
prevention of Sagopilone-induced peripheral neuropathy
M09PSO Phase II randomised, double blind, multicentre study GS Sonke II 13/08/2009 2
to assess the efficacy of AZD2281 in the teatment of (10/12/2009)
patients with platinum sensitive serous ovarian cancer
following two or more platinum containing regimens
N05CGO Randomized clinical pharmacological dose-escalation JHM Schellens I 08/09/2005 21
study to explore both safety and preliminary efficacy
and pharmaco-kinetics, -dynamics and -genomics of
fixed dose rate gemcitabine and 30-minute standard
gemcitabine infusion both administered in combination
with carboplatin as second-line
* = not registered at trialbureau

183
CLINICAL TrIALS
Type of Title Study Phase Activated No pts.
cancer study coordinator (closed) in NKI-
(nick name) in NKI-AVL AVL per
15/12/
heAd ANd NeCK
2009
M02STA The effect of a statin on the progression of the FJP Hoebers III 28/01/2003 79
intima-media thickness induced by radiotherapy
M05PET FDG-PET for avoidance of futile direct laryngoscopies M van de Brekel other 11/05/2005 *
(RELAPS) under general anaesthesia with taking of biopsies (02/03/2009)
in patients with suspicion on recurrent laryngeal
carcinoma after radiotherapy
M07CMD Treatment of myogenic cranio mandibular FJM Hilgers III 29/08/2007 *
dysfunction (CMD): a prospective randomised
clinical trial, comparing a mechanical stretching
device (Therabite) with standard physiotherapy
M07LRC A randomized, double blind, placebo controlled, JP de Boer II 10/06/2008 5
multicentre phase II study of oral lapatinib in
combination with concurrent radiotherapy and
cisplatin versus radiotherapy and cisplatin alone,
in subjects with stage III and IVA,B squamous
cell carcinoma of the head and neck (SCCHN)
M07MSH Molecular staging of surgically treated head and M van de Brekel other 12/02/2008 *
neck cancer patients: towards rationalized (01/01/2009)
postoperative clinical management
M08CON A randomized study of docetaxel/cisplatin/5- JP de Boer II 02/02/2009 14
(CONDOR) fluorouracil (TPF) 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
M08DDL Docetaxel versus Docetaxel and Lapatinib in JP de Boer II 12/02/2009 6
(TYVTAX) recurrent or 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 IB Tan other 29/04/2009 *
recurrent nasopharyngeal carcinome (NPC) using
(anti-) EBV based tumor markers in The Netherlands
M08MSH Measuring shoulder disability after neck dissection; M Stuiver other 25/03/2009 *
a comparison of 4 self report scales
M08SNU Ultrasound guided fine needle aspiration cytology M van de Brekel other 26/06/2008 1
(SNUS) and sentinel node biopsy in the detection of occult
lymph node metastases of early oral and oropharyngeal
cancer (SNUS)
M09HZT Efficacy of adding hyperbaric oxygen therapy to LM Smeele III 23/11/2009 0
(HBOT) the treatment of late radiation damage of the
lower jaw (osteonecrosis)
M09NST Clinical feasability of a new surgical tool for primary FJM Hilgers I/II 03/12/2009 *
or secondary tracheoesophageal puncture and voice
prosthesis insertion for prosthetic voice rehabilitation
after total laryngectomy
* = not registered at trialbureau

184
CLINICAL TrIALS
Type of Title Study Phase Activated No pts.
cancer study coordinator (closed) in NKI-
(nick name) in NKI-AVL AVL per
15/12/
2009
M09OSA Prospective cohort study on the prevalence of M van de Brekel 02/12/2009 *
obstructive sleep apnea in patients with head
and neck cancer treated wither either radiotherapy,
chemoradiation or surgery
M09PDT A multi centre cost-effectiveness evaluation of a IB Tan other 06/08/2009 *
novel treatment option in the netherlands: photo
dynamic therapy with temoporfin for the treatment
of advanced incurable head and neck cancers,
for whom prior conventional treatments have failed
N02SNL Early detection of lymph node metastasis of PJFM Lohuis Pilot 03/04/2002 8
squamous cell carcinoma of the larynx with (02/03/2009)
lymphatic mapping and sentinel node biopsy
N04LFV Longfunctie na toediening van medicatie FJM Hilgers III 07/02/2005 *
(salbutamol-ipratropiumbromide dosisaerosol) (02/03/2009)
met en zonder voorzetkamer bij gelaryngectomeerden
N04RTB Effecten van bestraling op bloedvaten NS Russell other 05/10/2004 70
N05HME De kortetermijninvloed van een Heat and Moisture JK Zuur other 01/09/2005 *
Exchanger op de endotracheale temperatuur en
luchtvochtigheid bij gelaryngectomeerden
N05TSP Prevention of trismus, swallowing and speech FJM Hilgers other 14/11/2005 *
problems in patients treated with chemoradiation
for advanced head and neck cancer
N07HME A prospective clinical feasibility study of a new FJM Hilgers other 16/05/2007 *
heat and moisture exchanger with bacteria, virus (02/03/2009)
and pollen filtration capabilities (HME-F)
N07MCP Microcirculatory changes during photodynamic MP Copper other 21/05/2007 *
therapy (PDT) in squamous cell carcinoma of the
oral cavity and oropharynx
N07SHT Subglottic humidity and temperature in nasal RJ Scheenstra other 14/05/2007 *
breathing and tracheotomy breathing with upper
airway occlusion
N07VIN Transoral resection of stage I-II carcinomas of the M van de Brekel II 12/03/2007 5
oropharynx by robot-assisted surgery: a feasability study
N08HHF Validation of hypoxia imaging of cancer in the WV Vogel II 22/10/2008 *
head and neck area with FAZA-PET
LeuKAemIA / mdS
M05H68 A randomized phase III study in previously M Kerst III 21/03/2006 2
(HOVON 68) untreated patients with biological high-risk CLL:
fludarabine + cyclophophamide (FC) versus FC +
low dose alemtuzumab
* = not registered at trialbureau

185
CLINICAL TrIALS
Type of Title Study Phase Activated No pts.
cancer study coordinator (closed) in NKI-
(nick name) in NKI-AVL AVL per
15/12/
LuNG
2009
E08062 Randomized phase II study of amrubicin as P Baas II 28/02/2008 0
single agent or in combination with cisplatin (19/11/2009)
versus etoposide-cisplatin as first-line treatment
in patients with extensive stage SCLC (ES).
E08072 Concurrent Once-daily Versus twice-daily JL Knegjens III 11/11/2009 0
(CONVERT) RadioTherapy: a 2-arm 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 P Baas III 18/02/2004 107
(NVALT 5) Thalidomide in patients with malignant pleural
mesothelioma after first line chemotherapy (NVALT5)
M05ATD Accurate target definition of non-small cell lung J Stroom other 05/01/2006 *
tumors using pathology-validated PET and CT
imaging for the optimization of radiation treatment
M05GCP Pharmacogenomic and pharmacokinetic study in JHM Schellens other 13/09/2005 124
patients with advanced non small-cell lung cancer
(NSCLC) treated with first-line gemcitabine / platinum
combination chemotherapy
M06NEL Efficacy of neoadjuvant erlotinib in patients with HM Klomp I/II 08/11/2006 38
(NEL) clinical stage I/II non-small cell lung cancer (NSCLC)
M07CCL Open-label, randomised multi-center study MM van den Heuvel I/II 13/03/2008 49
(Raditux) investigating Cetuximab, in combination with
concurrent chemo-radiotherapy in locally advanced
non-small cell lung carcinoma (RADITUX)
M07ESU Randomized double blind phase 2 study of erlotinib P Baas II 29/05/2007 10
(ESU) with or without SU11248 in the treatment of (19/05/2009)
metastatic non small cell lung cancer
M07OSM A phase III randomized, double blind, placebo P Baas II/II 09/10/2007 41
(OSM) controlled trial of 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- MM van den Heuvel III 13/12/2007 6
(START) blind placebo-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 MM van den Heuvel I 09/04/2009 6
(Selectikine) safety, 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, first-line chemotherapy
* = not registered at trialbureau

L
186
CLINICAL TrIALS
Type of Title Study Phase Activated No pts.
cancer study coordinator (closed) in NKI-
(nick name) in NKI-AVL AVL per
15/12/
2009
M09AVT A randomized open label multinational phase III JA Burgers III 19/08/2009 1
comparing amrubicin versus topotecan in patients with
extensive or limited and sensitive or refractory small
cell lung cancer after failure of first line chemotherapy
M09BPL Open label study of bevacizumab maintenance JA Burgers III 14/12/2009 0
(AVAPERL) therapy with or without permetrexed after a first line
treatment chemotherapy with bevacizumab-cisplatin
pemetrexed in patients with advanced, metastatic of
recurrent non-squamous non-small cell lungcancer
M09CRE Randomized trial on chest irradiation in extensive JL Knegjens III 19/03/2009 4
(CREST) disease small cell lung cancer
M09LST Evaluation of molecular sputum test diagnostics JA Burgers other 18/05/2009 37
for lung cancer
M09N10 A randomized phase II study of erlotinib compared JA Burgers II 11/09/2009 8
(NVALT10) to single agent chemotherapy-erlotinib combination
in pretreated patients with advanced NSCLC
(NVALT10 study)
M09PBO Dose escalation by boosting radiation dose within JSA Belderbos II 26/11/2009 0
(PET-BOOST) the primary 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 JSA Belderbos III 20/10/2009 1
(NVALT 11) in radically treated patients with stage III non-small
cell lung cancer: a phase III randomized study (NVALT 11)
M09ROS A randomized clinical trial of radiosurgery JSA Belderbos III 28/04/2009 0
(ROSEL) (stereotactic radiotherapy) or surgery in patients with
IA NSCLC who are fit to undergo primary resection.
N00ALF Endoscopic detection op pre-neoplastic lesions P Baas Pilot 30/06/2000 41
and carcinoma in the bronchial tree with deltaaminolevunic
acid fluorescence
N04LSN Feasibility of lymphoscintigraphy and sentinel node HM Klomp Pilot 08/09/2004 5
biopsy in patients with non-small lung cancer
N05MIB 99mTc-methoxyisobutylisonitrile (MIBI) for imaging RA Valdés Olmos other 07/06/2005 *
of apoptosis and prediction of tumour respons to
chemotherapy and/or radiotherapy in cancer patients
N07NRA A phase I/II study with NAMI-A, a Novel JHM Schellens I/II 31/07/2008 10
Ruthenium Anticancer Agent, and gemcitabine
combination second-line therapy in NSCLC patients
N08CPA A randomized phase I/II study of standard P Baas I/II 22/05/2009 5
chemotherapy (cisplatin and pemetrexed) with or
without Axitinib in patients with malignant
mesothelioma: interim biopsy analysis to
determine efficacy
* = not registered at trialbureau

187
CLINICAL TrIALS
Type of Title Study Phase Activated No pts.
cancer study coordinator (closed) in NKI-
(nick name) in NKI-AVL AVL per
15/12/
LymPhomA - hodGKIN’S dISeASe
2009
E20012 BEACOPP (4 cycle’s escalated + 4 cycle’s baseline) vs JW Baars III 29/03/2004 1
ABVD (8 cycle’s) in Stage III & IV Hodgkin’s Lymphoma
E20051 The H10 EORTC/GELA randomized Intergroup JW Baars III 23/11/2006 6
(H10) trial on early 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 RLM Haas III 02/02/2004 6
and involved field radiotherapy in patients with localized,
stages I and II, low grade non-hodgkin’s lymphoma
M05H55 Efficacy of maintenance therapy with rituximab JP de Boer III 20/07/2005 2
after induction therapy (R-CHOP versus R-FC) for
elderly patients with mantle cell lymphoma not
suitable for autologous stem cell transplantation
M06CMV Cytomegalovirus (CMV) - specific T cell immunity M Kerst other 28/09/2006 *
in B cell malignancies (19/11/2009)
M06H77 Efficacy and safety of a single dose of 14.8 MBq/kg JW Baars II 23/05/2006 0
(Hovon 77) 90Y-ibritumomab tiuxetan (Zevalin) in elderly (19/11/2009)
patient with diffuse large B-cell lymphoma and
FDG-PET positive partial remission following
first-line R-CHOP therapy. A phase II clinical trial
M07H80 Phase II study on the feasability and efficacy of JW Baars II 10/09/2007 1
(HOVON 80) R-DHAP-MTX 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 JW Baars III 22/01/2008 3
(HOVON-84) intensification of rituximab in combination with
2-weekly CHOP chemotherapy followed by rituximab
maintenance in elderly patients with DLBCL
M09TAM Treatment induced alterations in microenvironment D de Jong other 27/05/2009 *
(TAMIL) in follicular lymphoma. A multicenter descriptive study
N03OFP A phase II study of eradication therapy with additional JP de Boer II 06/10/2003 9
oral fludarabine in t(11;18) positive gastric MALT
lymphoma
N03RIM Radioimmunotherapy (131I-antiCD20 monoclonal JW Baars Pilot 25/06/2003 1
antibody) as consolidation treatment after remission
induction for patients with relapsed or refractory
CD20+ B-cell NHL
N05MIB 99mTc-methoxyisobutylisonitrile (MIBI) for imaging RA Valdés Olmos other 07/06/2005 *
of apoptosis and prediction of tumour respons to
chemotherapy and/or radiotherapy in cancer patients
N07RIT Determination of the pharmacokinet ics of rituximab JW Baars other 14/04/2008 28
and Ab to rituximab
* = not registered at trialbureau

188
CLINICAL TrIALS
Type of Title Study Phase Activated No pts.
cancer study coordinator (closed) in NKI-
(nick name) in NKI-AVL AVL per
15/12/
2009
N08IRP Iodine-124-rituximab positron emission tomography WV Vogel Pilot 17/12/2008 *
(IR-PET) (IR-PET) for molecular imaging of CD20 expression
meLANomA / SKIN
M05MSL A phase III multicenter randomized trial of sentinel OE Nieweg III 11/09/2006 8
(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 JBAG Haanen I 09/01/2009 4
tumor-specific immunity in stage IV melanoma
patients: a phase I clinical study
M09ADM A phase II, double-blind randomised study to assess JBAG Haanen II 06/10/2009 0
(AZD6244) the efficacy of AZD6244 (Hyd-Sulfate) in combination
with Dacarbazine compared with Dacarbazine alone
in first line patient with BRAF mutation positive
advanced cutaneous or unknown primary melanoma
N03LAM Longitudinal analysis of melanoma-specific immunity JBAG Haanen II 22/08/2003 10
in stage III and IV melanoma patients
N06TIS Integrated analyses of melanoma-T cell interactions; JBAG Haanen other 29/08/2006 14
relevance for immunotherapy
mISCeLLANeouS
M09XLT An international, randomized, double-blinded, JP de Boer III 08/07/2009 1
phase III efficacy study of XL184 versus placebo
in subjects with unresectable, locally advanced
or metastatic medullary thyroid cancer
N01RIT Identifications of molecular mechanisms NS Russell other 07/05/2002 35
involved in radiation-induced telangiectasia
N03THY Therapeutic management of thymoma and thymic LGH Dewit Pilot 25/11/2003 16
carcinoma: - a prospective registration study based
on preoperative risk assessment of local failure
N05CHO The efficacy of cordotomies in patients with chest A Lukas II 17/01/2006 *
pain due to primariy localized malignancies of the chest
N09DRF Intraoperative real time imaging with a dual RA Valdés Olmos other 08/06/2009 *
radioactive/fluorescence modality for sentinel
node localization. A feasibility study including
reproducibility of multimodality lymphatic
mapping with a cocktail tracer containing 99mTc
nanocolloid/ICG
SofT TISSue / oSTeoSArComA
E62012 Randomised trial of single agent doxorubicin versus S Rodenhuis III 09/07/2003 24
doxorubicin plus ifosfamide in the first line treatment
of advanced or metastatic soft tissue sarcoma
* = not registered at trialbureau

189
CLINICAL TrIALS
Type of Title Study Phase Activated No pts.
cancer study coordinator (closed) in NKI-
(nick name) in NKI-AVL AVL per
15/12/
2009
E62063 A phase III randomized study evaluating surgery F van Coevorden III 18/02/2009 1
of residual disease in patients with metastatic
gastro-intestinal stromal tumor responding to
Imatinib mesylate
E62072 A randomized double blind phase III trial of Pazopanib M Kerst III 18/11/2008 7
(PALETTE) versus placebo in patients with soft tissue sarcoma
whose disease has progressed during or following
prior therapy
M01ROS Phase II study of rosiglitazone in advanced liposarcoma S Rodenhuis II 24/08/2001 5
uro-GeNITAL
E30072 A phase III randomised double blind study comparing M Kerst III 21/07/2009 0
sorafenib with placebo in patients with resected
primary renal cell carcinoma at high or intermediate
risk of relapse
E30983 Randomized phase II/III study of Taxol-BEP versus BEP M Kerst II/II 07/09/1999 4
in patients with intermediate prognosis germ cell cancer (25/08/2009)
M00LMT Identification of occult lymph node metastases in S Horenblas Pilot 14/11/2000 *
testicular cancer to select patients for adjuvant treatment.
Feasability of a laparoscopic selective retroperitoneal
lymphadenectomy
M06HFP Hypofractionated irradiation for prostate cancer: F Pos III 08/03/2007 147
(HYPRO) a randomized multicenterphase III study (HYPRO)
M06LAN Late Adverse effects in Dutch testicular cancer M Kerst other 12/02/2007 *
(LANCE) survivors: a Nationwide case-control study on
Cardiovascular Events
M06SIL Phase I dose-escalation trial for the combination JBAG Haanen I 20/11/2006 6
of sorafenib with interleukin-2 treatment in
patients with clear cell renal carcinoma
M07PGC Phase II trial of paclitaxel, gemcitabine and cisplatin M Kerst II 08/01/2008 0
in patients with relapsing germ cell cancer after first
line chemotherapy
M08PZD A phase III, placebo-controlled, double-blind study HG van der Poel III 09/03/2009 2
(M15) to assess the efficacy and safety of once-daily orally
administered ZD4054 10 mg in non-metastatic
hormone-resistant prostate cancer patients
M08ZDP A phase III randomized double blind study to assess HG van der Poel III 08/09/2008 9
(ENTHUSE M1) the efficacy and safety of 10 mg ZD4054 versus (11/03/2009)
placebo in patients with hormone resistant prostate
cancer and bone metastasis who are painfree and
mildly symptomatic
M09ALC Double-blind, randomized phase II study to evaluate JHM Schellens II 13/07/2009 4
(REASON) the safety and efficacy of Acetyl-L-Carnitine in the (14/10/2009)
prevention of Sagopilone-induced peripheral neuropathy
* = not registered at trialbureau

190
CLINICAL TrIALS
Type of Title Study Phase Activated No pts.
cancer study coordinator (closed) in NKI-
(nick name) in NKI-AVL AVL per
15/12/
2009
M09PRG Evaluation of PROTEX absorbable injectable hydrogel B van Triest other 20/07/2009 5
(protex) when used 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 JBAG Haanen III 27/05/2009 5
(COMPARZ) sunitinib in the treatment of subjects with locally
advanced and/or metastatic renal cell cancer
M09RRC An open label multi center expanded access study C Blank IV 28/04/2009 10
(REACT) of RAD001 in 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
M94SAL Salvage regimen incorporating repeated ablative S Rodenhuis II 04/07/1994 24
chemotherapy with autologous PSCT, a phase II study
N06MPM Samarium-153-EDTMP (QUADRIMET) versus docetaxel HG van der Poel II 14/03/2007 9
(QUADRIMET) for multiple painful oseous metastases in prostate cancer
N06SNR Feasability of lymphoscintigraphy and sentinel node A Bex other 09/10/2007 *
biopsy in patients with renal cell carcinoma (23/11/2009)
N06SUN Sunitinib prior to nephrectomy in patients with metastatic A Bex II 16/05/2007 18
renal cell carcinoma and the primary in situ
N07TAP Evaluation of the Transversus Abdominis Plane - block DR Buitelaar other 08/01/2008 107
(TAP-block) (TAP-block) in preventing painful urine bladder spasm (14/04/2009)
in patients undergoing Robot Assisted Laparoscopic
Radical Prostatectomy under general anesthesia,
a prospective randomized controlled trial
N08API Analysis of prostate-specific immunity in stage III JBAG Haanen other 22/01/2009 1
and IV prostate cancer patients
N08SNR Site and distribution of sentinel lymph nodes in renal A Bex II 19/03/2009 0
cell carcinoma, a phase II study
N09IGF Pilot study on the use fluorescence imaging of lymph HG van der Poel Pilot 14/07/2009 *
nodes during laparoscopic pelvic sentinel node
dissection for prostate cancer using indocyanine green
N09QPB Quantative FDG-PET/CT of primary bladder cancer WV Vogel Pilot 08/06/2009 *
N09VDU Urethral suspension using vas deferens for prevention HG van der Poel II 11/06/2009 61
(RALP 2009) of urin incontinence after robot assisted laparascopic
prostatectomy (RALP)
* = not registered at trialbureau

InvIted speakers 2009
Craig Allred, St. Louis, MO, USA
The estrogen receptor and breast cancer
Mario Amendola, Milan, Italy
New lentiviral vectors for co-ordinate transgene
expression and multiple miRNA/siRNA delivery
Eelco van Anken, San Francisco, CA, USA
Endoplasmic reticulum stress signaling from oligomeric Ire1
Cecile Arrieumerlou, Basel, Switzerland
Spatial propagation of pro-inflammatory signals during
bacterial infection
David Baltimore, Pasadena, CA, USA
MicroRNAs in inflammation and cancer
Boris Bastian, San Fransisco, CA, USA
Genetic diversity in melanoma: implications for disease
classification and therapy
Patrick Bauerle, Munchen, Germany
Cancer therapy by T cell-engaging antibody constructs
Boudewijn Burgering, Utrecht, The Netherlands
FOXO transcription factors, mediators of oxidative stress
in lifespan and disease
Stephen Baylin, Baltimore, MD, USA
The cancer epigenome: towards epigenetic therapy
Peter Carmeliet, Leuven, Belgium
Oxygen sensors as regulators of tumor vessel
morphogenesis
Karen Cichowski, Boston, MA, USA
Unexpected mechanisms of Ras activation in metastatic
prostate cancer and gliomagenesis: epigenetics and more
Lena Claesson-Welsh, Uppsala, Sweden
Signal transduction in angiogenesis
George Cotsarelis, Philadelphia, PA, USA
Cutaneous epithelial stem cells and skin regeneration
Lisa Coussens, San Francisco, CA, USA
Pro-tumor immunity and solid tumor development
Kylie Creig, Parkville, Victoria, Australia
The transcription factor c-Myb is essential for early B cell
development
Peter Creswell, New Haven, CT, USA
Antigen cross-presentation: how do external proteins get in?
Jeroen Demmers, Rotterdam, The Netherlands
Functional proteomics of gene regulation networks
Eric Deutsch, Villejuif, France
Overview of thoracic models developed at IGR
Julian Downward, London, UK
Ras and PI 3-kinase signaling networks in cancer
Laura Esserman, San Francisco, CA, USA
ISPY2, a targeted drugs neo-adjuvant adaptive trial
design
191
InvIted speakers
Sydney Evans, Philadephia, PA, USA
What we can learn about tumor physiology and clinical
behavior from tumor oxygen maps
Dean Felsher, Stanford, CA, USA
Molecular and mathematical modeling of oncogene
addiction
Michael Feuerstein, Bethesda, MD, USA
Cognitive limitations and work performance in
occupationally active brain and breast cancer survivors
Riccardo Fodde, Rotterdam, The Netherlands
Cancer stem cells: are you a Feyenoord or Ajax
supporter?
Margaret Frame, Edinburgh, UK
Focal adhesion kinase in integrin signalling and disease
Iain Fraser, Bethesda, MD, USA
Analysis of mammalian signaling networks: lessons
from a research consortium
Helge Grosshans, Basel, Switserland
Life and death of microRNAs - genetic-biochemical
dissection of miRNA pathways
Jun-Lin Guan, Ann Arbor, MI, USA
Integrin signaling through FAK in mammary stem cells
and breast cancer
Iain Hagan, Manchester, UK
Coordinating cell division by integrating signaling
networks at the centrosome/spindle pole
Adriana Haimovitz-Friedman, New York, NY, USA
A ceramide rheostat balances angiogenesis and antiangiogenesis
Per Hall, Stockholm, Sweden
Incidence and prognosis of contralateral breast cancer
Harald Herrmann, Heidelberg, Germany
Structure and organization of nuclear lamin filaments:
Impact of laminopathic mutations
Steve Jackson, Cambridge, UK
Cellular responses to DNA damage: molecular insights
and new strategies for cancer therapy

192
InvIted speakers
Penny Jeggo, Brighton, UK
Impact of heterochromatin on DNA repair and damage
response signalling
Wilhelm Krek, Zürich, Switserland
VHL: linking microtubules to primary cilia
maintenance, chromosome stability and tumour
suppression
Toby Lawrence, London, UK
Re-educating tumour-associated macrophages
Fred van Leeuwen, Amsterdam, the Netherlands
Chromatin dynamics
Michael Leitzmann, Regensburg, Germany
Adiposity and cancer from an epidemiologic perspective
Zvi Livneh, Rehovot, Israel
Molecular insight into error-prone DNA repair in
mammalian cells: A mutagenic process that protects us
against cancer
Yohan Loriot, Villejuif, France
BCL2 targeting using an antisense in a SCLC model
Christian Luber, Munich, Germany
In vivo-proteomics: Label-free analysis and dendritic cell
subsets
Matthias Mann, Martinsried, Germany
Technology of SILAC-based quantitative teomics and
potential clinical applications
Muriel Moser, Brussels, Belgium
Naturally occurring regulatory T cells control the CD70/
CD27 pathway of T helper 1 cell differentiation
Paolo Michieli, Torino, Italy
Control of invasive growth in physiology and disease by
scatter factors and their tyrosine kinase receptors
Pierre Mordant, Villejuif, France
Dual blockade of mTor and RAF pathways in NSCLC
Harold Moses, Nashville, TN, USA
TGF-beta regulation of the tumor microenvironment
Andrea Musacchio, Milan, Italy
Feedback control of mitosis
Marcin Nowotny, Warsaw, Poland
Structural studies of RNases H – from substrate
binding to catalysis
Stefan Offermans, Heidelberg, Germany
Biological roles of heterotrimeric G-proteins revealed by
genetic mouse models
Håkan Olsson, Lund, Sweden
Risk factors and tumor biology of breast cancer
Michele de Palma, Milan, Italy
Monocyte and macrophage heterogeneity in blood and
tumor microenvironment
Peter Parker, London, UK
PKC and the control of spatially resolved signals
Salvatore Pece, Milan, Italy
The molecular profile of mammary stem cells provides a
new outlook on the cellular and molecular heterogeneity
of breast cancers
Helen Pickersgill, Vienna, Austria
An insider’s guide to getting your paper published:
Insights from a Science editor
David Piwnica-Worms, St. Louis, MO, USA
Dynamic molecular imaging of signal transduction
pathways in vivo
Helen Piwnica-Worms, St. Louis, MO, USA
Translation of fundamental cell cycle principles to novel
breast cancer treatments
Hidde Ploegh, Cambridge, MA, USA
Immune technology
Jeff Pollard, New York, NY, USA
Macrophages promote tumor progression and metastasis
Sandro Prato, Melbourne, Australia
Analysis of the mechanisms involved in the impairment
of tumour- specific CD8 T cells in a mouse model of
non-Hodgkin lymphoma
Alain Puisieux, Lyon, France
Inactivation of safeguard mechanisms by Twist
oncoproteins
Anne Ridley, London, UK
Rho GTPases and signalling in cell migration
Rafael Rosell, Barcelona, Spain
Prospective studies in the adjuvant and metastatic
settings of non-small-cell lung cancer: The Spanish Lung
Cancer Group SCAT and BREC trials (emphasis on
EGFR mutation status)
Lenhard Rudolph, Ulm, Germany
Checkpoint response to telomere dysfunction in aging
stem cells
Fred de Sauvage, San Francisco, CA, USA
Targeting the hedgehog pathway in cancer: from bench
to clinic
Wulf Schneider, Regensburg, Germany
TNF internalisation and adenovirus inhibition of TNFmediated
apoptosis

Joachim Schulze, Bonn, Germany
At the cross roads of genomics and the tumor
microenvironment: How can we use genomic
technologies to decipher inhibitory networks in the
tumor microenvironment
Peter Sicinski, Boston, MA, USA
Requirement for cyclin A function in stem cells
Marc Symons, New York, NY, USA
Role of Rho family GTPases in tumor invasion
Li-Huei Tsai, Cambridge, MA, USA
Epigenetic regulation of memory formation in health
and disease
Michiel Vermeulen, Utrecht, The Netherlands
Applying high accuracy quantitative mass spectrometry
to study epigenetic (de-)regulation of gene expression
Karen Vousden, Glasgow, Scotland, United Kingdom
Functions of p53 in tumour suppression and progression
Yosef Yarden, Rehovot, Israel
EGFR and HER2 in cancer: signaling networks and
targets for therapy
Jon Yewdell, Bethesda, MD, USA
Serendipity strikes again: Adventures in MHC Class I
antigen processing lead to discovery of methionine tRNA
misacylation
Lars Zender, Hannover, Germany
Integrative oncogenomic approaches for accelerated
cancer gene discovery in hepatocellular carcinoma
193
InvIted speakers

194
ProjEcTS
ProjEcTS SuPPorTEd
by ThE duTch caNcEr SociETy
Principal Number of Title Started Ended/Ends
investigator project
Aaronson, Neil KWF 2003-2977 Psychological and behavioral issues in cancer genetics 01-10-2003 01-12-2009
Aaronson, Neil KWF 2006-3470 Cognitive behavioral therapy (CBT) and physical exercise for 01-09-2006 01-09-2010
climacteric symptoms in breast cancer patients experiencing
treatment-indiced menopause
Aaronson, Neil KWF 2009-4299 A-CaRe Project 2: Effectiveness of physical exercise during 01-08-2009 01-08-2013
chemotherapy to improve physical firness and reduce fatigue:
A randomized trial
Agami, Reuven KWF 2005-3387 Identification and characterization of human tumor suppressor 01-09-2005 01-09-2009
genes in the p53 oathway.
Agami, Reuven KWF 2007-3908 In vivo functional screens for oncogenic and tumorsuppressive 01-08-2008 14-09-2009
microRNAs.
Agami, Reuven KWF 2006-3558 Genetic screens to identify cancer related functions of human 01-11-2006 01-11-2010
microRNAs
Agami, Reuven KWF 2007-3881 Role of miRNA genes in etiology of malignant germ cell tumors. 01-03-2007 01-03-2011
Bergman, André KWF 2009-4356 Investigating the Role of Inflammation in Prostate Cancer 01-08-2009 01-08-2015
Development
Bernards, René KWF 2005-3375 The Role of PRAME in oncogenesis 01-08-2005 31-07-2009
Bernards, René KWF 2008-4042 Identification of enzymes involved in regulatory ubiquitination in 01-01-2008 01-01-2012
TNF a–stimulated signalling by loss-of-function screens
Bernards, René KWF 2009-4337 Identification of genetic modifiers of sensitivuty to mTOR pathway 01-08-2009 01-08-2013
inhibition in breast cancer
Berns, Katrien KWF 2008-4027 Understanding resistance to HER2-targeting therapy in human 01-01-2008 01-01-2012
breast cancer through functional genetics
Berns, Ton KWF 2005-3390 Mouse models for Small Cell Lung Cancer: Genotype-phenotype 01-05-2005 31-07-2009
correlations as a basis to design better therapeutic intervention
strategies
Berns, Ton KWF 2008-4253 Designing and testing new intervention therapies for lung canccer 01-05-2009 01-05-2015
and mesotheliomas
Blank, Christian KWF 2008-3988 Blockade of PD-1/D-L1 interaction to improve T cell mediated 01-01-2008 01-01-2012
immunotherapy of cancer
Bleiker, Eveline KWF 2004-2987 Long-term physosocial impact of genetic testing among familial 01-11-2004 01-11-2009
adenomatous polyposis(fap) families
Bleiker, Eveline KWF 2005-3209 Psychosocial aspects of genetic testing in families at high risk of 01-11-2005 31-01-2010
multiple tumors at various sites and ages
ProjEcTS

Principal Number of Title Started 195 ProjEcTS
Ended/Ends
investigator project
Bleiker, Eveline KWF 2008-4016 Identification of psychosocial problems and perceived need for
support in cancer genetics
01-01-2008 01-01-2012
Borst, Jannie KWF 2003-2859 Improving anti-tumor immunity via TNF receptor family member 01-08-2003 01-09-2009
CD27 and its ligand
Borst, Jannie KWF 2004-3079 Tumor therapy with ionizing radiation and death ligand Trail: 01-07-2004 01-09-2009
efficacy and key molecular determinants.
Borst, Jannie KWF 2004-3087 Manipulation of the CD70 locus to monitor and modulate the 14-06-2004 01-09-2009
anti-tumor immune response and tumor development
Borst, Jannie KWF 2008-4028 T cell programming at the dendritic cell interface: impact on 01-01-2008 01-01-2012
anti-tumor immunity
Borst, Jannie KWF 2008-4110 Impact of TRAIL death receptor trafficking on pro-apoptotic 01-03-2008 01-03-2012
signaling
Borst, Piet KWF 2005-3379 A study of drug resistance mechanisms using large scale RNA 01-06-2005 01-06-2010
interference screens
Borst, Piet KWF 2006-3566 Mechanisms of chemotherapy resistance in spontaneous tumors 01-07-2006 15-07-2010
of genetically modified mice
Collard, John KWF 2007-3753 The par complex in cell polarity and tumor progression 01-07-2007 01-07-2011
Dalesio, Otilia KWF Datamanagement KWF Datamanagement 01-01-1982 01-01-2010
Dannenberg, KWF 2007-3978 Genetic analysis of class I HDACs in development and treatment 01-01-2008 01-01-2102
Jan-Hermen of cancer
Elkhuizen, Paula KWF 2009-4389 Properative accelerated partial breast irradiation (PAPBI): 01-01-2010 01-01-2014
defining radiotherapy sensitivity
Haanen, John KWF 2007-3943 HPV 16 E6/E7 DNA prime and E6/E7 long peptide boost 01-01-2008 01-01-2010
vaccination for multifocal Vulvar Intraepithelial Neoplasia (VIN).
Jacobs, Heinz KWF 2008-4112 A cancer genome atlas of the activation-induced cytidine 01-03-2008 01-03-2012
deaminase: novel insights into the pathogenesis and prognosis of
b cell lymphoma
Jacobs, Jacqueline KWF 2005-3458 Novel factors involved in chromosome end protection by telomeres 01-01-2006 01-01-2010
Jacobs, Jacqueline KWF 2007-3907 Dissecting the telomere damage response 01-05-2008 01-05-2010
Jalink, Kees KWF 2007-3733 TRMP7, a novel regulator of cytoskeletal tension: implications for 01-09-2007 01-09-2011
cancer progression, invasion and metastasis.
Jonkers, Jos KWF 2006-3486 Dissection of the role of E-cadhering loss-of-function in breast cancer 13-02-2006 12-02-2010
development and metastasis
Jonkers, Jos KWF 2006-3715 Functional assessment of innate and adaptive immunity in breast 01-01-2007 01-01-2011
cancer development
Jonkers, Jos KWF 2007-3772 Preclinical validation of chemical inhibitors of poly (ADP-ribose) 01-02-2007 01-02-2011
polymerase (PARP) in conditional mouse models for BRCA-
associated breast cancer.
Jonkers, Jos KWF 2008-4116 Impact of BRCA muntations on breast tumorigenesis and treatment 01-01-2008 01-01-2012
response: functional analysist of defined truncation mutants and
unclassified variants

196
ProjEcTS
ProjEcTS
Principal Number of Title Started Ended/Ends
investigator project
Linn, Sabine KWF 2006-3706 Towards patient-tailored systemic therapy in breast cancer:
a combined approached of translational research and mouse
01-01-2007 01-01-2013
model systems
Linn, Sabine KWF 2009-4435 In kaart brengen van actieve Estrogen Receptor binding sites bij 01-11-2009 01-11-2013
tamoxifen en aromatase inhibitor resistente cellijnen en
borsttumoren, om zo prodictieve markers te definiëren
Marijnen, Corrie KWF 2007-3945 Avoidance of overtreatment with radiotherapy in rectal cancer patients 15-09-2007 15-09-2009
Michalides, Rob KWF 2005-3388 Molecular basis of resistance to anti-estrogens in estrogen-receptor 01-10-2005 01-10-2009
positive breast cancer
Moolenaar, Wouter KWF 2005-3392 Autotaxin, a secreted lysophospholipase D: role in tumor progression 01-07-2005 01-07-2010
Moolenaar, Wouter KWF 2005-3383 PIP4K-beta/PtdIns5P/p53: A cellular stress regulated pathway and its 01-11-2005 31-10-2010
implication in oncogenesis
Nederlof, Petra KWF 2007-3749 Specific chromosomal imbalance in breast carcinomas as a marker 15-08-2007 15-08-2011
for breast cancer susceptibility
Neefjes, Jacques KWF 2005-3391 TPPII and the role in antigen presentation by MHC Class I molecules 01-10-2005 01-10-2009
Neefjes, Jacques KWF 2007-3883 Anti-cancer drugs and their effects on the ubiquitin cycle 01-04-2007 01-04-2011
Ovaa, Huib KWF 2005-3368 Activity and distribution of proteasome inhibitors 01-07-2005 01-01-2010
Peeper, Daniel KWF 2005-3382 The role of the neurotrophic tyrosine kinase receptor TrkB in anoikis 11-07-2005 10-07-2009
resistance, inavsion and metastasis of human tumors
Peeper, Daniel KWF 2006-3505 Human melanocytic nevi: oncogene-induced senescence as a 01-08-2006 01-08-2010
potential tumor-suppressing mechanism
Peeper, Daniel KWF 2007-3957 Towards improved melanoma treatment: gene identifcation, in vivo 01-07-2008 01-07-2014
modeling and drug target discovery
Remeijer, Peter KWF 2008-4024 Image guided radiotherapy for breast cancer 20-10-2008 20-10-2012
Rookus, Matti KWF 2004-3088 Gene-environment interactions in BRCA1/2 breast/ovarian cancer 06-09-2004 05-03-2009
families, a project of the Netherlands Collaborative Group on
Hereditary Breast Cancer
Rookus, Matti KWF 2007-3756 Heterogeneity of risk of breast and ovarian cancer in BRCA1 and 15-11-2007 15-11-2009
BRCA2 mutayion carriers
Rottenberg, Sven KWF 2009-4303 Analysis of tumor recurrence in a mouse model for hereditary 01-10-2009 01-10-2013
breast cancer: how do tumor-initiating cells escape eradication
by chemotherapy
Schagen, Sanne KWF 2002-2771 The incidence, nature and etiology of cognitive problems following 18-11-2002 01-09-2009
chemotherapy for cancer
Schagen, Sanne KWF 2009-4284 Structural, biochemical and functional indices of chemotherapy- 15-08-2009 15-08-2013
induced cognitive deficits in cancer patients
Schinkel, Alfred KWF 2007-3764 OATP1A/1B (SLCO1A/1B) drug uptake transporters in anticancer 01-12-2007 01-12-2011
drug pharmacokinetics and toxicity risks; possible strategies for
therapy optimization

Principal Number of Title Started 197 ProjEcTS
Ended/Ends
investigator project
Schmidt, Marjanka KWF 2007-3839 Genetics determinants of survival and second breast cancer
development in premenopausal breast cancer patients
01-01-2010 01-01-2011
Schmidt, Marjanka KWF 2009-4363 Breast cancer outcome: genetic destiny 01-07-2009 01-07-2015
Schumacher, Ton KWF 2003-2860 Feasibility and limitations of T cell receptor gene transfer 01-08-2004 01-01-2009
Schumacher, Ton KWF 2009-4581 UV-gevoelig MHC peptide uitwisselbare streptameer 01-07-2009 01-07-2010
Schumacher, Ton KWF 2006-3530 Generation of human tumor specific T cells with high affinity T cell 01-12-2006 01-12-2010
receptors
Schumacher, Ton KWF 2007-3825 SNP-based genome-wide identification of hematopoiesis-restricted 01-07-2007 01-07-2011
minor histocompatibility antigens
Schumacher, Ton KWF 2009-4282 Preclinical development of TCR gene therapy for prostate carcinoma 01-12-2009 01-12-2013
Sixma, Titia KWF 2006-3476 Structural and functional analysis of a novel E3 ubiquitin ligase 01-01-2010 01-08-2010
consisting of the polycomb proteins Bmi1 and Ring 1b (RNF2)
Sixma, Titia KWF 2008-4014 Comparative study of Ubiqutin-specific proteases 01-02-2008 01-02-2012
Sonke, Jan-Jakob KWF 2005-3378 High precision radiotherapy in the presence of anatomical changes 14-11-2005 14-11-2009
Sonke, Jan-Jakob KWF 2006-3545 Imaged guided correction strategies to optimize the precision 01-08-2006 01-08-2010
of radiotherapy
Sonnenberg, Arnoud KWF 2008-3996 A mouse stem cell model for skin cancer: Dual role of the integrin 01-08-2008 01-08-2012
a6B4 in tumor initiation and progression
Stewart, Fiona KWF 2005-3370 The role of TGFb and Notch signaling in the development of 01-04-2005 01-04-2009
radiation-induced telangiectasia
Stewart, Fiona KWF 2005-3373 Radiation-induced atherosclerosis: mechanisms and preventive 01-06-2005 01-06-2009
intervention strategies
Stewart, Fiona KWF 2008-3993 The role of endoglin in induction and repair of cardiovascular 01-06-2008 01-06-2012
damage after radiation alone or in combinatione with trastuzumab
(Herceptin)
Tan, Bing KWF KWF Ontwikkelingssamenwerkingsprogramma 01-01-2008 01-01-2012
Tan, Bing KWF 2008-4233 Early detection of primary and recurrent nasopharyngeal carcinoma 01-09-2008 01-09-2013
(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 modification to study the role of the mismatch repair 15-11-2004 14-11-2009
complex MSH2/MSH6 in mutation avoidance and tumor suppression
te Riele, Hein KWF 2006-3589 Significance of the Fanconi anemia caretaker pathway in 01-01-2007 01-01-2011
development and treatment of cancer
te Riele, Hein KWF 2007-3790 Role of the G2 restriction point in tumor development and behavior 01-04-2007 01-04-2011
van Blitterswijk, Wim KWF 2005-3377 Lipid rafts as novel targets for anti-cancer therapy and induction 01-02-2005 01-01-2010
of apoptosis.
van de Vijver, Marc KWF 2002-2575 Microarray analysis of breast cancer as a diagnostic tool to guide 01-02-2002 01-11-2009
optimal treatment

198
ProjEcTS
Principal Number of Title Started Ended/Ends
investigator project
van den Brekel, Michiel KWF 2007-3941 Prediction of local control after radiotherapy in head and neck
cancer
01-11-2007 01-11-2009
van Driel, Willemien KWF 2006-16 Phase III randomised clinical trial for stage III ovarian carcinoma 14-03-2007 14-03-2010
randomising between secondary debulking surgery with or without
hyperthermic intraperiotoneal chemotherapy (OVHIPEC-1)
van Herk, Marcel KWF 2007-3895 Probability based treatment planning with biological objectives for 01-11-2007 01-11-2011
high-dose high-precision radiotherapy of prostate cancer
van Herk, Marcel KWF 2007-3751 High precision image-guided radiotherapy for bladder cancer 01-01-2008 01-01-2012
van Leeuwen, Fijs KWF 2009-4344 Targeted CXCR4 with multimodal imaging agents as a means to 01-09-2009 01-09-2015
improve the efficacy of breast cancer surgery via combined pre-,
intra- and postoperative imaging
van Leeuwen, Flora KWF 2004-3068 Long-term risk of second cancers and cardiovascular disease 01-11-2004 01-11-2009
following treatment of Hodgkin’s disease
van Leeuwen, Flora KWF 2006-3631 Long-term risk of cancer after ovarian stimulation for in vitro 01-04-2007 01-04-2011
fertilization
van Leeuwen, Flora KWF 2008-3994 Cardiovascular morbidity and mortality in breast cancer survivors 27-10-2008 27-10-2012
van Lohuizen, Maarten KWF 2005-3376 Investigating the role of the Polycomb- group gene BMi in 01-07-2005 01-07-2009
medullosblastomas and in neural cancer stem cell maintenance
van Lohuizen, Maarten KWF 2006-3476 Structural and functional analysis of a novel E3 ubiquitin ligase 01-01-2010 01-08-2010
consisting of the polycomb proteins Bmi1 and Ring 1b (RNF2)
van Lohuizen, Maarten KWF 2007-3877 Epigenetic regulation by the deubiquitinating enzyme USP3: 01-03-2007 01-03-2011
impact on genome stability and tumorgenesis
van Lohuizen, Maarten KWF 2007-3803 Cancer stem cells and breast cancer: a polycomb connection 01-04-2007 01-04-2011
van ‘t Veer, Laura KWF 2007-3839 Genetics determinants of survival and second breast cancer 01-01-2010 01-01-2011
development in premenopausal breast cancer patients
Verheij, Marcel KWF 2007-3939 Improvement of chemoradiation response in head and neck cancer 01-02-2008 01-02-2010
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 01-11-2008 01-11-2012
amphiphilic anti-cancer drugs
Wolthuis, Rob KWF 2007-3789 Targeting Mitotic Protein Destruction as an approach for cancer 01-09-2007 01-09-2011
therapy
Wolthuis, Rob KWF 2008-4135 Divergent control of Cyclin B1-Cdk1 in cancer cells: a key role in 01-09-2008 01-09-2012
therapy

Major Projects
suPPorted by other organizations
199 Projects
Principal granting agency title started ended/ends
investigator
Aaronson, Neil EORTC Methods and measures for assessing the HRQL of mid to long term 01-05-2009 01-05-2011
survivors of testicular and prostate cancer
Aaronson, Neil St Nuts Ohra Spoed - erfelijkheidsonderzoek bij borstkanker: invloed op 01-08-2007 01-08-2011
behandelingskeuzes en welbevinden / Behavioral and psychosocial
effects of rapid genetic counseling and testing in newly diagnosed breast
cancer patients: a multicenter study
Agami, Reuven CBG CBG 3e fase: Identification of cancer-relevant genes using a functional 01-01-2009 01-01-2014
genetic approach
Agami, Reuven Dr. Josef Steiner Dr Josef Steiner Cancer Award 2007 01-05-2007 01-05-2011
Krebsstiftung
Agami, Reuven EEG-CEC / EU Mutant p53 as target for improved cancer treatment (mutp53) 01-02-2004 01-08-2009
Agami, Reuven EEG-CEC / EU Indentifying novel regulatory mechanisms of miRNA functions 01-10-2008 01-10-2013
Agami, Reuven NWO Genome wide search for DNA damage checkpoint functions in human 15-02-2005 14-02-2010
cells (Euryi-award)
Agami, Reuven NWO Combined miRNA profiling and genetic screens to identify and 01-10-2007 01-10-2012
characterize cancer-related miRNAs
Beijersbergen, Roderick Johnson en Johnson Johnson en Johnson 01-12-2006 15-01-2010
Bernards, René Astra Identification of novel activity modulators of small molecule mTOR 07-12-2007 07-12-2009
kinase inhibitos in breast cancer
Bernards, René CBG CBG 3e fase: Identification of cancer-relevant genes using a functional 01-01-2009 31-12-2013
genetic approach
Bernards, René CBG CBG 3e fase: Identification of cancer-relevant genes using a functional 01-01-2009 31-12-2013
genetic approach
Bernards, René CGC CGC 2008-2012 01-01-2008 01-01-2013
Bernards, René CGC CGC 2008-2012 01-01-2010 01-01-2013
Bernards, René EEG-CEC / EU Breast Cancer Biomarkers and Functional mediators : harnessing the
New Wealth of Omic Data (Target Breast) 13-02-2006 12-02-2010
Bernards, René EEG-CEC / EU EG Rubicon Annex I EC 01-01-2006 01-01-2011
Bernards, René EEG-CEC / EU EG Rubicon Annex I EC 01-01-2010 01-01-2011
Bernards, René NIH System-based predications of responses to cancer therapy 01-09-2005 01-02-2010
Bernards, René NWO Spinozapremie 26-09-2005 26-09-2010
Bernards, René TI Pharma Kinases in cancer (TI Pharma) 01-07-2007 01-07-2011

200
Projects
Principal granting agency title started ended/ends
investigator
Berns, Ton CBG CBG 3e fase: Identification of cancer-relevant genes using a functional 01-01-2009 31-12-2010
genetic approach
Berns, Ton CBG CGC 2008-2012 01-01-2010 01-01-2013
Berns, Ton EEG-CEC / EU Lung stem cells and small-cell carcinoma; Marie Curie International 01-01-2007 01-01-2009
Incoming Fellowships
Berns, Ton EEG-CEC / EU MUGEN: Identification of Novel Targets for Cancer Therapy. 01-01-2005 01-01-2010
Translating basic knowledge of functional oncogenomics into cancer
diagnosis and treatment
Berns, Ton Merck Merck Res Coll LKR 25006 01-02-2006 01-02-2010
Berns, Ton Merck Merck Res Coll LKR 25006 01-01-2010 01-02-2010
Berns, Ton NWO Characterizing oncogenes and tumor suppressors by low noise 01-01-2009 31-12-2009
expression arry datasets
Berns, Ton TI Pharma Kinases in Cancer (TI Pharma) 01-07-2007 01-07-2011
Bleiker, Eveline Dutch Gastr Res pancreas project (rotterdam) 01-11-2008 01-01-2010
Foundation
Bleiker, Eveline Polyposis PPC coordinatie 01-08-2009 01-01-2010
Contactgroep
Borst, Jannie NWO A novel type of ubiquitination regulates apoptosis signaling by 01-11-2008 01-11-2012
BH3-only protein Bid
Borst, Jannie TI Pharma TNF ligands in cancer 01-03-2006 01-03-2010
Borst, Piet EEG-CEC / EU Molecular mechanisms underlying chemotherapy resistance, 01-02-2007 01-02-2012
therapeutic escape, efficacy and toxicity (CHEMORES)
Borst, Piet ZonMw Characterization of the physiological roles of Multidrug Resistance 01-01-2008 01-01-2012
Protein (MRP) 1-6 by in vivo screening for their substrates (TOP subsidie)
Burgers, Sjaak CTMM Personalized chemo-radiation of lung and head and neck cancer. 01-01-2009 01-01-2014
WP5: Image-guided adaptive treatment (evaluation during treatment)
Cats, Annemieke Roche Ondersteuning junior medical trials coordinator. CRITICS-studie en 01-10-2007 01-01-2010
rectum-carcinoom projecten. ML20482
Cohen, Serge NWO NWO Veni 700.55.405 01-02-2006 31-01-2009
Collard, John EEG-CEC / EU An integrated concept of tumor metastasis: implications for therapy 01-04-2008 01-04-2012
Dalesio, Otilia EEG-CEC / EU ‘eTEN’ Programme - Call identifier 01-11-2006 01-05-2010
Dalesio, Otilia IKA IKA kankerregistratie 01-09-1990 01-01-2010
Dalesio, Otilia NVALT Statistical Center NVALT Clinical Trials in oncology 01-05-2004 01-01-2010
Dalesio, Otilia VIKC Verdeling voor CKTO projecten VIKC 01-07-2007 01-01-2010
Dannenberg, Jan-Hermen NWO-ALW The role of class I HDACs in normal physiology, tumorigenesis and 01-08-2007 01-08-2012
transcriptional regulation
Elsakkers, Peter AMC Poli Familiare Tumoren 01-01-2008 01-01-2010

Principal
investigator
granting agency title started 201 Projects
ended/ends
Filippo, Ronald Koninklijk Ontwikkeling KNGF-standaard Beweeginterventie oncologie 01-10-2009 01-08-2010
Nederlands
Genootschap voor
Fysiotherapie
(KNGF)
Filippo, Ronald VU medisch centrum Cytofys (EVA project) 01-09-2007 01-09-2011
Fornerod, Maarten NWO-ALW Connecting chromatin to the plasma membrane 01-01-2007 01-04-2010
Gilhuijs, Kenneth CTMM Neoadjuvant drug treatment for breast cancer - response-prediction 01-10-2008 01-10-2013
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 CTMM Breast Care WP0: Immalytics workstation for fusing of images 01-10-2008 01-10-2013
Haanen, John Cogem Gentherapie met naakt DNA, mogelijkheden voor deregulering 01-08-2009 01-02-2010
Haanen, John NWO ZonMW 432-00-001 01-09-2006 01-09-2009
Haramis, Anna Pavlina NWO NWO VIDI Haramis 917-56-322 01-01-2005 31-12-2009
Haramis, Anna Pavlina NWO SFN 2.2.33 Startgeld (bij VIDI Haramis, project 57350) 01-01-2005 31-12-2009
van Harten, Wim CvZ Operations/Lean Management 01-01-2002 01-12-2009
van Harten, Wim CvZ CvZ Array Raster 3 (fase 2) 01-01-2006 31-12-2009
Hauptmann, Michael EEG-CEC / EU Prospective cohort studies of children with substantial medical 01-09-2008 01-12-2009
diagnostic exposure
van Herk, Marcel CTMM CTMM AIR FORCE WP6: Data integration ‘Oncology Research 01-12-2008 01-07-2013
Workstation’ (ORW)
van Herk, Marcel CTMM CTMM AIR FORCE WP3: Development and validation of PET-CT 01-09-2008 01-09-2013
imaging tools: software.
van Herk, Marcel EOS / EOS 01-05-2000 01-05-2010
voorheen Philips
Hilgers, Frans ATOS Development and evaluation of a third generation ProvoxTM voice 01-06-2003 01-01-2010
prosthesis
Hilgers, Frans ATOS ATOS Medical Collaboration 30-06-2003 01-01-2010
Hofland, Linda Biolitec PDT Biolitec: Photodynamic Therapy 01-01-2005 01-01-2010
Horenblas, Simon Stichting Aanstelling Arts Onderzoeker subsidie via J.C. vd Veen Stichting 01-02-2008 01-01-2010
J.C. van Veen
Innocenti, Metello CGC CGC 2008-2012 junior Group Innocenti 01-05-2009 31-12-2012
Jacobs, Heinz ZonMw Role of translesion DNA synthesis in immunity and cancer 01-01-2005 01-01-2010
development
Jacobs, Jacqueline NWO NWO Vidi Jacobs 01-02-2007 01-02-2012
Jacobs, Jacqueline NWO SFN matching bij Vidi beurs Jacobs 01-02-2007 01-02-2012

202
Projects
Principal granting agency title started ended/ends
investigator
Jalink, Kees SenterNovem MEM-FLIM: Modulated Electron-Multiplied all-solid-state camera for 01-03-2008 01-03-2012
Fluorescence Lifetime Imaging Microscopy
Jalink, Kees ZonMw ZonMW 9120.6110 01-06-2006 01-06-2009
Jonkers, Jos AICR Genome-wide insertional mutagenesis screens to identify cancer genes 01-10-2007 01-10-2010
that collaborate with E-cadherin loss in mammary tumourigenesis
Jonkers, Jos AstraZeneca Targeting the P13K network in genetically engineered mouse models 01-11-2008 01-11-2010
of invasive lobular breast cancer
Jonkers, Jos CTMM Neoadjuvant drug treatment for breast cancer - response prediction 01-10-2008 01-10-2013
and response monitoring (BREAST CARE); SP (sub-workpackage)
2: Genetically engineered mouse models for response prediction and
molecular imaging of ER positive breast cancer
Jonkers, Jos CTMM Neoadjuvant drug treatment for breast cancer - response prediction 01-10-2008 01-10-2013
and response monitoring (BREAST CARE); SP (sub-workpackage)
4: Molecular imaging of PR- or ER-positive breast cancers using
radiolabeled PR- or ER-antagonists
Jonkers, Jos EEG-CEC / EU The role of Polycomb genes in normal stem cell and breast cancer 01-03-2009 01-03-2011
stem cell self-renewal and maintenance
Jonkers, Jos EEG-CEC / EU Eurosystem 01-03-2008 01-03-2012
Jonkers, Jos Kudos NKI-Kudos Collaborative Res. 01-07-2006 01-07-2009
Jonkers, Jos Merck Merck Res Coll LKR 25006 01-01-2010 01-02-2010
Jonkers, Jos NWO Mammary tumorigenesis in conditional tumor suppressor gene 01-02-2003 31-01-2010
knockout mice: disease progression, gene discovery and cellular
pathways
Jonkers, Jos TI Pharma Kinases in cancer (TI Pharma) 01-07-2007 01-07-2011
Kerkhoven, Ron CBG Deep Sequencing project CBG 2009-2013 (onderdeel van het CBG project) 01-01-2009 01-01-2013
Klomp, Houke Roche Roche HQ Neo adjuvant Tarceva: Tumor remission rate with neoadjuvant 01-07-2006 01-01-2010
erlotinib in operable patients with clinical stage I/II non-small cell lung
cancer (NSCLC) (M06NEL)
Klomp, Houke Vrolijk Stichting Vrolijk (NSCLC); aanstelling voor arts-onderzoeker 04-08-2006 01-07-2010
van Leeuwen, Fijs CTMM Neoadjuvant drug treatment for breast cancer-response-prediction 01-10-2008 01-10-2013
and response monitoring (BREAST CARE). SP (sub-workpackage)
3: Improved assays for analysis of multidrug resistance transporter
activity
van Leeuwen, Fijs EEG-CEC / EU HYPERImage: Hybrid PET-MR system for concurrent ultra-sensitive 01-04-2008 01-04-2011
imaging
van Leeuwen, Fijs STW Imaging agents for multimodal visualization of ductal carinoma in situ 01-09-2006 01-09-2009
(DCIS)
van Leeuwen, Flora AZG Cardiovascular morbidity in testicular cancer survivors: case-control 18-02-2005 31-12-2009
study of risk factors and assessment of pharmacogenomic
determinants of toxicity
van Leeuwen, Flora CTSU Heart-breast and heart-Hodgkin case-control studies 01-01-2010 01-09-2010

Principal granting agency title started ended/ends
investigator
van Leeuwen, Flora EEG-CEC / EU EG GENE-Rad-Risk: Radiation exposures at an early age: impact of 01-06-2005 30-11-2009
genotype on breast cancer risk.
van Leeuwen, Flora NIH Risk and prognosis of uterine corpus cancer after tamoxifen treatment 25-09-2007 01-09-2010
for breast
van Leeuwen, Flora Pink Ribbon Invloed van lichaamsbeweging en andere leefstijlaspecten op kwaliteit 01-08-2007 01-01-2009
van leven en prognose bij borstkanker
van Leeuwen, Flora Rathenau Instituut Onderzoekrapport voor Rathenau Instituut 01-12-2008 28-02-2009
van Leeuwen, Flora Vlissingen Van Vlissingenfonds: polikliniek overlevers Hodgkin 01-08-2009 01-08-2012
Lymfoomfonds
van Leeuwen, Flora ZonMw HEBON research facility for individuals tested for BRCA 1/2 mutations 23-10-2009 23-10-2013
van Leeuwen, Fred NPC NPC T1.4 Chromatin dynamics and epigenetic changes 01-01-2009 01-07-2013
van Leeuwen, Fred NWO The role of histone methylation in silencing and transcriptional memory. 01-02-2005 01-02-2010
van Leeuwen, Fred NWO A multifunctional genome-wide screen to unravel the roles of the 16-11-2007 16-05-2010
conserved epigenetic regulator Dot1 in chromosome organization
Linn, Sabine Agendia B.V. Unrestricted research grant to fund the work of Dr. Michael Knauer 01-09-2008 01-09-2009
van Lohuizen, Maarten CBG CBG 3e fase: Identification of cancer-relevant genes using a functional 01-01-2009 31-12-2013
genetic approach
van Lohuizen, Maarten Dijkzigt Stem cells in Development and Disease 01-01-2004 31-12-2009
van Lohuizen, Maarten EEG-CEC / EU Platforms for biomedical discovery with human ES cells 01-08-2006 01-08-2010
van Lohuizen, Maarten EEG-CEC / EU Eurosystem 01-03-2008 01-03-2012
van Lohuizen, Maarten Ernst Schering Regulation of histone H2A monoubiquitination byRing1b/Bmi1 and 01-09-2007 01-09-2009
Foundation USP3 in DNA damage response and oncogenic transformation
van Lohuizen, Maarten Merck Merck LKR #40778 PO X21000363 02-06-2006 01-02-2012
van Lohuizen, Maarten NPC NPC T4.2 Properties of pluripotency in mouse ESCs and iPS 01-01-2009 30-06-2013
van Lohuizen, Maarten NWO-ALW Genome-wide Spatial Profiling of Polycomb Domains in Drosophila 01-12-2007 01-12-2010
melanogaster
van Lohuizen, Maarten NWO-ALW Maintenance of genome integrity by histone (de)ubiquitinating enzymes 01-11-2008 01-11-2013
van Luenen, Henri NWO L1 and Alu elements: Structural and functional analysis of human 01-10-2004 01-10-2009
non-LTR retrotransposons
Michalides, Rob TI Pharma TI Pharma 3.7: Nuclear receptors in targeted cancer therapy: 01-11-2006 01-11-2010
improved methods for candidate selection
Mijnheer, Ben STW 3-D Dosimetric verification of advanced irradiation techniques for 01-01-2006 01-07-2009
cancer patient treatment
Moolenaar, Wouter CBG CBG 3e fase: Identification of cancer-relevant genes using a functional 01-01-2009 01-01-2011
genetic approach
Neefjes, Jacques CBG CBG 3e fase: Identification of cancer-relevant genes using a functional 01-01-2009 31-12-2013
genetic approach
203 Projects

204
Projects
Principal
investigator
granting agency title started ended/ends
Neefjes, Jacques EEG-CEC / EU Systems biology of phagosome formation and maturation - modulation 01-11-2008 01-11-2011
by intracellular pathogens
Neefjes, Jacques KNAW Tissue responses to radiotherapy and development of immunotherapy 15-11-2007 15-11-2011
for successful radioimmunotherapy of human melanoma
Neefjes, Jacques NPC NPC E2.5 Kinome and phosphatasome knock-down libraries 01-01-2009 30-06-2013
Neefjes, Jacques NWO Manipulating the MHC class II system to control immune responses 01-10-2007 01-10-2012
in autoimmunity
Neefjes, Jacques NWO The molecular mechanism of the degradation of large molecular 01-01-2010 01-01-2013
complexes
Neefjes, Jacques NWO-ALW Defining the MHC class II loading microdomain to understand 01-11-2005 01-11-2009
immune responses
Oldenburg, Hester Pink Ribbon Werkhervatting en seksualiteit na borstkanker 01-09-2008 01-09-2011
Ovaa, Huib Dana Farber SPORE in Myeloma: Effect of Proteasome Inhibitors on 11-06-2007 01-06-2010
Cancer Institute Immunoproteasome Activity in Multiple Myeloma
(Career Development Award)
Ovaa, Huib EEG-CEC / EU From Receptor to Gene: structures of complexes from signalling 01-01-2010 01-07-2010
pathways linking immunology, neurobiology and cancer (SPINE 2)
Ovaa, Huib EEG-CEC / EU Construction of Well-defined Ubiquitin Conjugates SP3 People 15-10-2008 15-10-2011
Ovaa, Huib NGI Commercial development of ubiquitinated peptides and screening 01-01-2010 01-01-2012
assays produced by high throughput robotic peptide synthesis
Ovaa, Huib NPC NPC E2.1 Tyrosine phosphatase inhitors, baits and ABPs 01-01-2009 30-06-2013
Ovaa, Huib NPC NPC V1 Commercial development of synthetic Nedd8 and 01-01-2009 01-07-2013
SUMO conjugates
Ovaa, Huib NWO Modulation of autotaxin activity by small molecules 01-11-2006 01-04-2010
Ovaa, Huib NWO Chemical biology of ubiquitin-like modifications 01-09-2005 01-09-2010
Ovaa, Huib NWO Development of conditional protein-ligand exchange applied to 01-09-2008 01-09-2012
immune technology; Onderzoeksprogramma: Integration of
Biosynthesis & Organic Synthesis (IBOS)
Ovaa, Huib NWO Chemische Pan- Dub Inhibitors to delineate DUB dependent biology and tools to 01-08-2009 01-08-2013
Wetenschappen study the ubiqultinated proteome
Ovaa, Huib NWO-ALW Post-translational transpeptidation and immunity 01-08-2009 01-08-2013
Ovaa, Huib Wellcome trust Probing the role of adjuvants in MHC-II antigen presentation 01-05-2009 01-11-2009
Ovaa, Huib ZonMw Immunoproteomics: antigen mapping through MHC epitope 01-11-2008 01-06-2010
exchange reactions
Peeper, Daniel EEG-CEC / EU EU FP6 Grant No.037758 ‘The anoikis suppressor TrkB as a target for 01-01-2007 01-01-2010
novel anti-cancer agents’
Peeper, Daniel EMBO EMBO Young Investigators 01-09-2006 01-09-2009

Principal granting agency title started ended/ends
investigator
Peeper, Daniel NWO Synthetic lethality: a novel tactic for selective anticancer drug target 01-06-2007 01-06-2012
discovery
Peeper, Daniel NWO-ALW Genome-wide identification and functional characterization of novel 01-01-2007 01-01-2009
tumor suppressor genes
Perrakis, Anastassis EEG-CEC / EU Integrating and strengthening the European Research Area FP6-2005 01-01-2007 01-01-2010
Lifescihealth 6 Proposal NO 037198
Perrakis, Anastassis EEG-CEC / EU A multidisciplinary approach to determine the structures of protein 01-02-2005 01-02-2010
complexes in a model organism
Perrakis, Anastassis EEG-CEC / EU From Receptor to Gene: structures of complexes from signalling 01-01-2010 01-02-2010
pathways linking immunology, neurobiology and cancer (SPINE 2)
Perrakis, Anastassis EEG-CEC / EU From Receptor to Gene: structures of complexes from signalling 01-07-2006 01-07-2010
pathways linking immunology, neurobiology and cancer (SPINE 2)
Perrakis, Anastassis NIH NIH grant 2R01 GM062612-05, EMBL Heidelberg 01-05-2006 01-05-2010
Perrakis, Anastassis NWO The NKI Protein Facility: Indentification, Production, Characterization 01-01-2009 31-12-2010
and interactions
Perrakis, Anastassis Pfizer Cristallization of Autotaxin 2230 03-06-2009 30-06-2011
Peters, Peter Aeras Global TB Public Private Partnership for research into and development of 01-05-2007 01-05-2011
Vaccine Foundation medicines, vaccines and diagnostic aids in the domain of AIDS,
tuberculosis and malaria
Peters, Peter EEG-CEC / EU EG AntePrion CT2006-019090 01-06-2006 01-06-2009
Peters, Peter EEG-CEC / EU EG AntePrion CT2006-019090 (M) Development of a pre-clinical blood 01-06-2006 01-06-2009
test for prion diseases Coordiantie
Peters, Peter EEG-CEC / EU EG ImmunoPrion Immunological and structural studies of prion diversity 01-06-2006 01-12-2009
Peters, Peter EEG-CEC / EU Understanding prion strains and species barriers and devising novel 01-11-2006 01-05-2010
diagnostic approaches (strainbarrier)
Peters, Peter EEG-CEC / EU Protecting the food chain form prions: shaping European priorities 01-10-2009 01-10-2013
through basic and applied research (KP 7)
Peters, Peter MIN OCW E-cadhering and cadhering-11 and their role in cancer migration’ binnen 01-01-2009 31-12-2011
het SmartMix programma NIMIC
Peters, Peter NIH Pathways of antigen presentation by CD 1 a, b and c 01-01-2005 31-12-2009
te Riele, Hein AFM 13280 Cellular factors that determine somatic (CTG)n hypermutability 01-07-2008 01-07-2009
in DM1: targets for therapy
te Riele, Hein NWO Horizon 050-71-051 01-12-2006 01-12-2011
te Riele, Hein NWO-ALW A novel procedure for enzymatic production of low-complexity RNAI 01-08-2009 01-08-2012
Libraries to identify coding- and non-coding turmor suppressor genes
Rodenhuis, Sjoerd CTMM Neoadjuvant drug treatment for breast cancer - response prediction 01-10-2008 01-10-2013
and response monitoring (BREAST CARE); SP (sub-workpackage)
5: Biomarker discovery and validation in patients
205 Projects
Rookus, Matti DES DES Schadefonds; Projectleiderschap: Matti Rookus en Floor van Leeuwen 15-06-2006 01-01-2011

206
Projects
Principal granting agency title started ended/ends
investigator
Rottenberg, Sven CTMM Neoadjuvant drug treatment for breast cancer - response prediction 01-10-2008 01-10-2013
and response monitoring (BREAST CARE); SP (sub-workpackage)
1: Response biomarker discovery for Cisplatin and Docetaxel in mouse
models of breast cancer
Ruers, Theo Philips Research Collaboration Agreement for Data Collection - Ex-vivo human 12-10-2009 12-01-2010
tissue study
Rutgers, Emiel EORTC EORTC breast group 01-01-2000 30-10-2009
Rutgers, Emiel EORTC EORTC Breast Group werkvergadering 01-03-2002 01-01-2010
van Sandick, Johanna Vrolijk Slokdarmkankeronderzoek Stichting Vrolijk 01-01-2008 01-01-2011
van Sandick, Johanna Vrolijk Lab slokdarmkankeronderzoek Stichting Vrolijk 01-09-2009 01-01-2011
Schagen, Sanne Pfizer Assessment of neuropsychological sequelae of tamoxifen and 01-06-2003 01-01-2009
exemestane in postmenopausal women with early breast cancer.
TEAM-neuropsychologiestudie
Schagen, Sanne Pink Ribbon Informatievoorziening over cognitieve klachten bij borstkankerpatienten 01-01-2009 01-01-2010
Schellens, Jan aCBG Conv. NKI-aCBG (Geneesm.) 01-08-2006 30-04-2010
Schellens, Jan Astra AstraZeneca Affinity of AZD1152 24-03-2005 01-12-2009
Schellens, Jan Astra Preclinical in vivo testing of AZD1152 to determine the effect of the 01-09-2007 01-01-2010
ABC-drug transporters P-glycoprotein (Pgp, Mdr1) and BCRP
(Bcrp1, Abcg2) on tissue distribution and clearance of AZD1152
Schellens, Jan Fonds NutsOhra Veiligheid- & kostenbesparingstudie voor DPYD2A genotypering bij 01-01-2008 01-01-2010
fluoropyrimidinetherapie
Schellens, Jan Fujifilm Fujifilm Manufacturing Europe BV 01-12-2007 01-01-2009
Manufacturing
Europe BV
Schellens, Jan Roche Support Roche clinical research associate Candersartan study M06HER 01-02-2009 01-01-2012
Schinkel, Alfred STW Generation and validation of cyctochrome P450 3A knockout and 01-06-2004 01-10-2009
transgenic mice as tools for improved drug development and research
Schoo, Hans EORTC Congres : Perspectives of Immunotherapy in GI cancer 01-07-2009 31-12-2009
Schumacher, Ton ACTS (NWO) Development of conditional protein-ligand exchange applied to 01-09-2008 01-09-2012
immune technology
Schumacher, Ton DSF (Danish Development and clinical evaluation of new strategies for adoptive cell 01-01-2009 01-01-2014
Council for transfer (ACT) in the treatment of cancer.
Strategic Research)
Schumacher, Ton EEG-CEC / EU Attack: Adoptive engineered T-cell targeting to activate cancer killing 01-11-2005 01-11-2010
Schumacher, Ton EEG-CEC / EU ATTRACT - Advanced Teaching and TRaining for Adoptive Cell Therapy. 01-10-2009 01-10-2012
FP7. Marie Curie
Schumacher, Ton Landsteiner Stichting MHC multimer-based adoptive T cell therapy. 01-05-2006 01-05-2009
Schumacher, Ton Landsteiner Stichting Graft versus Host disease by TCR-engineered T cells: mechanisms and 01-01-2009 01-01-2012
means for prevention. LSBR. Dossiernr 0804

Principal
investigator
granting agency title started 207 Projects
ended/ends
Schumacher, Ton Melanoma Research Platform For MHC-Exchange Based T-Cell therapy For Melanoma 01-09-2008 01-09-2010
Alliance
Schumacher, Ton NWO Dissecting metabolic T-Cell dysfunction in cancer 01-02-2009 01-02-2010
Schumacher, Ton UCL, University LRF New Grant Award 06037 toegekend aan Dr Gavin Bendle: 01-03-2007 01-03-2009
College London Adjuvant strategies in T cell receptor gene therapy
Schumacher, Ton ZonMw T cell receptor gene therapy of metastatic melanoma: 01-04-2005 31-03-2010
a phase I clinical trial
Sixma, Titia CBG CBG 3e fase: Identification of cancer-relevant genes using a functional 01-01-2009 01-01-2014
genetic approach
Sixma, Titia EEG-CEC / EU MCRTN Ubiregulators: Signal Transduction by Ubiquitination, 01-01-2007 01-01-2011
a Matter of Location
Sixma, Titia EEG-CEC / EU EG Rubicon Annex I EC 01-01-2010 01-01-2011
Sixma, Titia EEG-CEC / EU Neurotransmitter Cys-loop recptors: structure, function and disease 01-02-2008 01-02-2012
Sixma, Titia EEG-CEC / EU Mismatch2model-Characterization and quantitative modeling of DNA 01-12-2008 01-12-2012
mismatch repair and its role in the maintenance of genomic stability
and cancer avoidance
Sixma, Titia NWO NWO CW PIO 700.51.401 01-02-2003 01-01-2009
Sixma, Titia TI Pharma New approaches for Ligand-Gated Ion Channel (LGIC) drug discovery. 01-01-2007 01-01-2011
TI Pharma D2-103
Sonke, Jan-Jakob CTMM Neoadjuvant drug treatment for breast cancer - response prediction 01-10-2008 01-10-2013
and response monitoring (BREAST CARE); SP (sub-workpackage)
9: Image guided radiation therapy
Sonke, Jan-Jakob EEG-CEC / EU HYPERImage: hybrid PET-MT system for concurrent ultra-sensitive 01-04-2008 01-04-2011
imaging
Sonnenberg, Arnoud nierstichting Function of tetraspanin-integrin complex Cd151- a3B1 in development 01-10-2008 01-10-2012
and maintenance of the glomerular filtration barrier. NSN project
Sonnenberg, Arnoud NWO NWO 815.02.002 01-11-2005 01-11-2009
Sonnenberg, Arnoud NWO-ALW Controlling hemidesmosome dynamics by phosphorylation of residues 01-03-2007 01-04-2011
on the integrin b4 subunit
Sonnenberg, Arnoud STW CGC A. Sonnenberg 01-06-2009 01-06-2012
van Steensel, Bas EEG-CEC / EU NET member of The Epigenome Network of Excellence: Epigenome, 01-06-2004 01-06-2009
epigenetic plasticity of the genome
van Steensel, Bas NWO Whole-genome analysis of the regulation of chromatin structure and 01-03-2005 01-03-2010
gene expression
van Steensel, Bas NWO Multi-gene chromatin domains 01-12-2006 01-12-2010
van Steensel, Bas NWO Chromatin Protein Discovery Project 01-02-2008 01-02-2012
van Steensel, Bas NWO-ALW Nuclear lamina genome interactions in mammalian cells 01-07-2009 01-07-2014

208
Projects
Principal
investigator
granting agency title started ended/ends
Stewart, Fiona EEG-CEC / EU Cardiorisk - The mechanisms of cardiovascular risks after low 01-02-2008 01-02-2011
radiation doses
Stroom, Joep Maastro Accurate target definition of non-small cell lung tumors using 01-09-2006 01-09-2010
pathology-validated PET and CT imaging for the optimization of
radiotion treatment
Tan, Bing Biolitec The cost-effectiveness study/ZonMw project Biolitec-NKI 01-12-2008 01-01-2012
Tan, Bing ZonMw A multi-centre cost-effectiveness evaluation of a novel treatment option 01-03-2009 01-03-2012
in the Netherlands: Photo Dynamic Therapy with temoporfine for the
treatment of advanced incurable head and neck cancers, for whom prior
conventional treatments have failed
Valdés Olmos, Renato EEG-CEC / EU Mammography with molecular imaging (MAMMI) Specific Targeted 01-01-2007 01-01-2011
project
Valdés Olmos, Renato ONCO Vision Dection of Deep Located Sentinel Nodes using Preoperative SPECT-CT 01-05-2008 01-05-2010
with an Hybrid System and Peroperative Real Time Imaging with a
Portable Small Camera
van ‘t Veer, Laura CBG CGC 2008-2012 01-01-2010 01-01-2013
van ‘t Veer, Laura EEG-CEC / EU Molecular signatures as diagnostic and therapeutic targets for 01-11-2005 01-05-2009
disseminated epithelial malignancies
van ‘t Veer, Laura EEG-CEC / EU Translating molecular knowlegde into early breast cancer management: 01-03-2004 01-03-2011
building on the BIG (Breast International Group) network for improved
treatment tailoring. TRANSBIG
van ‘t Veer, Laura EEG-CEC / EU Collaborative Oncological Gene-environment Study 01-05-2009 01-05-2013
van ‘t Veer, Laura TI Pharma Predicting drug responses in breast cancer. TI Pharma T3-108 01-07-2007 01-07-2011
van de Vijver, Marc Astra Neoadjuvante registratiestudie BOOG 01-01-2006 01-01-2013
van de Vijver, Marc CTMM Neoadjuvant drug treatment for breast cancer - response prediction 01-10-2008 01-10-2013
and response monitoring (BREAST CARE); SP (sub-workpackage)
7: Identification and validation of predictive markers for drug response
in metastatic breast cancer
de Visser, Karin AstraZeneca Functional assessment of CSF-1 receptor signalling in de novo breast 01-11-2009 01-04-2010
cancer development and metastatis information
de Visser, Karin ZonMw The inflammatory tumor microenvironment and its impact on breast 01-01-2009 01-01-2014
cancer development and therapy
Wessels, Lodewyk AstraZeneca Pathway-based tumour characterisation & alignment of cell panels 01-02-2010 01-02-2012
against tumour samples
Wessels, Lodewyk CTMM Neoadjuvant drug treatment for breast cancer - response-prediction 01-10-2008 01-10-2013
and response monitoring (breast care); SP (sub-workpackage)
10: Biomarkers informatics
Wessels, Lodewyk MIN OCW NBIC Biorange 01-01-2004 01-01-2010
Wessels, Lodewyk NBIC NBIC / BioAssist / HTHC Programmeur 01-01-2008 01-01-2011
Wessels, Lodewyk NBIC NBIC-II BioRange BR4.9 01-01-2009 31-12-2012
Wolthuis, Rob NWO-ALW Coordination of cell division by regulated protein destruction 01-01-2007 31-12-2011

Personnel Index
Aalbers, Arend 155
Aaronson, Neil 93
Aarts, Marieke 65
Abdallah, Abdallah 37
Abrao Possik, Patricia 87
Achachah, Mohamed 105
Ackerstaff, Annemieke 155
Adriaansz, Sandra 120
Adriaensen, Gwijde 155
Agami, Reuven 49
Aguilar, Helena 79
Ait Moha, Daoud 105
Ajouaou, Abderrahim 105
Albers, Harald 35
Alderden, Carolien 120
Alderliesten, Maaike 30
Alderliesten, Tanja 133
Aleman, Berthe 99, 132
Alendar, Andrej 85
Amore, Alessia 35
Anirudhan, Gireesh 87
Argenzio, Elisabetta 24
Ariotti, Silvia 60
Atsma, Douwe 104
Aukema, Tjeerd 104
Axwijk, Priscilla 105
Baank, Saskia 104
Baars, Danny 169
Baars, Joke 119
Baars, Philippe 104
Baas, Paul 45, 119
Baas-Vrancken Peeters, Marie-Jeanne 155
Bakker, Martine 104
Bakker, Sandra 119
Bakker, Sietske 65
Balague Ponz, Olga 104
Balm, Alfons 155
Banishki, Nikola 67
Bartelink, Harry 132
Batteau, Lukas 106
Beaudry, Jean Bernard 83
Beekman, Chantal 106
Beelen, Karin 71, 119
Beers-Bauhuis, Carolien 104
Begg, Adrian 43
Beijersbergen, Roderick 79
Beijnen, Jos 47, 119
Bejaoui, Inès 90
Belderbos, José 95, 132
Bendle, Gavin 58
Benraadt, Tinie 169
Bentin Toaldo, Cristiane 34
Bergman, André 119
Berkers, Celia 35
Bernards, René 77
Berns, Anton 85
Berns, Katrien 77
Besnard, Peter 105
Bessels, Frauwkje 169
Betgen, Anja 133
Bex, Axel 156
Bies, Laura 58
Bijos, Dominika 51
Bin Ali, Rahmen 85
Blank, Christian 64, 119
Blanken, Roel 120
Bleiker, Eveline 97, 105
Blitz, Johanna 85
Bloemers, Monique 132
Blom, Marleen 83
Blom, Marie-José 99
Boekel, Naomi 99
Boekhout, Annelies 120
Boer, Mandy 89
Boerrigter-Barendsen, Lucie 104
Boessenkool-Pape, Juliët 99
Bonarius, Marja 119
Bonfrer, Hans 104
Boogerd, Willem 95, 119
Boon, Ute 69
Boot, Henk 119
Borkner, Lisa 64
Borst, Gerben 132
Borst, Jannie 56
Borst, Piet 67
Bos, Arnold 49
Bos, Melanie 119
Boshuizen, Rogier 119
Bosma, Astrid 39
Boss, David 119
Boucher, Audi 169
Boutmy-de Lange, Majella 105
Bouwman, Peter 69
Braaf, Linde 39
Brandsma, Dieta 119
Brandwijk, Kim 39
Braumuller, Tanya 69
Braunschweig, Ulrich 51
Brink-de Vries, Nienke 119
Broeks, Annegien 39
Brohet, Richard 99
Brouwers, Elke 119
Bruin, Natascha 104
Bruin, Sjoerd 39, 155
Bucholtz, Karina 120
Buckle, Tessa 106
Buikhuisen, Wieneke 119
Buil, Levi 104
Buitelaar, Dirk 156
Buning-Kager, Marian 104
Burgers, Sjaak 119
Burylo, Artur 47
Caillat, Christophe 20
Calbo, Joaquim 85
Canisius, Sander 75
Carreno, Monique 169
Cats, Annemieke 97, 119
Celie, Patrick 20
Chen, Chun 133
Chen, Wei 105, 133
Chin, Patrick 105
Christodoulou, Evangelos 20
Ciampricotti, Metamia 69
Citterio, Elisabetta 83
Claassen, Jolien 169
Clijsters, Linda 81
Cohen, Serge 20
Collard, John 26
Cooke, Saskia 169
Coquet, Jonathan 56
Cordia, Igor 156
Cornelissen, Paulien 83
Cornelissen, Sten 39
Courrech Staal, Ewout 155
Cox, Roy 95
Dalesio, Otilia 169
Damen, Carola 119
Damen, Eugène 132
Dannenberg, Jan-Hermen 90
De Boer, Jan Paul 119
De Boer, Johan 133
De Boer, Roel 132
De Bois, Josien 133
De Bruin, Marieke 99
De Groot, Dirk-Jan 22
De Haas, Marcel 67
De Jong, Annemieke 35
De Jong, Daphne 104
De Jong, Gerda 169
De Jong, Johann 75
De Jong, Monique 43, 132
De Jong, Rianne 133
De Leeuw, Renée 34
De Marco, Valeria 20
De Punder, Karin 37
De Ridder, Jeroen 75
De Ronde, Jorma 41, 75
De Ruiter, Michiel 95
De Visser, Karin 69
De Vreeze, Ronald 155
De Vries, Evert 56
De Vries, Hilda 85
De Vries, Nienke 83
De Vries, Sandra 65
De Waal, Marjolijn 169
Deenen, Maarten 47, 119
Dees-Ribbers, Hermine 133
Dekker, Marleen 65
Dekker, Rob 65
Dellemijn, Trees 60
Delzenne-Goette, Elly 65
Deurloo, Wilma 169
Devriese, Lot 47, 119
Dewit, Luc 132
Didraga, Mihaela 39
Diemer, Frederiek 99
Dirac, Annette 77
209
Personnel Index

210
Personnel Index
Doodeman, Barry 133
Doornebal, Chris 69
Dorlo, Thomas 120
Douma, Kirsten 93, 97
Douma, Sirith 87
Doumont, Gilles 69
Drost, Brigitte 156
Drost, Jarno 49
Drost, Rinske 69
Dubbelman, Anne Charlotte 120
Dubbelman, Ria 120
Dufournij, Brigitte 169
Duijts, Saskia 93
Duppen, Joop 133
Durmus, Selvi 73
Efthymiou, Katina 156
Eggermond, Anja 99
Eijzenga, Willem 93, 97
Ekhart, Corinne 120
Ekkebus, Reggy 35
El Oualid, Farid 35
Elkhuizen, Paula 132
Elkon, Rani 49
Ellenbroek, Saskia 26
Elouarrat, Dalila 24
Elshof, Lotte 105
Epping, Mirjam 77
Evers, Bastiaan 69
Evers, Danny 155
Fabius, Armida 79
Faesen, Alex 18
Fan, Lin 104
Feenstra, Christel 104
Fernandez Salcedo, Ernesto 169
Filion, Guillaume 51
Fish, Alex 18
Fles, Renske 39
Floot, Ben 45
Foekema, Joke 120
Fornerod, Maarten 55
Frederiks, Floor 53
Frederikx, Geert 47, 120
Frijns, Evelyne 22
Gadiot, Jules 64
Gallenne, Tristan 87
Galovic, Magda 28
Gargiulo, Gaetano 83
Garstka, Gosia 32
Gasparini, Alessia 133
Gast, Marie-Christine 120
Gebretensae, Abadi 120
Gehring, Karin 93
Geiger, Thomas 87
Genest, Paul-André 91
Gerlach, Carmen 58
Gerritsen, Bram 75, 79
Gerritsma, Miranda 93
Geumann, Ulf 56
Geurts, Tom 155
Geutjes, Ernst-Jan 77
Gilhuijs, Kenneth 105
Godsave, Sue 37
Goey, Andrew 120
Gomez, Raquel 60
Gomez Benito, Maria 49
Gonggrijp, Maaike 67
Graafland, Niels 156
Greig, Kylie 87
Groothuizen, Flora 18
Gundy, Chad 93, 95, 97
Haanen, John 60, 119
Haas, Rick 132
Hage, Joris 156
Hagenaars, Christiane 169
Hahn, Christoph 156
Hahn, Daniela 97, 105
Halonen, Pasi 79
Hamming-Vrieze, Olga 132
Hansen, Fleur 155
Haramis, Anna-Pavlina 92
Harms, Emmy 120
Hau, Song-Hieng 169
Hau, Tissee 69
Hauptmann, Michael 75
Haussman, Jens 20
He, Youji 39
Heemsbergen, Wilma 75, 132
Heemskerk, Bianca 60
Heidebrecht, Tatjana 20
Heideman, Marinus 62
Heijens, Claudia 120
Helgason, Helgi 120
Henneman, Linda 31
Heynen, Guus 77
Hibbert, Rick 18
Hiemstra, Annelies 169
Hijmans, Marielle 77
Hilarius, Doranne 93
Hilgers, Frans 155
Hilkens, John 89
Hilkmann, Henk 35
Hillebrand, Michel 120
Hoebers, Frank 132
Hoefnagel, Cornelis 104
Hoekstra, Paula 169
Hofland, Ingrid 43
Hogenbirk, Marc 62
Hogervorst, Frans 39, 104, 105
Hollmann, Birgit 132
Holstege, Henne 69
Holtkamp, Marjo 120
Hölzel, Michael 77
Hömig, Cornelia 87
Hompes, Daphne 155
Honnef, Joeri 133
Hooijkaas, Anna 64
Hoopman, Rianne 93
Hoppes, Rieuwert 35
Horenblas, Simon 156
Houben, Anna 24
Houben, Diane 37
Hoving, Saske 45, 105
Huang, Sidong 77
Huitema, Alwin 119
Hulshoff, Lenie 156
Hulsman, Danielle 83
Hummel, MJM 103
Iden, Sandra 26
Innocenti, Metello 28
Iskit, Sedef 87
Isogai, Tadamoto 28
Iusuf, Dilek 73
Ivanov, Eduard 99
Jacobi, Irene 155
Jacobs, Heinz 62
Jacobs, Jacqueline 91
Jalink, Kees 29
Jan, Sabrina 67
Jansen, Edwin 132
Jansen, Hans 37
Jansen, Robert 120
Janssen, Esther 99
Janssen, Lennert 32
Janssen, Luuk 155
Janssens, Tine 120
Jaspers, Janneke 69
Jeanson, Kiki 99
Jenal, Mathias 49
Jong, Eefje 120
Jongsma, Johan 85
Jongsma, Maikel 24
Jongsma, Marlieke 32
Jonkers, Irene 169
Jonkers, Jos 41, 69
Jonkman, Joop 169
Joosten, Krista 20
Joosten, Robbie 20
Jorritsma, Annelies 60
Kaiser, Andrew 58, 64
Kalisvaart, Robin 133
Kalverda, Bernike 55
Kant, Josien 169
Kaplon, Joanna 87
Kappers, Ingrid 155
Karakullukcu, Baris 155
Kasiem, Mobien 105
Kedde, Martijn 49
Keessen, Marianne 120
Keizer, Ron 120
Kenter, Gemma 156
Kersbergen, Ariena 67
Kerst, Martijn 119
Kerver, Emile 119
Ketema, Mirjam 22
Kimmings, Nikola 155
Kind, Jop 51
Klarenbeek, Jeffrey 29

Klarenbeek, Sjoerd 69
Klijn, Christiaan 69, 75
Klinkenberg, Astrid 95
Klokman, Willem 99
Klomp, Houke 155
Klop, Martin 155
Klous, Marjolein 119
Kluijt, Irma 97, 105
Klümpen, Heinz Josef 120
Knauer, Michael 71, 104
Knegjens, Joost 132
Knols, Ruud 93
Koenen, Annemieke 120
Kok, Marleen 39, 71
Kolmschate, Lies 169
Kolodziej, Katy 51
Kool, Jaap 83, 85
Koolen, Stijn 120
Koopman-Kroon, Ciska 120
Koops, Wim 105
Koornstra, Rutger 71
Koppelmans, Vincent 95
Koppens, Martijn 83
Korse, Tiny 104
Koudijs, Marco 69
Kranenburg, Andor 85
Krap, Menno 155
Kreeft, Annemarijn 155
Kreft, Maaike 22
Kregel, Eva 69
Kreike, Bas 132
Krewinkel, Han 132
Krijger, Peter 62
Krimpenfort, Paul 85
Kristel, Petra 41
Kröger, Robert 105
Król, Magdalena 31
Kruis, Matthijs 133
Krumpochova, Petra 67
Kuenen, Marianne 93, 95, 99
Kuijl, Coen 32
Kuiken, Johan 79
Kuikman, Ingrid 22
Kuil, Joeri 105
Kuilman, Thomas 87
Kuiper, Maria 120
Kujala, Pekka 37
Kumar, Prasanth 77
Kunst, Peter 119
Kwint, Margriet 133
Kwon, Min-chul 85
Lagas, Jurjen 73, 120
Lambooij, Jan Paul 85
Lammens, Chantal 93, 97
Lancini, Cesare 83
Lange, Charlotte 105
Lange, Jan 156
Langeslag, Michiel 29
Lankheet, Nienke 120
Le, Chau 104
Le Sage, Carlos 49
Lebesque, Joos 132
Leijen, Suzanne 47
Leijte, Joost 156
Lenain, Christelle 87
Léveillé, Nicolas 49
Lieftink, Cor 79
Linders, Dorothé 104
Linn, Sabine 39, 71, 95, 119
Linnemann, Carsten 58
Lips, Esther 39, 41
Littler, Dene 20
Liu, Zhen 28
Lodder, Wouter 155
Lohuis, Peter 156
Loo, Claudette 105
Lopez, Marta 75
Lorist, Lisette 169
Lubsen–Brandsma, Lottie 156
Lucas, Luc 120
Lukas, Anne 119
Lutkenhaus, Lotte 106
Maas, Chiel 56
Maas, Marnix 133
Mager, Alet 119
Mahn, Marianne 169
Majewski, Ian 77
Mallo, Henk 120
Mandjes, Ingrid 169
Mans, Anton 133
Marchetti, Serena 47, 119
Margadant, Coert 22
Mattiroli, Francesca 18
McDermott, Leah 133
Meijerman, Irma 47
Meijnen, Philip 132
Meinhardt, Wim 156
Meissl, Katrin 87
Melis, Jacoline 93
Melo, Carlos 49
Mencarelli, Angelo 133
Menendez, Victoria 32
Menning, Sanne 95
Mertens, Sander 26
Meuleman, Wouter 51, 75
Meulenaar, Jelte 120
Mexner, Vanessa 133
Michalak, Ewa 69
Michalides, Rob 34
Mijnheer, Ben 132
Mikolajewska, Izabela 39
Minderhoud, Tom 133
Minkema, Danny 133
Mitsiki, Eirini 20
Mittempergher, Lorenza 39
Modder, Carla 169
Moes, Johannes 120
Molloy, Tim 39
Mooij, Thea 99
Mook, Stella 39, 105, 132
Moolenaar, Wouter 24
Moonen, Luc 132
Morris, Ben 79
Mulder, Lennart 39, 41
Mullenders, Jasper 77
Muller, Pietje 169
Muller, Saar 104, 105
Muusers, Rick 104
Nacerddine, Karim 83
Nagel, Remco 49
Nagtegaal, Tanja 93, 97
Naik, Shalin 58
Nan-Offeringa, Lianda 120
Navran, Arash 132
Nederlof, Petra 39, 41, 99, 104, 105
Neefjes, Jacques 32
Neijenhuis, Sari 43
Nieuweboer-Krobotova, Inka 156
Nieuwenhuis, Lotte 155
Nieweg, Omgo 155
Nijkamp, Jasper 133
Nijkamp, Wouter 79
Nijwening, Jeroen 79
Nol, Annemarie 120
Nooijen, Willem 104
Nowee, Marlies 132
Numan, Rachel 155
Nunnink, Chantal 156
Nuyen, Bastiaan 119
Nyst, Heike 156
Ogink, Janneke 81
Oldenburg, Hester 93, 155
Olijve, Albert 120
Olsthoorn-Ooms, Marije 105
Olszewska, Agnieszka 133
Onderwater, Suzanne 120
Ong, Nico 37
Oostendorp, Roos 47, 120
Oosterhuis, Koen 60
Oosterkamp, Rianne 77
Opperdijk, Vera 132
Oude Vrielink, Joachim 49
Ouwens, Gabey 99
Ovaa, Huib 35
Paape, Anita 106
Pagie, Ludo 51
Pameijer, Frank 105
Pang, Baoxu 32
Panneman, Carmen 133
Pasca, Edoardo 105
Paul, Petra 32
Paulus van Pauwvliet, Cecile 169
Pawlitzky, Inka 83
Pecharroman Gallego, Raul 133
Peeper, Daniel 87
Pengel, Kenneth 105, 133
Peperzak, Victor 56
211
Personnel Index

212
Personnel Index
Peric-Hupkes, Daniel 51
Perrakis, Anastassis 20
Peters, Carolien 133
Peters, Peter 37
Peuscher, Marieke 91
Piek-den Hartog, Marianne 155
Pierson, Jason 37
Pieterse, Mark 69
Pijpe, Anouk 99
Pindyurin, Alexey 51
Ploeger, Lennert 133
Plug, Rob 105
Pluim, Dick 47
Polle, Bas 155
Poller, Birk 73
Ponsioen, Bas 24, 29
Pool, Bert 104
Poppema, Boelo 119
Ports, Michael 22
Pos, Floris 132
Postel, Ruben 22
Postema, Jan Willem 104
Pramana, Jimmy 156
Prasetyanti, Pramudita 69
Prevoo, Warner 105
Pritchard, Colin 85
Pronk, Loes 169
Proost, Natalie 85
Pruntel, Roelof 105
Qi, Wen 105
Quaak, Susanne 60, 120
Rasch, Coen 132
Rehorst, Harriet 169
Reichgelt, Babs 132
Reinders, Anneke 169
Relyveld, Germaine 156
Remeijer, Peter 132
Remmelzwaal, Jolanda 169
Retèl, Valesca 103
Reumer, Annet 18
Rifes, Pedro 22
Rit, Simon 133
Roberts, Daniel 169
Rodenhuis, Sjoerd 41, 119
Rodenko, Boris 35
Roelvink, Marja 120
Röling, Michael 55
Ronday, May 156
Roodbergen, Rita 95
Rookus, Matti 99
Roos, Ed 31
Roosjen, Edwin 133
Rooswinkel, Rogier 56
Rooze, Lyandra 104
Rosado, Arantxa 51, 87
Rosenberg, Efraim 104, 105
Rosing, Hilde 119
Roskam, Marielle 169
Rossi, Maddalena 106, 133
Rottenberg, Sven 67
Rucktooa, Prakash 18
Ruers, Theo 155
Ruijs, Marielle 105
Ruijter-Schippers, Henrique 105
Russell, Nicola 45, 99, 132
Rutgers, Emiel 93, 99, 155
Sachs, Norman 22
Sadaka, Charlotte 32
Sahtoe, Danny 18
Salomon, Izhar 32
Salverda, Govert 132
Sanders, Marloes 103
Sani, Musa 37
Sapthu, Sunny 67
Scanu, Tiziana 32
Schaap, Jeffrey 155
Schaapveld, Michael 99
Schaefer, Henning 49
Schaefer, Ronny 26
Schagen, Sanne 95
Scharpfenecker, Marion 45
Scheenstra, Alize 133
Scheenstra, Renske 156
Scheerman, Esther 105
Schellens, Jan 47, 119
Schilder, Christien 95
Schinkel, Alfred 73
Schippers-Gillissen, Carla 105
Schmidt, Marjanka 39, 99, 105
Schmitz, Alexander 106
Schmitz, Annemarie 105
Schneider, Christoph 132
Schol, Joke 120
Schot, Margaret 120
Schotte, Remko 58
Schrier, Mariëtte 49
Schumacher, Ton 58
Schutte, Peter 156
Schuurman, Karianne 35
Secades, Pablo 22
Seemann, Ingar 45, 105
Seigers, Riejanne 95
Seto, Azusa 18
Shanmugham, Anitha 35
Shu, Jenny 58
Siedschlag, Christian 105
Sinaasappel, Michiel 104, 105
Sivro-Prndelj, Ferida 104
Sixma, Titia 18
Smeele, Ludi 156
Smit, Judith 18
Smit, Marjon 87
Smits, Marianne 119
Smitsmans, Monique 133
Snoek, Margriet 85
Sol, Wendy 67
Sombroek, Cherita 99
Sonke, Gabe 93, 119
Sonke, Jan-Jakob 132
Sonneborn, Mariska 104
Sonnenberg, Arnoud 22
Sparmann, Anke 83
Speijer, Gabrielle 132
Srámek, Michael 156
Staiger, Christine 75
Steeghs, Neeltje 119
Steenbakkers, Roel 132
Steuten, L 103
Stewart, Fiona 45
Storm, Dea 169
Stornaiuolo, Mariano 18
Straver, Marieke 155
Stroom, Joep 132
Stulemeijer, Iris 53
Stuurman, Rick 120
Sutherland, Kate 85
Swart, Erwin 58
Sznajder, Beata 169
Taal, Babs 119
Talhout, Wendy 51
Tan, Bing 156
Tanger, Ellen 83
Tanis, Pieter 155
Te Poele, Johannes 45
Te Riele, Hein 65
Teertstra, Jelle 105
Ten Cate, Julia 156
Ten Hoeve, Jelle 75
Ter Brugge, Petra 41, 69
Ter Heine, Rob 120
Ter Riet, Bas 81
Terweij, Marit 53
Tesselaar, Margot 119
Teunissen, Bas 120
Thijssen, Bas 120
Tibben, Matthijs 120
Tielen, Ivon 105
Tielenburg, René 133
Tilgenkamp, Ria 169
Timmer-Arents, Linda 103
Timmers, Adriaan 156
Tjin, Esther 60
Toebes, Mireille 58
Tolhuis, Bas 83
Tomasoa, Brenda 132
Tomova, Cveta 37
Topolnjak, Rajko 133
Torres Acosta, Alex 169
Tran, Ly 104, 120
Tripathi, Pankaj 67
Tromp-van Driel, Catrien 119
Tulner, Linda 120
Uno, Naoki 87
Urbanus, Jos 58
Uren, Anthony 85
Uyterlinde, Wilma 120
Valdés Olmos, Renato 104

Valkenet, Ludy 169
Van As-Brooks, Corina 156
Van Bemmel, Joke 51
Van Berkel, Peter 103
Van Beurden, Marc 93, 155, 156
Van Blitterswijk, Wim 30
Van Boven, Hester 104
Van Coevorden, Frits 155
Van Dam, Frits 95
Van de Ahé, Fina 85
Van de Kasteele, Willeke 60
Van de Kooij, Bert 56
Van de Noll, Ruud 120
Van de Poel, Henk 156
Van de Steeg, Evita 73
Van de Velde, Tony 169
Van de Ven, Marieke 69
Van de Vijver, Marc 104
Van de Vrugt, Henri 65
Van de Wetering, Koen 67
Van de Wiel, Bart 104
Van Deemter, Liesbeth 67
Van den Belt-Dusebout, Sandra 99
Van den Berg, Joost 60, 120
Van den Berk, Paul 62
Van den Boer, Cindy 155
Van den Brekel, Michiel 155
Van den Brink-de Vries, Nienke 104
Van den Broek, Sandra 99
Van den Haak, Marjolijn 169
Van den Heuvel, Michel 119
Van den Hoorn, Tineke 32
Van den Hoven, Jolanda 120
Van der Berg, Marieke 156
Van der Berg, Nienke 106
Van der Burg, Eline 69
Van der Donk, Emile 169
Van der Gulden, Hanneke 69
Van der Hage, Jos 155
Van der Heijden, Ingrid 69, 71
Van der Heijden, Michiel 77
Van der Horst, Gerda 56
Van der Kammen, Rob 28
Van der Kant, Rik 32
Van der Kolk, Lizet 105
Van der Maas, Martin 60
Van der Molen, Lisette 156
Van der Ploeg, Iris 155
Van der Sar, Jana 120
Van der Steeg, Evita 120
Van der Torre, Jaco 91
Van der Veen, Wietze 156
Van der Velden, Sophie 105
Van der Velden, Yme 92
Van der Wal, Anja 65
Van der Weide, Marchien 119
Van der Wel, Nicole 37
Van Deventer, Sjoerd 32
Van Dijk, Pim 18
Van Dongen, Miranda 77, 105
Van Driel, Willemien 156
Van Dyk, Ewald 75
Van Esch, Anita 73
Van Gijn, Roel 119
Van Haaften, Gijs 49
Van Harten, Wim 103
Van Hasselt, Coen 120
Van Heijst, Jeroen 58
Van Hell, Albert 30
Van Herk, Marcel 132
Van Hien, Richard 39
Van Huizum, Martine 156
Van Kasteren, Sander 35
Van Kouwenhove, Marieke 49
Van Kranen, Simon 133
Van Kuijen, Anne-Marije 155
Van Leeuwen, Fijs 45, 104, 105
Van Leeuwen, Flora 39, 99
Van Leeuwen, Fred 53
Van Leeuwen, Marieke 93
Van Lent, Wineke 103
Van Lohuizen, Maarten 83
Van Luenen, Henri 67
Van Monsjou, Hester 156
Van Montfort, Erwin 85
Van Mourik, Anke 133
Van Netten, Gabry 169
Van Noord, Janet 91
Van Pel, Renée 104
Van Rens, Anja 105
Van Riel, Helma 26
Van Rooij, Nienke 58
Van Rossum-Schornagel, Quirine 119
Van Sandick, Johanna 155
Van Steensel, Bas 51
Van Tellingen, Olaf 104
Van Tinteren, Harm 169
Van Triest, Baukelien 132
Van Turnhout, Arnoud 156
Van ’t Veer, Laura 39, 99, 104, 105
Van Velthuysen, Loes 104
Van Vliet, Mariska 120
Van Vliet-Vroegindeweij, Corine 132
Van Vugt, Huub 83
Van Waardenberg, Wil 169
Van Waart, Hanna 93
Van Waterschoot, Robert 73, 120
Van Welsem, Tibor 53
Van Werkhoven, Erik 169
Van Winden, Annemieke 120
Van Zeijl, Leonie 24
Van Zon, Maaike 37
Van Zon, Wouter 81
Van Zwienen, Marieke 132
Vanneste, Ben 132
Vargas, Mark 18
Vendel, Brian 132
Vens, Conchita 43
Veraar, Elise 56
Verbrugge, Inge 56
Vergouwe, Ingeborg 156
Verhagen, Caroline 43, 156
Verheij, Marcel 30, 132
Verheus, Martijn 99
Verhoef, Senno 39, 93, 97, 99, 105
Verhoeven, Els 83
Verloop, Janneke 99
Vermeeren, Lenka 104
Vermeulen, Eric 93, 99
Verschueren, Karijn 132
Verwaal, Vic 155
Verwijs, Manon 43
Verwoerd, Desiree 34
Verzijlbergen, Kitty 53
Vincent, Andrew 169
Visser, Daan 29
Visser, Dick 120
Visser, Nils 45
Vissers, Joep 83
Vlaming, Marijn 73
Voets, Erik 81
Vogel, Celia 87
Vogel, Wouter 104
Vollebergh, Marieke 71
Von Castelmur, Eleonora 20
Voncken, Francine 132
Vormer, Tinke 65
Vos, Matthijn 37
Vredeveld, Liesbeth 87
Vreeswijk, Sandra 133
Vrijaldenhoven, Suzan 119
Vroonland, Colinda 104
Vyth-Dreese, Florry 60
Wagenaar, Els 73
Wals, Anneke 169
Wang, Bruce 92
Wassenaar, Moniek 132
Wendling, Markus 133
Wesseling, Jelle 41, 104
Wessels, Lodewijk 41, 75
Westerman, Bart 83
Wever, Lidwina 169
Wevers, Marijke 93, 97
Wevers, Marion 95
Wielders, Camiel 65
Wielders, Eva 65
Wientjens, Ellen 69
Wieringa-Ariaens, Aafke 105
Wigbout, Gea 105
Wijdeven, Ruud 32
Wijnands, Yvonne 31
Wildeman, Melchert 156
Willemse, Els 169
Wilting, Roel 90
Winterwerp, Herrie 18
Wit, Niek 62
Witte, Marnix 133
213
Personnel Index

214
Personnel Index
Witteveen, Thelma 132
Wittkämper, Frits 132
Woerdeman, Leonie 97, 156
Wolthaus, Jochem 133
Wolthuis, Rob 81
Wouters, Michel 155
Xiao, Yanling 56
Xu, Guotai 67
Xylourgidis, Nikos 55
Yang, Jonathan 133
Yanover, Eva 90
Zandbergen, Jeroen 169
Zander, Serge 67
Zerp, Shuraila 30
Zevenhoven, John 85
Zhang, Hua 133
Ziblat, Lonny 169
Zijp, Lambert 133
Zlotorynski, Eitan 49
Zupan-Kajcovski, Biljana 156
Zuur, Charlotte 156
Zuurmond, Kirsten 106
Zwaagstra, Annabel 89
Zwart, Wilbert 34

THE
NETHERLANDS
CANCER
INSTITUTE
SCIENTIFIC
ANNUAL
REPORT 2009
The Netherlands Cancer Institute
Antoni van Leeuwenhoek Hospital
Plesmanlaan 121
1066 CX Amsterdam
www.nki.nl