2008 - NKI / AvL

THE
NETHERLANDS
CANCER
INSTITUTE
SCIENTIFIC
ANNUAL
REPORT 2008
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SCIENTIFIC ANNUAL REPORT 2008
3

4
Patron HM The Queen Beatrix

SCIENTIFIC ANNUAL REPORT 2008
THE NETHERLANDS CANCER INSTITUTE
CANCER RESEARCH LABORATORY AND CANCER HOSPITAL
www.nki.nl
5

6 COPYRIGHT
Scientific Annual Report 2008
Illustrations and unpublished data in these reports may not be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
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 31
Division of Experimental Therapy 38
Division of Gene Regulation 46
Division of Division of Immunology 53
Division of Molecular Biology 62
Division of Molecular Carcinogenesis 75
Division of Molecular Genetics 79
Division of Psychosocial Research and Epidemiology 89
Division of Diagnostic Oncology 100
Division of Medical Oncology 116
Division of Radiotherapy 126
Division of Surgical Oncology 143
Biometrics Department 156
Clinical Trials 161
Invited Speakers 177
Projects 180
Personnel Index 193
7

President of Board of Governors W Kok
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
9
BOARD MEMBERS
BOARD OF DIRECTORS
AJM Berns, chairman and director of research
S Rodenhuis, director clinical research and development
WH Van Harten, director organization and management
BOARD OF GOVERNORS
W Kok, president
HCJ Van der Wielen, vice-president
T De Swaan, treasurer
FH Schröder
D Sinninghe Damsté
MC Smeets
PC Van der Vliet
GP Vooijs
SCIENTIFIC ADVISORY COUNCIL
AJM Berns, chairman
JJ Neefjes, secretary
S Rodenhuis
T Sixma
M Verheij
J Borst

10 RESEARCH DIVISIONS
RESEARCH DIVISIONS
Biochemistry
Titia Sixma (head)
Anastassis Perrakis
Franciska Manuputty (office manager)
Cell Biology I
Arnoud Sonnenberg (head)
Wim van Blitterswijk
John Collard
Kees Jalink
Wouter Moolenaar
Ed Roos
Cell Biology II
Jacques Neefjes (head)
Rob Michalides
Huib Ovaa
Peter Peters
Marieke van der Velde (office manager)
Experimental Therapy
Laura van ’t Veer
Adrian Begg
Jan Schellens
Fiona Stewart
Thea Eggenhuizen (office manager)
Gene Regulation
Reuven Agami
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
Rob Wolthuis
Tom de Knegt (office manager)
Linda Römer (secretary)
Molecular Carcinogenesis
René Bernards (head)
Roderick Beijersbergen
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
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
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
Senno Verhoef
Wouter Vogel
Jelle Wesseling
Christine Arkes (secretary)
Carla van Tiggelen (secretary)

Medical Oncology
Sjoerd Rodenhuis (head)
Joke Baars
Paul Baas
Jos Beijnen
André Bergman
Christian Blank
Willem Boogerd
Henk Boot
Dieta Brandsma
Sjaak Burgers
Annemieke Cats
Jan Paul de Boer
Leo Gualtherie van Weezel
John Haanen
Alwin Huitema
Martijn Kerst
Sabine Linn
Anne Lukas
Jan Schellens
Jan Schornagel
Marianne Smits
Gabe Sonke
Babs Taal
Wim ten Bokkel Huinink
Margot Tesselaar
Michiel van den Heuvel
Marchien van der Weide
Nico van Zandwijk
Mariëlle de Kwant (secretary)
Radiotherapy
Marcel Verheij (head)
Berthe Aleman
Harry Bartelink
José Belderbos
Eugene Damen
Roel de Boer
Luc Dewit
Paula Elkhuizen
Rick Haas
Olga Hamming-Vrieze
Guus Hart
Wilma Heemsbergen
Frank Hoebers
Edwin Jansen
Han Krewinkel
Joos Lebesque
Corrie Marijnen
Harry Masselink
Ben Mijnheer
Luc Moonen
Floris Pos
Coen Rasch
Babs Reichgelt
Peter Remeijer
Nicola Russell
Govert Salverda
Christoph Schneider
Joep Stroom
Bart van Bunningen
Marcel van Herk
Baukelien van Triest
Corine van Vliet
Marieke van Zwienen
Frits Wittkämper
Patricia Haye-Fewer (management assistant)
Surgical Oncology
Theo Ruers (head)
Marc van Beurden
Michiel van den Brekel
Alfons Balm
Annemieke Ackerstaff
Axel Bex
Biljana Zupan-Kajcovski
Christiaan Keijzer
Dirk Buitelaar
Emiel Rutgers
Frans Hilgers
Frits van Coevorden
Henk van de Poel
Hester Oldenburg
Houke Klomp
Bing Tan
Ingeborg Vergouwe
Inka Nieuweboer-Krobotova
Joris Hage
Johanna van Sandick
Julia ten Cate
Katina Efthymiou
Leonie Woerdeman
Lotti Lubsen–Brandsma
Ludi Smeele
Marc van Beurden
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
Sanne Vos
Simon Horenblas
Tino Stoppa
Vic Verwaal
Wietze van der Veen
Willemien van Driel
Wim Meinhardt
Annemieke Hoogland (office manager)
Biometrics Department
Otilia Dalesio
Financial Administration
Didier Dohmen
General Facilities, ICT and Personnel Department
Eric De Wilde
General Research Coordination
Jacques Neefjes, deputy scientific director
Henri van Luenen, director of operations
11
RESEARCH DIVISIONS

12
INTRODUCTION
Director of Research Ton Berns
INTRODUCTION
I am pleased to present our Scientific Annual Report
2008. 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
overview of our activities with illustrations can be found
in our brochure (available on our website)
The NKI-AVL is a Comprehensive Cancer Center,
combining hospital and research laboratories under one
roof in a single independent organization. The hospital
comprises 180 beds, an outpatient clinic and a large
radiotherapy department. Facilities for clinical research
include a large patient database, clinical data
management, extensive diagnostic facilities, a pharmacy
with a production unit for experimental drugs, and
active research groups in pharmacy, epidemiology and
psychosocial oncology. The laboratory covers all major
areas of cancer research, with special emphasis on cellbased
screens, mouse tumor models, cell biology,
structural biology, and immunology. Translational
research is an integral activity of many research groups
and is fostered by collaboration between clinical and
basic scientists. This scientific report deals mainly with
the basic and clinical science in the NKI-AVL.
Information on patient-care is described in our General
Annual Report.
In 2008 most of our research groups relocated to the
renovated research labs that were constructed in the
former hospital building. We have taken this opportunity
to restructure the divisions in order to enhance
interactions between the research groups. As a result,
two new divisions were formed while two existing
divisions were combined. The transition to the new labs
went very smoothly thanks to the excellent preparation
and organization of the whole process.
Our clinical activities again exhibited growth, although
not as much as expected due to the space limitations in
the outpatient clinics and the operating rooms. In 2009,
we will add an extra operating room specially equipped
for minimally invasive surgery. We also foresee
additional, temporary facilities to relieve the pressure on
our outpatient facility. Filling current vacancies for
nursing staff, however, is at present our most serious
concern. Expansion of our radiotherapy department is
planned to take place through the establishment of two
off-site satellites at clinical centers elsewhere. In
addition, we are planning, together with the Erasmus
University, the University Medical Center Leiden and the
Delft Technical University, a proton/heavy ion radiation
center for treating specific groups of patients that will
benefit from the more targeted delivery of radiation.
The transition to a new reimbursement system in the
health care system is progressing, and this enables us to
further expand our clinical activities. However, we foresee
harsher financial challenges due to the current economic
crisis. This is a more general problem we are facing,
although we do not expect major setbacks in the coming
year. We concluded 2008 with a small profit for the
hospital. Also our research expenditure remained within
budget in 2008. We expect to balance the budget for
both the hospital and the research in 2009 as well,
despite steep increases in some expenses beyond our
control.
In 2008 our PhD students organized, together with
Cancer Research UK, an international scientific meeting
for PhD students only. The meeting was held at the NKI
and was much appreciated by the participants.
The International Scientific Advisory Board spent two
full days at the Institute to critically evaluate our research
portfolio. This was also meant as a rehearsal for the site
visit evaluation that will take place in the Fall of 2009
in which an international site visit team appointed by
the Royal Netherlands Academy of Arts and Sciences
will evaluate the Cancer Institute on behalf of the
Dutch Cancer Society and the Ministry of Health,
the organizations that provide the core funding for
The Netherlands Cancer Institute. Our International
Scientific Advisory Board was very positive about the
quality of our research program, but it also gave valuable
advice with regard to policies for recruiting new staff
members, coaching young group leaders, and fostering
translational research. Many of their suggestions have
already been implemented in the course of 2008.
Table 1
Core research funding NKI-AVL from the Cancer Society and the Ministry of Health in period 1993-2008 in million Euro’s
Year 1993 1996 1999 2001 2002 2003 2004 2005 2006 2007 2008
Cancer Society 4.1 5.6 7.0 8.7 8.7 9.1 8.9 8.6 8.3 8.8 9.3
Ministry Health 7.3 8.1 8.8 9.4 9.9 10.1 9.1 9.3 9.4 9.5 9.9
Total* 11.4 13.7 15.8 18.1 18.6 19.2 18.0 17.9 17.7 18.3 19.4
In addition to the core funding the NKI acquired support through external grants, donations, and research agreements.
The contribution of this latter income to the total budget has been steadily increasing over the years and represents roughly
60% of the total budget in 2008.

The staff retreat that will be held in January 2009 will
focus on how to further enhance the synergy between
the preclinical and clinical research within the NKI-AVL.
In 2008, we continued to expand collaborative efforts
and signed an agreement with the Technical University
Twente to work together in developing minimally
invasive surgical techniques. We also concluded an
agreement with Astra Zeneca to serve as one of their
preferred academic partners both with regard to
preclinical and clinical research. We signed an
agreement with the Karolinska Institutet in Stockholm
and the Institut Gustave Roussy in Paris to collaborate
more closely on innovative clinical trials and exchanging
expertise in specific diagnostic areas. This collaborative
effort between three European cancer centers will give
access to powerful technological platforms of genomics/
proteomics assays. These are important to refine
diagnosis and measure responses to therapy, thereby
assisting in defining more effective combination
therapies. These initiatives should help us to advance
personalized medicine. We are actively involved in
establishing a broader alliance of European
Comprehensive Cancer Centers of Excellence, which can
also serve as a more influential companion of the
pharmaceutical industry.
Highlights
Our institute continues to have a strong scientific
output; 2008 was again a productive year. It is always
difficult to estimate the impact of research when the
results are still fresh. The research highlights
summarized here serve primarily as a sampling of work
currently on-going in the 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).
Nuno Rocha, Rik van der Kant and Coenraad Kuijl from
the Neefjes lab (Division of Cell Biology) performed an
in-depth cell biological study explaining the contribution
of cholesterol to lysosomal behavior as detected in
various genetic diseases like Gaucher and Niemann Pick
disease. They showed that a unique cholesterol sensor
on lysosomes attracts a protein from the ER to remove a
motor protein on late endosomes. This is the first study
showing how different intracellular compartments
communicate to control each other’s behavior.
Eva Wielders in the group of Hein te Riele (Division of
Molecular Biology) has used a novel technique,
oligonucleotide-directed gene modification, to recreate
allelic variants of the central mismatch repair gene
MSH2 in mouse embryonic stem cells. By studying the
phenotype of mutant ES cells she could unambiguously
classify two MSH2 variants of uncertain significance as
innocent polymorphisms and a third one as a deleterious
mutation causative of hereditary non-polyposis colorectal
cancer. This work will help medical geneticists in the
counseling of (suspected) HNPCC families.
Breast cancers that are deficient in homologous
recombination are exquisitely sensitive to bifunctional
alkylating agents, such as were given in the high-dose
chemotherapy programs in the late 20 th century.
13
INTRODUCTION
Marieke Vollebergh in the group of Sabine Linn
(Divisons of Medical Oncology and Molecular Biology),
in collaboration with Petra Nederlof and Jelle Wesseling
(Division of Diagnostic Oncology), has shown that
sporadic tumors more likely respond to such regimens
and can be recognized by either establishing a BRCA1
signature in an array-CGH test or by finding an
expanding growth pattern at microscopic examination of
a resection specimen.
The group of Bas van Steensel (Division of Gene
Regulation) mapped some 1,300 large domains in the
genome that associate with Lamin (LADs). These LADs
represent a repressive chromatin environment,
demonstrating that the human genome is divided into
large, discrete domains that are units of chromosome
organization within the nucleus.
The group of Fred van Leeuwen (Division of Gene
Regulation) developed a novel assay in yeast to
determine protein turnover in vivo and applied it to
histones. They found that histones throughout the
genome are subject to extensive replication-independent
exchange, demonstrating that histones and their posttranslational
marks are not permanent residents in
chromatin.
Daniel Peeper’s group (Division of Molecular Genetics)
has previously found that cells can respond to the
activation of oncogenes by entering a long-term
proliferative arrest, known as Oncogene-Induced
Senescence (OIS). As such, OIS serves as a potent
mechanism protecting us against cancer. Recently, they
uncovered an unanticipated role for secreted proteins,
including interleukins, in this process. Interleukins
are known for their important role in the immune
system, but Peeper’s group found that they are also
indispensable for OIS. These secreted factors,
collectively called the Senescence-Messaging Secretome
(SMS), allow for communication (‘SMS-ing’) between
cells under stress (like senescent cells that carry an
activated oncogene) and their environment.
Alexandra Pietersen in the lab of Van Lohuizen
(Division of Molecular Genetics) described an important
role for the Polycomb gene Bmi1 in the regulation of
differentiation and proliferation of mammary epithelial
stem cells and progenitors
Anthony Uren and Jaap Kool (Berns and Van Lohuizen
labs, Division of Molecular Genetics) in collaboration
with the groups of Lodewyk Wessels (Division of
Molecular Biology) and David Adams (Sanger Centre,
UK), highlighted the power of high throughput
insertional mutagenesis screens as a cancer gene
network discovery platform in cancer-predisposed
p19ARF and p53 deficient mice.
The group of Laura van ‘t Veer (Division of Experimental
Therapy) found that primary colorectal tumors that
develop liver metastasis have a unique chromosomal
signature that might be suitable to identify patients at
risk to develop liver metastasis. The observed genomic
aberrations also provide an inroad to understand
the underlying mechanism and may lead to more
individualized disease management.

14
INTRODUCTION
The group of Jan Schellens (Division of Medical
Oncology) has documented a promising anticancer
activity of the combination of the PARP inhibitor
AZD2281 alone and in combination with carboplatin in
early clinical studies. PARP inhibitors selectively target
tumors that are defective in DNA repair by homologous
recombination, such as breast cancer in BRCA1 or
BRCA2 carriers.
Adrian Begg and coworkers (Division of Experimental
Therapy) tested the predictive potential of signatures
known or suspected to affect response to radiotherapy.
The putative stem cell marker CD44 was the most
significant predictor of outcome (local control), with an
acute hypoxia signature showing a strong trend. CD44
also emerged as the most significant gene in a datadriven
approach searching for any set of genes
distinguishing cures from recurrences.
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 since the beginning of the 1980’s
(Table 2). It is satisfying to note that the Institute is
retaining an internationally prominent position in
cancer research, despite its limited budget that is
relatively modest in comparison with many comparable
institutes abroad.
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. Sjoerd Rodenhuis and colleagues
successfully competed for a large grant together with
industry from the Center for Translational Molecular
Medicine (CTMM) aimed at identifying new diagnostic
markers for therapy response. Several groups in the
Institute also participate with university groups in a
CTMM program on lung cancer. Bas van Steensel
received a VICI grant from NWO, and VIDI awards were
given to Karin de Visser and Elisabetta Citterio. I myself
Table 2
Short-term citations and impact of scientific articles published by the NKI research staff 1981 - 2008
Publication year Publications Citations* Citations/publication Impact**
1982 155 706 3.2 295
1983 192 986 4.6 365
1984 277 1797 5.1 616
1985 270 1575 6.5 549
1986 272 1628 5.8 650
1987 296 2036 6.0 765
1988 375 2040 5.5 742
1989 283 1822 6.4 764
1990 319 2704 8.5 854
1991 268 2933 10.9 910
1992 269 2721 10.1 911
1993 245 3033 12.3 958
1994 307 4380 14.2 1292
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 270 3551 13.2 1659
2001**** 298 3991 13.3 1582
2002 349 7436 21.3 2455
2003 333 5240 15.3 2122
2004 327 5267 16.1 1882
2005 368 6350 17.3 2461
2006 436 6330 14.5 2608
2007 412 2629
2008# 422 2396
* Citations in the two years after publication, excluding self-citations. Starting 1989 citation analysis has been carried out
online. This allows detection (and elimination) of all self-citations. Before 1989 this pruning was limited to first authors.
** The impact factor is the average number of citations per year of an article in a given journal. The total impact is the sum
of the impact of all articles published that year.
*** As from 1997 publication year of articles is the criterion, instead of Scientific Report-year listing.
**** Self-citations not excluded for publications 2001 and later.
# From 2008 the online publication date is used as criteria for the year of publication.

eceived a program award from the Dutch Cancer
Society. The quality of research of the groups in each
division is evaluated approximately every 5 years by an
external site visit team. Because of the reshuffling in
2008 of research groups as a result of the relocation to
the new research building and the fact that our
International Scientific Advisory Board conducted an
overall evaluation of the research in 2008, no site visit of
individual research groups was held in 2008.
Honors 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 2008, 27 staff
members had professorships at one of the Dutch
Universities. John Haanen was appointed as Professor of
Translational Immunotherapy of Cancer at the Leiden
University, and Daniel Peeper as professor of Functional
Oncogenomics at the Free University in Amsterdam.
Harry Bartelink received a doctorate honoris causa from
the Academia of Medicine in Gdansk, Poland. Titia
Sixma became member of the Royal Netherlands
(Dutch) Academy of Arts and Sciences. Bas van Steensel
and Daniel Peeper were elected as EMBO members. The
AvL-prize 2008 for young, promising NKI-scientists was
awarded to Wilbert Zwart for his work on tamoxifen
resistance mechanisms.
There were substantial changes in our clinical staff
mostly due by retirements. Harry Masselink, Joos
Lebesque and Bart van Bunningen retired as radiation
oncologists after serving the Institute for periods
ranging from 27 to 37 years. Corrie Marijnen left the
Radiotherapy Department to become the new chair of
the subsection Radiation Oncology at the Leiden
University Medical Center. Jan Schornagel, Wim ten
Bokkel Huinink, Nico van Zandwijk retired as medical
oncologists and Jaap van der Sande as a neurooncologist.
Fortunately, we have been able to recruit excellent
successors. Sanne Schagen and Eveline Bleiker were
appointed as tenured staff members in the Division of
Psychosocial and Epidemiological Research, and
Maarten Fornerod and Jos Jonkers became tenured staff
members in the Division of Gene Regulation and
Molecular Biology, respectively.
Staff of the NKI-AVL fulfilled numerous functions in
national and international organizations, on boards of
scientific journals, as members of study sections, and as
organizers or co-organizers of scientific meetings,
workshops and congresses.
15
INTRODUCTION
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. Collectively, we have the scientific skills and
expertise, as well as the drive and motivation to
effectively compete for resources to support our
activities. In the institute-wide evaluation that will take
place later this year, we aim to convince the site visit
team that the NKI is a unique institution that should be
given full support by the Dutch Cancer Society and the
Ministry of Health that provide the core funding for the
Institute. Thus far, we have been very successful in
securing support through a variety of funding
mechanisms, and with the on-going initiatives to
combine forces with other institutions and
pharmaceutical industries within and outside the
Netherlands we are in a good position to raise additional
funds for our research. But we also need to access new
sources to fund our research. The Antoni van
Leeuwenhoek Research Fund that we founded together
with the Dutch Cancer Society is one such endeavour.
This fund can also help us to further increase our
visibility to industry and private sponsors as an
organization worth investing in. Real breakthroughs are
more likely to come from of the combination of basic
understanding of the underlying mechanisms and stateof-the-art
implementation, and offer the best
perspectives to substantially improve cure rates and
long-term survival rates of cancer patients. This is an
area in which the NKI has a strong record. I am
convinced that breakthroughs in cancer diagnosis and
treatment can be realized with approaches that are now
broadly applied in the NKI.
Our scientific output has been very robust over the years,
indicating that the organization is stable and capable of
making substantial contributions to reduce significantly
the burden of cancer. This, together with the drive and
enthusiasm that is so characteristic of the individuals
that shape our institute, should convince donors that the
NKI-AVL is an institution at the international forefront
of cancer research and therefore worth supporting.
In closing, I want to end by thanking 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 Institute and has provided the funds for a
new hospital and 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, student training
takes place at various levels, from Master, PhD to postdoctoral
level and involves medical workers, technical
personnel and scientists. Research and clinical staff as
well as their group members are involved in theoretical
and practical training. A number of staff members have
joint appointments as professors at Dutch universities
and also contribute to the regular curriculum at these
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 Jannie Borst (chair and dean Master students),
Hein te Riele (general affairs), Roderick Beijersbergen
(Master course), John Hilkens, John Collard (HLO
students and publicity), Titia Sixma (dean PhD students)
and Fons Balm (clinical teaching). Peter Peters
functions 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 four months.
In 2008, 31 Dutch university students completed a
placement of 5-12 months at the biomedical research
divisions. As in previous years, the students came
primarily from the Amsterdam Universities UvA (7) and
VU (9), as well as Utrecht University (7), but also from
Nijmegen (4), Wageningen (2), Leiden (1) and Delft (1).
The new Life Science and Technology Masters is
particularly suited to the biomedical research
environment in the NKI. 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 as 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 2008, the institute had 144 PhD students
registered with the OOA. Of these, 22 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 participate in 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. Also, students with no prior background in
cancer research can participate in the Experimental
Oncology course.
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
Table 1 – Courses in Experimental Oncology
Epidemiology Flora van Leeuwen
Surgery Emiel Rutgers
Radiodiagnostics Renato Valdes Olmos,
Michiel Sinaasappel
Pathology* Daphne de Jong
Molecular diagnostics** Ron Kerkhoven,
Sabine Linn
Drug therapy Sjoerd Rodenhuis
Radiotherapy* Marcel Verheij
DNA damage response
and apoptosis** Adrian Begg, Jannie Borst
Signal transduction** Wouter Moolenaar
Cell cycle** René Bernards,
Daniel Peeper
Cell Division Rob Wolthuis
Cell senescence and
genomic instability** Roderick Beijersbergen,
Hein te Riele
Cell adhesion** Ed Roos
Tumor microenvironment Karin de Visser
Mouse models of cancer** Jos Jonkers
Immunology and
immunotherapy** Ton Schumacher,
John Haanen
Analysis of protein structure Titia Sixma
Rational drug development
and drug delivery** Jos Beijnen,
Alfred Schinkel
* including tour
** including tutorial

Table 2 – OOA graduate student courses 2008
9-12 March The Macroscopic and Pathologic
Anatomy of the mouse
WH Lamers, CJF van Noorden
April English Writing and Presenting
Ann Bless
14-16 April Epigenetics
R Steenbergen, T vd Elsen, J Kooter
11-20 May Signalling
Wouter Moolenaar and
Arnoud Sonnenberg
Spring Stem cells and Cancer
Jan Paul Medema
14-16 October Annual Graduate Student Retreat
Titia Sixma
October Inflammation and Cancer
Ed Roos en Karin de Visser
November English Writing and Presenting
Ann Bless
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
2008, this event was hosted by the NKI and the excellent
organization was performed by four NKI graduate
students (Floor Frederiks, Bernike Kalverda, Jasper
Mullenders and Daan Peric Hupkes). Participants from
British CrUK institutes, the Italian IFOM and the
Spanish CNIO contributed 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.
Each student has a supervisory committee that meets
once a year to monitor scientific progress. The committee
discusses progress with the supervisor and student
separately and participates in a joint discussion of the
research. The students are represented in the OIOcouncil
that meets with the Dean of graduate student
affairs on a regular basis, as well as upon special request.
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 often 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.
Still, there are many other options available for an
interesting career after a successful postdoc period.
Many postdocs are unaware of these possibilities, or
they may consider it too great a step to go into biotech,
industry, journalism or a government position. Since
only 20% of postdocs are likely to get a permanent
position in academia, it can be of interest to have a good
look around during your postdoc period and to find out
17
EDUCATION IN ONCOLOGY
about all the possibilities for the next step in your career.
This year we were very happy to be able to receive a grant
from the ministry of economic affairs to professionalize
our ten year NKI-AVL history of postdoc activities in
order to create an organization which we called PCDI for
Postdoc Career Development Initiative with a nationwide
activity. We have not only offered new perspectives, but
also helped improving skills that will increase career
choices, both within and outside academia. The activities
we have offered can be visited at www.pcdi.nl.
The PCDI organized two postdoc retreats at Kapellerput,
which were held from 9-11th of April and from 5-7th
of November 2008. Each time we reached hundred
participants and were overbooked. Thanks to the
organizing committees we had great inspirational
speakers, among which Bruce Alberts, Hans Clevers,
Peter Friedl, Bart Noordam but also young successful
scientists, business managers and entrepreneurs.
The workshops on transferable skills were well received,
and we look back at stimulating networking days with
enthusiastic ex-postdocs and other representatives
working in academia, companies and non-profit
organizations. The retreats scored in an anonymous
evaluation between very good and excellent, which will
hopefully inspire the participating postdocs and last year
PhD students to make the right choices with regard to
their career.

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
Andrea Alfieri 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
Pim Van Dijk Technical staff
Herrie Winterwerp Technical staff
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 in eukaryotes. 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. Mismatch binding triggers
the uptake of ATP in the MutS nucleotide-binding domain and it is this mismatchdependent
ATP state that authorizes repair by recruitment of additional mismatch
repair components. In collaboration with Robert van den Heuvel in Utrecht we have
used native mass spectrometry to study MutS DNA complexes. In a collaboration
with Serge Cohen and Tassos Perrakis a novel method was developed that will allow
us to analyze the detailed nucleotide status of these large complexes. In collaboration
with Peter Friedhoff and Martin Marinus we studied a dimeric mutant of the MutS
protein. This mutant allows detailed kinetic studies and structure determination of
the full-length protein, alone and in complexes. Analysis of these data is beginning to
yield interesting insight into the mechanism of mismatch discrimination (figure 1).
Figure 1: Surface plasmon
resonance analysis of DNA
mismatch repair protein MutS
biniding to mismatched DNA.
Use of a dimer mutant allows
kinetic fitting of DNA binding.
Interestingly, it is not the search
process, but the off-rate that
provide discrimination between
mismatch and homoduplex DNA.
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
contribute to the development of novel drugs wich are directed towards critical

pathways in ubiquitin and SUMO conjugation. The process of conjugation by
ubiquitin-(like) proteins involves covalent linking of one or more of the 76-aminoacids
within ubiquitin 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, many questions on selectivity and specificity remain.
We study distinct E2/E3 complexes involved in conjugation of ubiquitin and the
related modifier SUMO as well as proteins involved in de-ubiquitination.
One interesting aspect of SUMO compared to ubiquitin is its specificity for certain
sequences. In collaboration with Klaus Schwamborn at Pepscan a chip was developed
that allows studying this specificity in an efficient manner (Schwamborn et al, 2008).
The E2 ubiquitin conjugating enzyme for sumoylation, Ubc9, can itself be modified
by SUMO (figure 2).
Figure 2: The activity for the E2 for sumoylation, Ubc9, can be modulated by SUMO itself in two different
manners: Right: Binding of SUMO to Ubc9 in a non-covalent interaction, the so-called back-side binding,
affects SUMO chain formation (Knipscheer et al, EMBO J. 2007). Left: The covalent SUMO modification
of mammalian Ubc9 on K14 affects the target choice, increasing the rate of modification of the SIM
containing target Sp100, but limits its activity against RanGAP (Knipscheer et al, Mol Cell 2008)
In collaboration with Andrea Pichlers group in Vienna, we showed that this
modification is important for target choice of the E2 enzyme (Knipscheer et al,
2008). It can enhance modification of the Sp100 transcription factor and we showed
that this enhanced modification is due to binding of the covalently bound SUMO to
the target via a SUMO Interaction Motif (SIM). Surprisingly the site of SUMO
modification on Ubc9 is equivalent to the site of SUMO modification of E2-25K, an
E2 enzyme for ubiquitin, but the consequence of the latter modification is radically
different (Pichler et al, 2005). Our crystal structure suggested that this difference
could be due to intramolecular interaction of the attached SUMO with a Ubc9specific
beta-hairipin and indeed, specific removal of the hairpin, by replacing it with
a di-glycine sequence causes a shift in the effect of E2 modification to resemble that
in E2-25K (Knipscheer et al, 2008).
DNA translesion synthesis (TLS) allows for tolerance to DNA damage and synthesis
beyond the mismatch during replication and avoids replication stallimg. This process
is initiated by ubiquitin modification of PCNA by the ubiquitin conjugating E2/E3
enzyme complex Rad6/Rad18. In our studies of the Rad6/Rad18 activity we used
thermodynamic analysis of the DNA binding properties. Studying the ZnF domain
we were able to compare the binding properties of ubiquitin and short chains of
ubiquitin (Crosetto et al, 2008).
Nicotinic Acetylcholine receptor homolog AchBP In collaboration with the
groups of Guus Smit and Iwan de Esch at the Free University in Amsterdam we
study the Acetylcholine Binding Proteins (AChBP) as tools for understanding the
ligand binding in pentameric ligand-gated ion-channels. 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.
Based on in silico screening we found a number of novel molecules that bind AChBP.
Structural analysis showed that binding was indeed present, but with considerable
variability in the binding. The analysis of these compounds on nAChRs indicate that
these compounds compete with bungarotoxin, implying binding to the ligand
binding site. In addition, however, they seem to bind to the transmembrane pore,
precluding further development as lead compounds. Further studies focus on
additional hits from the in silico screen.
Publications
19
BIOCHEMISTRY
Bertaccini EJ, Lindahl E, Sixma T,
Trudell JR. Effect of cobratoxin binding on
the normal mode vibration within
acetylcholine binding protein. J Chem Inf
Model. 2008;48:855-60
Cohen SX, Ben Jelloul M, Long F, Vagin A,
Knipscheer P, Lebbink J, et al. ARP/wARP
and molecular replacement: the next
generation. Acta Crystallogr D Biol
Crystallogr. 2008;64:49-60
Crosetto N, Bienko M, Hibbert RG,
Perica T, Ambrogio C, Kensche T, et al.
Human WRNIP1 is localized in replication
factories in a UBZ-dependent manner. J
Biol Chem. 2008
Knipscheer P, Flotho A, Klug H, Olsen JV,
van Dijk WJ, Fish A, et al. Ubc9
sumoylation regulates SUMO target
discrimination. Mol Cell. 2008;31:371-82
Schwamborn K, Knipscheer P, van Dijk E,
van Dijk WJ, Sixma TK, Meloen RH, et al.
SUMO assay with peptide arrays on solid
support: insights into SUMO target sites.
J Biochem. 2008;144:39-49
van Leuken RJ, Luna-Vargas MP, Sixma TK,
Wolthuis RM, Medema RH. Usp39 is
essential for mitotic spindle checkpoint
integrity and controls mRNA-levels of
aurora B. Cell Cycle. 2008;7:2710-9

20
BIOCHEMISTRY
BIOCHEMISTRY
Group leader Anastassis Perrakis
Anastassis Perrakis PhD group leader
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
Wijnand Mooij PhD Post-doc
Eirini Mitsiki PhD Post-doc
Jens Hausmann MSc PhD student
Evangelos Christodoulou Technical staff
Tatjana Heidebrecht Technical staff
Diederick De Vries Technical staff
Publications
van de Weerdt BC, Littler DR, Klompmaker R,
Huseinovic A, Fish A, Perrakis A, et al.
Polo-box domains confer target specificity to
the Polo-like kinase family. Biochim
Biophys Acta. 2008 Jun;1783(6):1015-22.
Repanas K, Fuentes G, Cohen SX, Bonvin AM,
Perrakis A. Insights into the DNA cleavage
mechanism of human LINE-1 retrotransposon
endonuclease. Proteins. 2008
Perrakis A, Romier C. Assembly of protein
complexes by coexpression in prokaryotic
and eukaryotic hosts: an overview. Methods
Mol Biol. 2008;426:247-56
Langer G, Cohen SX, Lamzin VS, Perrakis A.
Automated macromolecular model building
for X-ray crystallography using ARP/wARP
version 7. Nat Protoc. 2008;3:1171-9
Joosten K, Cohen SX, Emsley P, Mooij W,
Lamzin VS, Perrakis A. A knowledgedriven
approach for crystallographic protein
model completion. Acta Crystallogr D Biol
Crystallogr. 2008;64:416-24
Cohen SX, Ben Jelloul M, Long F, Vagin A,
Knipscheer P, Lebbink J, et al. ARP/
wARP and molecular replacement: the next
generation. Acta Crystallogr D Biol
Crystallogr. 2008;64:49-60
STRUCTURAL BIOLOGY
This year our biology focus remained around mechanisms for correct DNA
inheritance: DNA replication and the subsequent chromatide separation between the
daughter cells. This interest is demonstrated by our work in the DNA replication
license and in spindle assembly checkpoint proteins.
Our involvement on three in-house collaborations remained active, trying to
determine structures that are of importance to in-house research: we work with
Wouter Moolenaar on ATX, with Piet Borst on JBP1 and with Ton Schumacher on
MHC class I structures.
Work in computation methods went on, with many steps taken towards new
algorithms for crystallographic model completion and consolidating the control
structures of the ARP/wARP software. A new side product of our computation effort
was a tool to facilitate the design of truncated proteins suitable for protein expression
experiments.
DNA replication licensing and the role of the Cdt1/Geminin complex This year
we finalized our work on the function of the Geminin: Cdt1 complex, an inhibitor 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, possibly representing a conformation
switch. In vivo experiments in mammalian cells and in Xenopus oocyte extracts
conclusively support our conclusions for the function of this molecular switch.
Structural studies of proteins involved in mitotic progression Our interest in
this area was initiated by the work on Polo-like kinases, together with the group of
Rene Medema. We have recently shown with biophysical measurements combined
with cell-based assays that the polo binding domains (PBDs) of Plk1-3 show
unexpected differences. We demonstrated a partial functional overlap but also
distinct roles of the different PBDs.
Last year we have initiated a high-throughput approach towards characterising
different proteins involved in mitotic progression and obtained the first crystals.
We have made good progress improving the crystals of a regulatory domain of BubR1
and we are about to determine the structure of a regulatory domain of TTK/Mps1.
We have also determined the structure of the DNA-binding domain the FoxM1
transcription factor (figure 3).
Figure 3: (A) A cartoon representation of the structure of the complex of the FoxM1 DNA binding domain
bound to DNA and the assumed position of the N- and C-terminal domains. (B) Binding of FOXM1 to
Random DNA, a singe recognition sequence and a double recognition sequence and (C) the occurrence of
FOXM1 DNA recognition sequnces in the 3,000 bp flanking regions of human genes.

FoxM1 controls the production of genes involved in cell cycle progression and
control, such as Cyclin B2, Plk1 and securin. FoxM1 is highly expressed during
development, but it is also known to be present in numerous cancer cell-lines. More
excitingly, in the laboratory the artificial-inhibition of FoxM1 has been shown to
noticeably reduce cell-viability in primary breast cancer cell-lines. This behaviour
makes a clear understanding of how FoxM1 functions at a molecular level highly
desirable. The structure of the domain of FoxM1 responsible for DNA-binding, tells
us much about its sequence specificity. We will extend this work to learn more about
the regulation of FoxM1’s transcriptional activity, which in the longer-term can aid the
development of inhibitors against this potential therapeutic target.
Structural studies of autotaxin Autotaxin (NPP1) hydrolyzes circulating
lysophosphatidylcholine. Engineering of a human cell line (HEK293 derivative) that
is stably expressing glycosylation mutants of rat autotaxin was the key for
crystallization success, resulting to crystals diffracting to high resolution. Structure
solution has been hampered by technical issues, such as low protein yield of Seleno-
Methionine substituted ATX and small crystal size. We are still in the process of
determining the structure.
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 major protozoal human pathogens such as T. brucei (sleeping sickness), T.
cruzi (Chagas’ disease) and Leishmania species (various types of Leishmaniasis).
Recently we were able to crystallize the DNA-binding domain of JBP1 in complex
with J-DNA and we will proceed with structure determination this year.
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 (H7) and Huib Ovaa (H3) we have generated a protocol
for the in crystallo exchange of a photo-cleavable 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. We have determined a few structures that
demonstrate that this strategy can produce good quality MHC class I - peptide
complexes.
Methods for high throughput Structural Biology The PiMS protein information
management system work was finished early this year, allowing the handling of
crystallization data. The end of the BIOXHIT network for methods development in
X-ray crystallography marks the end of our work in this area.
We carried on with an in-house development focused on the creation of a collection
of versatile ligation independent cloning (LIC) vectors suitable for E. coli, insect and
mammalian cells expression. This development allows a single PCR product to be
directly transferred to the vector of choice for all available expression systems. As part
of the 3D-Repertoire network we are participating in a large-scale test for the
recombinant production of binary complexes, using our vectors. As part of the
SPINE-2 network we have also benchmarked our vectors together with similar
systems for recombinant expression, with encouraging results.
As part of the effort to be able to design easily cloning experiments for the expression
of truncation variants of our proteins of interest, we have also created the
proteinCCD server for enabling the design of oligo-nucleotides for such experiments
(http://xtal.nki.nl/CCD)
Methods for X-ray crystallography The development within the ARP/wARP
framework, focused on developing ‘conditional restraints’ for enhancing hybrid
model refinement at a low resolution, density based validation through statistical
targets, some fundamental work on algorithms for extracting density features from
electron density regions of predefined shapes, and a new loop building algorithm.
All these development will allow better model building in the future versions of
ARP/wARP.
21
BIOCHEMISTRY
Figure 4: Crystals of ATX, FoxM1/DNA,
JBP/DNA with and without polarize filters,
two MHC class I complexes after soaking,
Mps1 and BubR1 kinases regulatory
domains.

22
MOLECULAR CARCINOGENESIS
CELL BIOLOGY I
Division head, group leader Arnoud Sonnenberg
Arnoud Sonnenberg PhD Group leader
Ruben Postel PhD Post-doc
Pablo Secades PhD Post-doc
Quint Le Duc MSc PhD student
Evelyne Frijns MSc PhD student
Mirjam Ketema MSc PhD student
Coert Margadant MSc PhD student
Norman Sachs MSc PhD student
Maaike Kreft Technical staff
Ingrid Kuikman Technical staff
Publications
van den Bout I, van Rheenen J, van
Angelen AA, de Rooij J, Wilhelmsen K,
Jalink K, Divecha N, Sonnenberg A.
Investigation into the mechanism regulating
MRP localization. Exp Cell Res
2008;314:330-41
van den Bout I. Identification and
functional analysis of genes regulated by b1
and b3 integrins. Academic thesis,
University of Amsterdam 2008
Huveneers S, Arslan S, van de Water B,
Sonnenberg A, Danen EH. Integrins
uncouple Src-induced morphological and
oncogenic transformation. J Biol Chem
2008;283:13243-51
Figure 1: Localization of zebrafish
Nesprin-3a at the nuclear envelope in
transiently transfected keratinocytes.
Confocal images of cells expressing GFPnesprin-3a
stained with DAPI to visualize
nuclei.
DIVISION OF CELL BIOLOGY I
RECEPTORS FOR MATRIX ADHESION
Torsin A influences the interaction between nesprin-3 and Sun proteins We
have previously identified a new member of the nesprin protein family, nesprin-3,
that binds to the cytoskeletal linker protein plectin, thereby establishing a link
between the intermediate filament system and the nucleus. The localization of
nesprin-3 at the outer nuclear membrane depends on its interaction with Sun
proteins present at the inner nuclear membrane. We have recently identified an
additional binding partner of nesprin-3, torsin A, which might influence the
localization of nesprin-3 by regulating the interaction of nesprin-3 with Sun proteins.
Torsin A is an AAA+ ATPase, which resides in the lumen of the endoplasmic
reticulum and the nuclear envelope, and mutation of torsin A causes the movement
disorder early onset torsion dystonia. The interaction between nesprin-3 and torsin A
does not require the Sun binding motif of the nesprin-3 C-terminal KASH domain
and is independent of the ATPase activity of torsin A. However, the ATPase activity of
torsin A seems to be involved in establishing the binding of nesprin-3 to Sun
proteins, since co-precipitation of Sun2 with nesprin-3 is reduced in the presence of
ATPase-deficient torsin A mutants. We are currently investigating the effect of torsin
A knock-down on the localization of nesprin-3 and its interaction with Sun proteins.
Furthermore, a family member of torsin A, torsin B, can interact with nesprin-3 in a
similar manner, indicating a potential redundancy in function.
Function of nesprin-3 in zebrafish In order to reveal the function of nesprin-3 in
vivo, we used the zebrafish as a model system. Cloning the full-length zebrafish
nesprin-3 mRNA revealed (as in the mouse) two variants: nesprin-3a and nesprin-3b.
The zebrafish nesprin-3a transcript encodes a protein of 1100 amino acids, which
contains the conserved plectin-binding domain at the N-terminus as well as the
C-terminal conserved KASH domain at the C-terminus. When overexpressed in cells
zebrafish nesprin-3a-GFP is localized at the nuclear envelope. The nesprin-3b
transcript appears to have used an alternative splice site in exon 1, resulting in an inframe
deletion of 7 amino acids in the plectin-binding domain. Additionally, we
observed that both the nesprin-3a and nesprin-3b transcripts become expressed
dynamically during the first days of development. Furthermore, in situ hybridization
with dig-labelled antisense nesprin-3 RNA revealed maternal and ubiquitous nesprin-3
mRNA expression during early embryonic development. Morpholino-mediated
knockdown of zygotic nesprin-3 in zebrafish resulted in blistering of the epidermis
already 2-3 days post fertilization. This suggests that loss of nesprin-3 in vivo affects
the complete cell morphology in epidermal cells. A detailed analysis of the epidermal
cell phenotype in these nesprin-3 knock-down zebrafish embryos is ongoing.
Modulating podocyte adhesion Cell adhesion to the extracellular matrix can be
regulated by altering the adhesive properties of integrins. A candidate protein in
modulating the adhesive strength of certain integrins is the tetraspanin CD151.
CD151 tightly associates with the class of laminin-binding integrins, especially a3b1.
Recently, we generated CD151 knockout mice which develop severe kidney failure
similar to that in patients with mutations in the CD151 gene primary glomerular
disease. Electron microscopy analysis revealed that CD151 as well as the integrin a3b1
are present at the basal side of podocyte foot processes. These highly specialized
structures anchor podocytes to the underlying basement membrane to withstand the
permanent transcapillary filtration pressure in the kidney glomerulus. Mice lacking
either CD151 or a3b1 specifically in podocytes show a remarkably similar kidney
phenotype to that seen in mice in which CD151 was totally knocked out, suggesting
that podocyte defects are the cause for the disease. In vitro analysis indeed revealed
that podocytes without CD151 detach from a laminin matrix at far lower forces than
required to detach podocytes with CD151. Whereas re-expression of wild-type CD151
rescued the adhesion deficit, mutant CD151 incapable of binding a3b1 did not.
Podocyte adhesion via a3b1 alone may be sufficient to withstand moderate filtration

pressures, but CD151 is necessary to cope with prolonged and/or high filtration
pressure. Furthermore, we show that mice lacking CD151 develop kidney failure only
on a pure FVB/N strain, but not on a pure C57Bl6/J strain. The latter strain is known
to have a lower blood and consequently filtration pressure. CD151 mutations in
humans may therefore predispose to kidney failure which is manifested when
transcapillary filtration pressure rises, for example due to hypertension. We are
currently investigating this hypothesis.
Figure 2: Glomerular abnormalities in CD151KO mice.
Electron micrograph of a 3-month-old CD151KO mouse
glomerulus reveals abnormal capillary loops characterized
by thickening of the basement membrane (*) and loss of
the regular pattern of podocyte foot processes (arrows).
These initial lesions are responsible for the later kidney
failure.
Conditional deletion of the Itgb4 integrin gene in Schwann cells leads to
delayed peripheral nerve regeneration Several different integrins participate in
the complex interactions that promote repair of the peripheral nervous system. In
collaboration with Dr. I van der Zee (NCMLS, Nijmegen), we have investigated the
role of the integrin a6b4 in peripheral nerve regeneration in mice by cre-mediated
deletion of the Itgb4 (b4) gene in Schwann cells. After a crush lesion of the sciatic
nerve, the recovery of motor, but not that of sensory, nerve function in b4 (-/-) mice
was delayed. Immunostaining of neurofilament-200 showed that there also is a
significant reduction in the number of newly outgrowing nerve sprouts in b4 (-/-)
mice. Morphometric quantitative measurements revealed that fewer axons are
myelinated in the nonlesioned b4 (-/-) nerves. After a sciatic nerve crush lesion, b4
(-/-) mice did not only have fewer myelinated axons compared with lesioned wild-type
nerve, but their axons also showed a higher g-ratio and a thinner myelin sheath,
pointing at reduced myelination. This study revealed that the b4 protein remains
expressed in the early stages of peripheral regeneration, albeit at levels lower than
those before the lesion was inflicted, and showed that laminin deposition is not
altered in the absence of b4. These results together demonstrate that integrin a6b4
plays an essential role in axonal regeneration and subsequent myelination.
Kindlin-1 is an essential activator of b1 integrin Kindler syndrome (KS) is a rare
autosomal recessive skin disorder mainly characterized by severe skin blistering. KS
patients all carry loss-of-function mutations on both alleles of a gene designated
KIND1, encoding the protein kindlin-1. Kindlin-1 is part of a protein family including
kindlin-2 (Mig-2) and kindlin-3 (URP-1). Whereas expression of kindlin-2 is more
ubiquitous, expression of kindlin-3 is restricted to haematopoietic cells, and kindlin-1
is predominantly expressed in the colon and the epidermis. Both kindlin-2 and
kindlin-3 have been shown to be essential for integrin activation. We have
investigated the function of kindlin-1 using normal human keratinocytes (NHK) and
kindlin-1 deficient cells isolated from 2 different KS patients. In both KS patients
there was a duplication of the basement membrane and there were microblisters
along the epidermal-dermal junction. We have previously established that deletion of
the integrin a3b1 in the epidermis causes the same phenotype in mice. In vitro, we
find that kindlin-1 is colocalized with b1 integrins in focal adhesions and is associated
with the actin, but not the tubulin or keratin cytoskeletal systems. Cell adhesion, cell
spreading, cytoskeletal organization, and proliferation on collagens and on laminins
are severely impaired in KS cells as compared to NHK. The defects are partially
rescued by outside-in activation of b1 integrins with the activating antibody TS2/16.
Further, the defects are reversed by stable reconstitution of KS cells with kindlin-1.
Summarizing, we have identified kindlin-1 to be an essential activator of b1 integrins
in the skin. Studies to identify the mechanism of activation are ongoing.
Publications (continued)
23
CELL BIOLOGY I
Huveneers S, Truong H, Fässler R,
Sonnenberg A, Danen EHJ. Binding of
soluble fibronectin to integrin a5b1 – link to
focal adhesion redistribution and contractile
shape. J Cell Sci 2008;121:2452-62
Huveneers S. The regulation of Rho and
Src signaling Academic thesis, University of
Amsterdam, 2008
Margadant C, Frijns E, Wilhelmsen K,
Sonnenberg A. Regulation of
hemidesmosome disassembly by growth
factor receptors. Curr Op Cell Biol
2008;20:589-96
Braam SR, Zeinstra L, Litjens S,
Ward-van Oostwaard D, van den Brink S,
van Laake L, Lebrin F, Kats P,
Hochstenbach R, Passier R, Sonnenberg
A, Mummery CL. Recombinant vitronectin
is a functionally defined substrate that
supports human embryonic stem cell self
renewal via avb5 integrin. Stem Cells
2008;26:2257-65
Yañez-Mó M, Barreiro O, Gonzalo P,
Batista A, Megias D, Genis L, Sachs N,
Sala-Valdés M, Alonso MA, Montoya MC,
Sonnenberg A, Arroyo AG,
Sánchez-Madrid F. MT1-MMP
collagenolytic activity is regulated through
association with tetraspanin CD151 in
primary endothelial cells. Blood
2008;112:3217-26
Nery FC, Zeng J, Niland BP, Hewett J,
Farley J, Irimia D, Li Y, Wiche G,
Sonnenberg A, Breakefield XO. TorsinA
binds the KASH domain of nesprins and
participates in linkage between nuclear
envelope and cytoskeleton. J Cell Sci
2008;121:3476-86
van der Zee CEEM, Kreft M, Beckers G,
Kuipers A, Sonnenberg A. Conditional
deletion of the Itgb4 integrin gene in
Schwann cells leads to delayed peripheral
nerve regeneration. J Neuroscience
2008;28:11292-303
Kanters E, van Rijssel J, Hensbergen PJ,
Hondius D, Mul FP, Deelder AM,
Sonnenberg A, van Buul JD, Hordijk P.
Filamin B mediates ICAM-1-driven
leukocyte transendothelial migration. J Biol
Chem 2008;283;31830-9

24
CELL BIOLOGY I
Division head, group leader Wouter Moolenaar
Wouter Moolenaar PhD Group leader
Maikel Jongsma PhD Post-doc
Dalila Elouarrat MSc PhD student
Anna Houben MSc PhD student
Bas Ponsioen MSc PhD student
Leonie Van Zeijl MSc PhD student
Trudi Hengeveld Technical staff
Adrian Rzadkowski Technical staff
LIPID GROWTH FACTOR SIGNALING
Our group has a longstanding interest in lipid growth factors, particularly
lysophosphatidic acid (LPA) and sphingosine 1-phosphate (S1P), their signaling
properties and their role in health and disease. LPA and S1P signal through specific
G protein-coupled receptors to stimulate cell migration, proliferation and survival.
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
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. We have developed ATX activity assays, which have successfully been
used for high-throughput screening of small-molecule compound libraries. Since
ATX is found overexpressed in various cancers, whilst LPA promotes tumor
progression in mouse models, ATX qualifies as a potential target for therapy. Our
work should lead to novel ways of interfering with LPA receptor signalling cells and
with inappropriate LPA production in the cellular microenvironment.
A second line of research is devoted to the regulation of cell-cell communication via
gap junctions, which is often defective in tumor cells.
LPA receptor signaling: Rho activation, gene expression and oncogenic
potential
The LPA-Rho activation pathway: identification of a new player
LPA receptors strongly couple to the G(13)-RhoA pathway to regulate the actin
cytoskeleton. Through live-cell imaging studies, we recently discovered that LPAinduced
RhoA activation is consistently accompanied by rapid recruitment of a
‘Chloride Intracellular Channel’ protein (CLIC) to the plasma membrane (figure 3).
CLIC family members are soluble proteins that have been proposed to auto-insert
into membranes to form chloride channels. Using patch-clamp technology, we are
currently examining whether LPA-induced CLIC translocation results in enhanced
chloride currents across the plasma membrane (collaboration Kees Jalink).
Figure 3: Rapid recruitment of CLIC4 to the plasma membrane in response to RhoA-activating receptor
agonists (LPA or TRP).
Expression profiling
To examine how LPA receptor signaling influences global gene transcription, we
subjected mouse embryonic fibroblasts (MEFs) to microarray analysis using 32K
oligo-arrays. About 900 PA-regulated genes could be clustered into groups based on
their temporal patterns and biological functions. In addition to genes involved in
growth stimulation and cytoskeletal reorganization, LPA induced many genes that
encode secreted factors, including chemokines, growth factors, cytokines, proangiogenic
factors, components of the plasminogen activator system and
metalloproteases, as confirmed by quantitative RT-PCR. The LPA-associated gene
signature showed a remarkable overlap with that induced by epidermal growth factor.
Our study highlights the importance of LPA in programming fibroblasts not only to
proliferate but also to produce many paracrine mediators of tissue remodeling,
angiogenesis, inflammation and tumor progression.
Oncogenic potential of LPA receptors
Using a functional genetic screen, Panthea Taghavi and colleagues (Division of
Molecular Genetics) have identified the LPA2 receptor as an oncogene that
collaborates with c-Myc and Tbx2 in MEF transformation. LPA1 and LPA4, but not
LPA3, could reproduce the transforming effect of LPA2. LPA-induced cell

transformation was dependent on the Gi-linked ERK and PI3K signaling pathways.
The transforming ability of LPA1, LPA2 and LPA4 was confirmed by tumor
formation assays in nude mice. These results show that LPA signaling can promote
cell transformation and tumor formation in the conjunction with c-Myc and Tbx2.
Through a similar genetic screen, Roderik Kortlever and colleagues (Division of
Molecular Carcinogenesis) showed that the LPA2 receptor induces senescence bypass
in MEFs in the presence of wild-type p53. This result uncovers an unexpected link
between LPA receptor signaling and the p53 tumor-suppressive pathway.
Autotaxin, a secreted lysophospholipase D implicated in vascular
development and tumor progression
Regulation of ATX and discovery of small-molecule inhibitors
ATX (or NPP2) is a unique ecto-nucleotide phosphodiesterase/pyrophosphatase
(NPP) that functions as a lysoPLD to produce LPA from extracellular
lysophosphatidylcholine. Through genetic studies in mice, we 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 David Egan). 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.
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.
ATX function in vivo
Having shown that ATX is essential for vascular development, our next step is to
establish the importance of ATX in metastasis and tumor angiogenesis, making use
of various mouse models. We find that knockdown of endogenous ATX in melanoma
cells reduces their metastatic capacity in a mouse model, supporting the view that
secreted ATX alters the microenvironment to promote tumor progression. Through
various collaborations with outside groups, we hope to learn more about ATX
function in specific tissues and to uncover the origin of ATX in the circulation. Newly
discovered small-molecule inhibitors of ATX will be tested in experimental animals
for their anti-tumor potential. In addition, we set out to analyze the role of ATX in
vascular development of zebrafish embryos, using the morpholino-based knockdown
approach (collaboration Stefan Schulte-Merker, Hubrecht Institute, Utrecht).
Preliminary results suggest that ATX-deficient zebrafish embryos suffer from
vascular defects reminiscent of those observed in ATX knockout mice.
Regulation of gap junctional communication Direct cell-cell communication
through gap junctions is essential for coordinated cell behavior; it is usually defective
in tumor cels. Gap junctions consist of clustered connexin subunits that form cell-tocell
channels for small molecules, with connexin43 (Cx43) being the most widespread
connexin. In fibroblasts, Cx43 gap junctional communication is inhibited by LPA and
other G-protein-coupled receptor agonists. We established the closure and reopening
of Cx43 channels is controlled by phosphatidylinositol 4,5-bisphosphate (PIP 2).
Our subsequent studies identified the ubiquitin E3 ligase Nedd4 as an essential
component in the pathway from activated receptor to Cx43 channel closure.
Following receptor activation, Nedd4 binds to Cx43 in a manner that depends on
Cx43 residue Tyr265. The challenge now is to define the link between PIP 2 hydrolysis
and Nedd4 recruitment to Cx43 gap junctional channels.
Publications
25
CELL BIOLOGY I
Kortlever RM, Brummelkamp TR,
van Meeteren LA, Moolenaar WH,
Bernards R. Suppression of the p53-
Dependent Replicative Senescence Response
by lysophosphatidic acid signaling. Mol
Cancer Res. 2008; 6:1452-60
Stortelers C, Kerkhoven R,
Moolenaar WH. Multiple actions of LPA
on fibroblasts revealed by transcriptional
profiling. BMC Genomics 2008; 9:387
Taghavi P, Verhoeven E, Jacobs J,
Lambooij J, Stortelers C, Tanger E,
Moolenaar WH, van Lohuizen M. In vitro
genetic screen identifies a cooperative role for
LPA signaling and c-Myc in cell
transformation. Oncogene 2008; 27:6806-16
Van Meeteren L, Brinkmann V,
Saulnier-Blache JS, Lynch KR,
Moolenaar WH. Anticancer activity of
FTY720: Phosphorylated FTY720 inhibits
autotaxin, a metastasis-enhancing and
angiogenic lysophospholipase D. Cancer
Lett. 2008; 266:203-8

26
CELL BIOLOGY I
Group leader John Collard
John Collard PhD Group leader
Audrey Gerard PhD Post-doc
Sandra Iden PhD Post-doc
Saskia Ellenbroek MSc PhD student
Amra Hajdo-Milasinovic MSc PhD student
Sander Mertens MSc PhD student
Saskia Amra Sander MSc PhD student
Rob Van der Kammen Technical staff
Publications
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 (in press)
Iden S, Collard JG. Cross-talk between
small GTPases and polarity proteins in cell
polarization. Nat Rev Mol Cell Biol, 2008;
9:846-859
Strumane K, Song J-Y, Baas I, Collard JG.
Increased Rac activity is required for the
progression of T-lymphomas induced by
Pten-deficiency. Leukemia Research, 2008;
32:113-120
Rygiel TP, Mertens AE, Strumane K,
van der Kammen RA, Collard JG. The Rac
activator Tiam1 prevents keratinocyte
apoptosis by controlling ROS-mediated
ERK phosphorylation. J Cell Sci, 2008;
121:1183-1192
GENETIC CONTROL OF INVASION AND METASTASIS
The aim of our research is to identify and characterize genes that play an essential
role in the acquisition of an invasive and metastatic phenotype of tumorigenic cells.
Insight into the molecular mechanisms that underlie the formation and progression
of tumors may lead to the development of novel diagnostic tools or improved
therapeutic strategies.
Function-based screens for invasion-inducing genes Using functional screens
we have identified different invasion-inducing genes by retroviral insertional
mutagenesis in combination with in vitro selection of invasive T-lymphoma cells.
These included the a 5- subunit of the a 5b 1 integrin, the LPA2 receptor, Rap1 and
Tiam1, all of which have been implicated in cell-matrix adhesion and/or signaling
pathways mediated by Rho GTPases. Interestingly, the Tiam1 gene encodes an
activator of the Rho-like GTPase Rac. Currently, we are studying by which molecular
mechanisms in particular Rho proteins play a role in normal development and tumor
formation and progression.
Rho family proteins are controlled by various regulatory proteins The invasioninducing
Tiam1 gene encodes an activator or guanine nucleotide exchange factor
(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.
Regulators of the activities of Rho proteins are GEFs and GTPase activating proteins
(GAPs). GEFs activate small GTPases by exchanging GDP for GTP, whereas GAPs
inactivate them by enhancing the intrinsic rate of hydrolysis of bound GTP to GDP.
In the cytoplasm, GTPases are kept in the GDP-bound form by association with
guanine nucleotide dissociation inhibitors (RhoGDIs). RhoGDIs bind and mask the
hydrophobic C-terminal region of GTPases, which is responsible for targeting them
to the plasma membrane.
Tiam1 regulates Rac-mediated downstream signaling GEFs are regulated by
intra-molecular inhibition, changes in intracellular localization, post-translational
modifications and interaction with other proteins. All of these regulatory
mechanisms seem to be involved in Tiam1 regulation. In particular, complex
formation with various scaffold proteins is an important mechanism to determine
signaling towards and downstream of Rac. For instance, oncogenic Ras controls Rac
signaling through direct interaction with the RBD domain of Tiam1. We found that
Tiam1 is indispensable for a3b1-mediated Rac activation and that Tiam1-deficient
keratinocytes do not adhere to and spread on a glass substrate, because they are
unable to deposit their own laminin-5 substrate. Tiam1-deficient keratinocytes are
impaired in activation of Rac upon adhesion to an exogenous laminin-5 substrate,
whereas other integrin-mediated signaling pathways are intact. Tiam1-deficiency
thereby hampers keratinocyte migration in vitro and re-epithelialization of excision
wounds in mouse skin in vivo. Furthermore, Tiam1 interacts with the p21-Arc subunit
of the Arp2/3 complex. As a result, Tiam1 co-localizes with the Arp2/3 complex at
sites of actin polymerization such as epithelial cell-cell contacts and membrane
ruffles. The Arp2/3 complex acts as a scaffold to localize Tiam1 and thereby Rac
activity, which both are required for activation of the Arp2/3 complex. The Arp2/3
complex drives actin polymerization primarily at the leading edge of cells. In
addition, Tiam1 interacts with the Par polarity complex and thereby regulates cell
polarity as discussed below.
Tiam1-Rac signaling and cell polarity Tiam1 associates with Par3 of the Par
polarity complex and thereby regulates polarity processes in various cell types and
different contexts. Tiam1 localizes to adherens junctions in epithelial cells and ectopic
expression of Tiam1 promotes E-cadherin-based cell-cell adhesions whereas knock
down of Tiam1 leads to loss of cell-cell adhesions and epithelial-mesenchymal
transition. Tiam1 controls tight junction (TJ) formation by activation of the Par
polarity complex, which is required for the establishment of apical-basal cell polarity

in epithelial cells. In the absence of cell-cell adhesions, epithelial cells develop frontrear
polarization and migrate in a directional persistent fashion involving signaling
by the Tiam1-Par complex. Lack of Tiam1, knock down of Par3, or reduced PKCz
activity impair front-rear polarization, persistent migration and chemotaxis of
keratinocytes. Tiam1 in conjunction with the Par complex also affects directional
protrusion outgrowth in astrocytes. In lymphocytes, Tiam1 is required for
chemokine- and S1P induced Rac activation and subsequent cell directional cell
migration. As a result, Tiam1-deficient T-cells show reduced chemotaxis in vitro, and
impaired homing, egress and contact hypersensitivity in vivo. Analysis of the T-cell
trans-endothelial migration cascade revealed that Par/PKCz/Tiam1/Rac signaling is
dispensable for T-cell arrest but is essential for the stabilization of polarization and
efficient crawling of T-cells on endothelial cells. Intriguingly, T-cells that lack Tiam1
predominantly transmigrate through individual endothelial cells (transcellular
migration) rather than at endothelial junctions (paracellular migration), suggesting
that T-cells are able to change their route of trans-endothelial migration according to
their polarization status and crawling capacity.
Tumorigenicity in Tiam1 mutant mice Although Tiam1 is expressed during
embryonic development, 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. We found that Tiam1 -/- mice
display resistance to DMBA/TPA-induced skin tumor formation. Although the
number and growth of tumors is strongly reduced in Tiam1 -/- mice, the frequency of
malignant conversion of the skin tumors that do develop is increased. We also
studied the function of Tiam1 in tumorigenesis induced by other oncogenic signaling
pathways and in different cell types. For instance, we found that Tiam1 is a Wntresponsive
gene and that its protein levels are increased in intestinal tumors
produced in APC-min mutant mice as well as in human colon tumors. Furthermore,
the initiation and growth of intestinal tumors was reduced in Tiam1-deficient APCmin
mice, suggesting that Tiam1 promotes b-catenin/TCF-induced intestinal tumor
formation. In contrast, we do not find differences in T-lymphomagenesis induced by
constitutive activation of the PI3-kinase pathway in wild-type and Tiam1-deficient
mice. Mammary tumor formation and progression induced by MMTV-c-neu appear
to be delayed and reduced in the absence of Tiam1, whereas mammary tumors
induced by MMTV-myc are not dependent on the presence of Tiam1. These studies
indicate that Tiam1 may contribute to the formation and progression of tumors
induced by various -but not all-oncogenic signaling pathways. During tumor
initiation, Tiam1-mediated Rac activation promotes survival signals and thereby
prevents apoptosis whereas the growth and the progression of tumors can be
promoted or inhibited by the presence of Tiam1 depending on the oncogenic pathway
involved and tumor cell type studied.
Opposing functions of Rac1 and Rac3 Rac1 and Rac3 are highly homologous
regulatory proteins of the Rho GTPase family. Rac1 is ubiquitously expressed and
regulates adhesion, migration and differentiation in various cell types. In contrast,
Rac3 is primarily expressed in the brain. We found opposing functions for Rac1 and
Rac3 in neuronal cells. Knock down of Rac1 by siRNA leads to decreased cell-matrix
adhesions and cell rounding in neuronal cells whereas knock down of Rac3
stimulates cell adhesion and neuritogenesis. The Rac1-opposing function of Rac3 is
not mediated by or dependent on components of the RhoA signaling pathway.
Further analyses revealed that Rac1 and Rac3 interact with Git1, a multifunctional
ArfGAP protein which regulates cell-matrix adhesion, cell spreading and endocytosis.
However, in contrast to Rac1, the Rac3/Git1 interaction is not mediated by bPix and
Rac3 severely attenuates Git1/paxillin interaction required for cell spreading.
Interestingly the ArfGAP domain of Git1 is required for Rac3 downstream signaling
and Arf6 activity is strongly reduced in Rac3 expressing cells. Our data suggests that
Rac3 and Rac1 oppose each other by differently modulating Git1 function.
27
CELL BIOLOGY I
Figure 4: Percentage of WT and Tiam1-/-
T-cells showing trans-endothelial migration
via the transcellular route by migrating
through individual endothelial cells.
Figure 5: Crawling and route of transendothelial
migration depend on PKC/
Tiam1-mediated polarization and crawling.
Polarized T-cells crawl on top of endothelial
monolayers and preferentially transmigrate
through endothelial junctions (paracellular
migration). Loss of Tiam1 and disturbed
Par/PKC-signaling prevents chemokineinduced
Rac activation leading to unstable
polarization and thereby impaired T-cell
crawling on top of endothelial monolayers.
As a result these T-cells preferentially cross
the endothelial monolayer through
individual endothelial cells (transcellular
migration).

28
CELL BIOLOGY I
Group leader Ed Roos
Ed Roos PhD Group leader
Joost Meijer MSc PhD student
Yvonne Wijnands Technical staff
Publications
Alvarez B, Stroeken PJ, Edel MJ, Roos E.
Integrin Cytoplasmic Domain-associated
Protein-1 (ICAP-1) promotes migration of
myoblasts and affects focal adhesions.
J Cell Physiol 2008;214:474-82
Meijer J, Ogink J, Kreike B, Nuyten D,
De Visser KE, Roos E. The chemokine
receptor CXCR6 and its ligand CXCL16
are expressed in carcinomas and inhibit
proliferation. Cancer Res 2008;68:4701-08
Meijer J, Ogink J, Roos E. Effect of the
chemokine receptor CXCR7 on proliferation
of carcinoma cells in vitro and in vivo.
Br J Cancer 2008;99:1493-1501
Figure 6: Role CXCR4 in metastasis During
outgrowth of metastases, cells are deprived of
glucose because of insufficient blood supply.
This becomes critical at a size of ~1000 cells,
long before the onset of angiogenesis that
comes about when tumors contain ~10 6
cells. A signal transduced by a CXCR4containing
complex induces adaptation of
cells, e.g. by changes in tumor cell
metabolism and by an increase in glucose
transporters in the plasma membrane. This
allows cells to survive and even proliferate in
low glucose conditions.
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
metastasis of a colon carcinoma but not involved in invasion. We next observed that
CXCR4 is required for continuation of growth when micrometastases are ~1000 cells
in size. The same is true for dispersed single cells in a s.c. Matrigel plug. To show
this, we used a CXCL12-KDEL ‘intrakine’ that downregulates CXCR4 but also the
newly discovered receptor CXCR7. Using RNAi and other intrakines we have now
demonstrated that CXCR4 is involved and not CXCR7. CXCR4 was also necessary for
outgrowth of KEP1 mammary carcinoma, a mouse model for human invasive lobular
mammary carcinoma. After reaching a size of ~1000 cells, CXCR4-supressed KEP1
tumors regressed. Surprisingly, CXCL12 is not involved, as demonstrated with
inhibitors and the K1R-CXCL12 antagonist. We have now established that CXCR4 is
essential for resistance against glucose deprivation, and that this depends on an
alternative CXCR4 ligand. This also involves several other surface proteins. Even in
medium without glucose and serum, KEP1 cells can survive when this ligand is
added. Apparently, tumor cells are not able to adapt to glucose deprivation when not
induced to do so by a signal from a CXCR4 signaling complex. It should be noted
that angiogenesis does not come about before tumors are 1 mm in diameter and
contain ~10 6 cells. Before that, tumors have no blood supply and can only grow when
they adapt their metabolism. We are presently exploring the option to generate
specific inhibitors of this phenomenon that should have therapeutic potential.
Chemokines as growth factors for carcinomas We have found that several
chemokine receptors can influence growth of carcinomas. CXCR7 is expressed on
many carcinomas and mediates strong enhancement of proliferation induced by
CXCL12. On some cells with lower CXCR7 levels, CXCR4 can also mediate this
effect. In contrast to reports by others, however, we observed no effect at all of stable
and complete CXCR7 suppression on growth of s.c. tumors and lung metastases.
Microarray analysis revealed expression of the chemokine receptor CXCR6 and its
ligand CXCL16 by all 170 human mammary carcinomas tested and in many
carcinoma cell lines, the latter confirmed by FACS analysis. So far, they had only
been detected on T-lymphocytes and macrophages, respectively. Soluble CXCL16
enhanced proliferation via CXCR6 in cells in which endogenous CXCL16 was
suppressed by RNAi. CXCL16 is special in that it is a transmembrane protein from
which the soluble chemokine can be cleaved off. This transmembrane form is
present on carcinoma cells. Unexpectedly, it reduced growth, since suppression of
either CXCR6 or CXCL16 by an intrakine and RNAi greatly promoted proliferation in
vitro and in vivo. The effect was confirmed using inhibitory CXCL16 antibodies and
by transfection of CXCL16 into CXCL16-negative cells. It was blocked by pertussis
toxin and therefore mediated by CXCR6. It is remarkable that carcinomas maintain
expression of an autocrine receptor-ligand pair that suppresses growth. This suggests
an essential function in tumor development.
Synaptotagmin-3: role in migration, antigen presentation and outgrowth of
carcinoma metastases We have previously shown that synaptotagmin-3 (Syt3) is
required for recycling of CXCR4 through MVB. Since antigen presentation by MHC
class II also involves passage through MVB, we tested involvement of Syt3 and found
that Syt3 inhibition suppressed the formation of MHC II-antigen complexes. Indeed,
transport of MHCII and in particular the associated invariant chain (CD74) to MVB
was blocked.
Syt3 and CD74 were found to be highly expressed by carcinomas. Based on the above
results, we tested their possible involvement in the function of CXCR4 in outgrowth
of metastases and of single tumor cells in Matrigel plugs. Indeed, inhibition of Syt3
had the same effect as suppression of CXCR4: tumors did not become larger than
~1000 cells. Growth of CT26 colon carcinomas was arrested whereas KEP1
mammary carcinomas regressed. Next, we found that knockdown of CD74 led to
greatly reduced surface levels of CXCR4, even though total protein levels were not
changed. Thus, we have shown that CD74 is required to retain CXCR4 on the
surface, quite likely by promoting recycling from MVB back to the plasma membrane.

BIOPHYSICS OF CELL SIGNALING
Our lab employs biophysical techniques to study cell signaling events with high
spatial and temporal resolution. Electrophysiological (e.g. patch clamping) and
advanced bio-photonic techniques (e.g. Fluorescence Resonance Energy Transfer
(FRET), Fluorescence Lifetime Imaging (FLIM), Fluorescence Cross Correlation
Spectroscopy (FCCS) and photorelease of caged compounds) are used in research
projects in our group as well as in a number of collaborations within and outside our
institute. Our lab also contributes to the development of hardware, software and
FRET sensors for various intracellular messengers.
Phosphatidylinositol bisphosphate (PIP 2) as a membrane-delimited
messenger Phosphoinositides in the plasma membrane regulate a wide variety of
cellular responses. We developed FRET-based assays for the lipids PIP 2 and PIP 3, and
use these to study the regulation of agonist-induced PIP 2 breakdown and its
subsequent resynthesis by PIPkinases. Novel techniques such as rapamycin-induced
recruitment of PIP 2-phosphatases to the membrane now also allow precise and
dosed breakdown of PIP 2 in living cells. Results from this ongoing line play an
important role in our other research lines and in collaboration with Dr W.
Moolenaars lab on the regulation of Gap Junctions.
The cation channel TRPM7 is a key regulator of podosomes Podosomes and
invadopodia are adhesive structures involved in migration and invasion. They consist
of an actin-rich core with associated regulatory and contractile proteins. Recently we
found that TRPM7, a non-selective cation channel with inherent kinase activity that is
involved in sensing mechanical forces, is a key regulator of podosomes. TRPM7overexpressing
cells have much increased adhesion and show enhanced migration in
transWell assays as well as matrigel invasion assays. TRPM7 mediates agonistinduced
cell adhesion through Ca 2+ influx and myosin heavy chain phosphorylation.
We discovered that PLC-activating agonists stimulate TRPM7 channels. Currently, we
study TRPM7 activation in detail by combining biophysical readout techniques with
mutational analysis of the C terminus. We found that TRPM7 and the Ca 2+ -sensitive
PLC isoform PLC 1 form a feedback loop that involves Ca 2+ influx through TRPM7.
In collaboration with the group of Dr FN van Leeuwen (Nijmegen) we have analyzed
recruitment and localization of GFP-tagged versions of Drebrin, actinin 4 and SIPA1.
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 studying FRET between GFP- and
mRFP-tagged constituent proteins. We also study regulation of the closely related
channel TRPM6, which is involved in Mg 2+ homeostasis.
Cytosol-membrane shuttling of Epac1 regulates cell adhesion During our
recent studies aimed at constructing new FRET sensors for cAMP based on Epac1
(short for Exchange Protein Activated by cAMP), we observed that stimulation of cells
with certain agonists causes translocation of Epac1 from the cytosol to the plasma
membrane. Further analysis of this phenomenon revealed that depending on the
agonist, two different signaling cascades can independently control Epac1
translocation to specific sites at the membrane. The relocalization of Epac1 to the
membrane is thought to be crucial for the localized activation of its effector, Rap1,
which regulates integrins (collaboration with Dr JL Bos, Utrecht).
Figure 7: Podosomes in N1E-115/
TRPM7 cells as visualized by Total
Internal Reflection Fluorescence (TIRF)
microscopy. TIRF is particularly suited
for imaging cell adhesion structures as it
is sensitive exclusively to fluorescence
within ~150 nm of the substrate. Scale
bar, 5 µm.
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
Ilona Vuist Trainee
Publications
29
CELL BIOLOGY I
Zwier JM, Oomen L, Brocks L, Jalink K,
Brakenhoff GJ. Quantitative image
correction and calibration for confocal
fluorescence microscopy using thin reference
layers and SIPchart-based calibration
procedures. J Microsc 2008;231:59-69
Vliem MJ, Ponsioen B, Schwede F,
Pannekoek WJ, Riedl J, Kooistra MR,
Jalink K, Genieser HG, Bos JL, Rehmann H.
8-pCPT-2'-O-Me-cAMP-AM: An Improved
Epac-Selective cAMP Analogue
Chembiochem 2008;9:2052-4
Van der Krogt GN, Ogink J, Ponsioen B,
Jalink K. A comparison of donor-acceptor
pairs for genetically encoded FRET sensors:
application to the Epac cAMP sensor as an
example. PLoS ONE 2008;3:e1916
Oomen LC, Sacher R, Brocks HH,
Zwier JM, Brakenhoff GJ, Jalink K.
Immersion oil for high-resolution live-cell
imaging at 37 degrees C: optical and
physical characteristics. J Microsc
2008;232:353-61
Jalink K, Van Rheenen J. FilterFRET:
quantitative imaging of FRET by sensitized
emission. In: FRET and FLIM methods,
Vol 33, 289-349. Th. Gadella, Ed. Elzevier,
New York (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
John De Widt BSc Technical staff
Shuraila Zerp BSc Technical staff
Jeffrey Klarenbeek MSc Technical staff
Publications
Verheij M, van Blitterswijk WJ.
Sphingomyelin metabolism: Implications
for cell signaling and drug uptake. In:
Reviews in Cancer Biology & Therapeutics
2007 (Kasis UN, Notario V, Haimovitz-
Friedman A, Bar-Eli M, Eds,), Chapter 3,
Transworld Research Network, pp 55-71
Zerp SF, Vink SR, Ruiter GA, Koolwijk P,
Peters E, van der Luit AH, de Jong D,
Budde M, Bartelink H, van Blitterswijk WJ,
Verheij M. Alkylphospholipids inhibit
capillary-like endothelial tube formation in
vitro: Anti-angiogenic properties of a new
class of anti-tumor agents. Anticancer Drugs
2008;19:65-75
Baldanzi G, Cutrupi S, Chianale F,
Gnocchi V, Rainero E, Porporato P,
Filigheddu N, van Blitterswijk WJ,
Parolini O, Bussolino F, Sinigaglia F,
Graziani A. Diacylglycerol kinase-a
phosphorylation by Src is required for
activation, membrane recruitment and
Hgf-induced cell motility. Oncogene
2008;27:942-56
Los AP, de Widt J, van Blitterswijk WJ,
Divecha N. Is there a role for diacylglycerol
kinase-zeta in cell cycle regulation? Adv
Enzyme Regul 2008;48:31-9
Van Blitterswijk WJ, Verheij M. Anticancer
alkylphospholipids: mechanisms of action,
cellular sensitivity and resistance, and
clinical prospects. Curr Pharm Des
2008;14:2061-74
LIPID METABOLISM IN SIGNAL TRANSDUCTION
Membrane lipids have both structural and signaling functions. Plasma membrane
sphingolipids play a role in apoptosis and they tend to self-associate and cluster with
cholesterol in dynamic microdomains known as lipid rafts. Inositol phospholipids
have signaling functions in vesicular trafficking, cellular stress and survival. In this
context we study mechanisms of tumor cell sensitivity and (cross-)resistance to
various apoptotic stimuli. In a second project, we investigate how exogenous shortchain
sphingolipids enhance the anti-cancer action of doxorubicin and related
amphiphilic drugs in vitro and in vivo.
Figure 8:
Interconversion of phosphoinositides by
PI3-kinase, PTEN and SHIP
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 is
accompanied by cross-resistance to other apoptotic stimuli such as DNA damage and
Fas/CD95 ligation. Resistant cells were defective in raft-mediated endocytosis and in
sphingomyelin synthesis (SMS1), and showed downregulated SHIP1, a PIP 3
5-phosphatase, resulting in upregulation of the PI3-kinase/PKB survival pathway and
increased tyrosine-phosphorylated proteins. We currently investigate the common
mechanism underlying the cross-resistance to multiple apoptotic stress stimuli.
For clinical applications of APLs, see the report by M 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 carcinoma cell lines (e.g. A431), but not in
normal cardiac myoblasts. We tested these liposomes in nude mice that were
subcutaneously inoculated with A431 cells. In a dose-escalation study, the anti-tumor
efficacy of GC-modified liposomal DOX was superior to standard liposomal DOX.
Increased drug delivery to tumor cells was also observed by intravital microscopy in
mice with a tumor implanted in a dorsal skin flap window chamber (collaboration
with G Koning, Erasmus MC, Rotterdam). This new liposomal formulation of DOX,
which we have patented, undergoes further testing and seems promising for clinical
application.
Figure 9: APL-resistant S49 AR cells show decreased
SHIP1 and Fas expression and constitutively activated
PKB/Akt (Western blots). Bottom panel shows decreased
cell surface expression of Fas (immunofluorescence).

CELL BIOLOGY II
ANTIGEN PRESENTATION BY MHC CLASS I AND MHC CLASS
II MOLECULES
Our aim is to understand how the adaptive immune system is regulated and to find
both targets and leads for modifying it. MHC class I and MHC class II molecules
control the activities of T cells. These T cells can then stimulate the immune system
or activate it to kill tumor cells and other cells. This is used in various immunotherapy
strategies to eliminate cancers. Our aim is to supply the fundamental framework
for such strategies. We use a large variety of state-of-the-art technologies like single
cell biochemistry, chemistry and high-throughput screening to achieve this goal.
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 genetically switched. This yields a living
pulse chase system that can be used to determine half-lives of (tagged) proteins.
We are crossing in yeast mutant strains to identify the cell ’s biological pathways
controlling degradation of the proteasome. Using two switchable tag systems, the
stability of large protein complexes can be determined. We have established this
system and are analyzing the stability within the 20S core of the proteasome and the
stability of the 26S proteasome by inserting the switch cassettes at different sites in
the molecule.
Gap junctions, apoptosis and cross-presentation The proteasome generates
peptides for MHC class I molecules. How classical antigen presentation by MHC
class I molecules is achieved is well established. However, peptides can be presented
by antigen presenting cells such as dendritic cells when made by other cells. This
process is called cross-presentation. We have shown that one way to get crosspresentation
is by transferring peptides from an infected cell to other cells through
gap junctions. We have tested whether antigen (cross)-presentation proceeds in cells
sentenced to apoptosis. 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. This provides one explanation for presentation of
antigens from apoptotic cells.
Radiation and adaptive immune responses, new combination therapies We
have shown that ionizing radiation induces various responses on cells including
protein degradation, mTOR activation and production of new proteins. Together this
results in higher MHC class I expression and better CTL responses against tumors in
tissue culture and in mice. We have identified four ‘radiation-specific’ peptides
including that of an Fbox protein. This Fbox protein supports cell growth under low
radiation exposure and silencing arrests cells in the G1-S phase. Since Fbox proteins
target substrates for ubiquitination and destruction, we are currently defining the
target(s) using proteomics combined with over-expression studies. This will yield
potential proteins responsible for selective sensitivity to radiotherapy in patients
which will be tested once the target is identified.
Endosomes and MHC class II antigen presentation MHC class II molecules
control the formation of antibodies and other aspects of the immune system like CTL
responses against infections and tumors. These MHC class II molecules meet
peptides for antigen presentation somewhere in the endocytic pathway. How
endosomes move and fuse is only partly understood. We have identified how the
dynein motor protein binds to endosomes (through the Rab7-RILP motor receptor)
and how this interaction is controlled (through ORP1L that can interact with the ER
protein VAP). The VAP-ORP1L interaction is controlled by cholesterol in late
31
CELL BIOLOGY II
Division head, group leader Jacques Neefjes
Jacques Neefjes PhD Group leader
Coen Kuijl PhD Post-doc
Victoria Menendez PhD Post-doc
Veronica Monserrat 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
Xiaohang Qiao MSc PhD student
Izhar Salomon MSc PhD student
Rik Van der Kant MSc PhD student
Lennert Janssen BSc Technical staff

32
CELL BIOLOGY II
Publications
Hodge JW, Guha C, Neefjes J, Gulley JL.
Synergizing radiation therapy and
immunotherapy for curing incurable
cancers. Opportunities and challenges.
Oncology (Williston Park) 2008;22:1064-70
Koppers-Lalic D, Verweij MC,
Lipinska AD, Wang Y, Quinten E,
Reits EA, Koch J, Loch S, Rezende MM,
Daus F, Bienkowska-Szewczyk K,
Osterrieder N, Mettenleiter TC,
Heemskerk MH, Tampe R, Neefjes JJ,
Chowdhury SI, Ressing ME, Rijsewijk FA,
Wiertz EJ. Varicellovirus UL 49.5 proteins
differentially affect the function of the
transporter associated with antigen
processing, TAP. PLoS Pathog
2008;4:e1000080
Neijssen J. The Fate of Intracellular
Peptides and MHC Class I Antigen
Presentation. Leiden University, 2008
Rocha N, Neefjes J. MHC class II
molecules on the move for successful antigen
presentation. EMBO J 2008;27:1-5
van den Hoorn T, Neefjes J. Activated
pDCs: open to new antigen-presentation
possibilities. Nat Immunol 2008;9:1208-10
endosomes and explains why high levels of endosomal cholesterol induce endosome
clustering as the result of dynein motor activity. In effect, our model is the first
explanation of control of motor protein activities on intracellular vesicles.
We are currently testing whether fusion and transport of endosomes could be
coupled processes and how these are controlled.
Genome-wide cell biology of new factors in MHC class II antigen presentation
To get more insight into the cell biological processes controlling MHC class II
antigen presentation, a FACS based genome-wide siRNA screen has been performed
using two antibodies labeled with Cy3 and Cy5 respectively. One antibody monitored
MHC class II expression levels whereas the other monitored the efficiency of antigen
loading. After some 100.000 2-color FACS analyses and cross-correlation with
expression profiles of primary human B cells, monocytes and dendritic cells
(either resting or activated), 255 hits were identified as being involved in MHC
class II expression or peptide loading.
To understand these hits (and as an additional way to validate our data), we have
established high-throughput techniques to arrive at pathways. To identify the hits
involved in controlling transcription of MHC class II molecules, they were again
silenced by siRNA and expression of the transcription master regulator CITA, its
substrate MHC class II and the invariant chain were determined by qPCR. To further
subdivide potential hits, transcription of MHC class I molecules was also determined
as hits may affect the MHC locus rather than MHC class II expression only. This
analyses yielded some 10 factors and cross-qPCR is currently been performed to test
whether pathways controlling the expression of transcription regulators (CIITA) can
be identified.
Along the same token, we have silenced the hits in cells expressing Golgi markers
and MHC class II-GFP molecules and subsequently fixed and stained early
endosomes. For color confocal images are made and analyzed by ‘CellProfiler’ and
other software programs to identify similar phenotypes. This yielded additional
clusters and first hints on novel signaling pathways controlling MHC class II
expression. In total we can explain the function of some 140 out of 255 hits most of
which cluster in only 4 networks. How these pathways work is currently studied
using yeast-2 hybrid, proteomics and other techniques.
As a spin-off, we identified some 30 secreted factors not expressed in human primary
immune cells. These factors may locally control MHC class II immune responses
and are currently made to test this on primary human immune cells.
Expanding the PKB/Akt1 pathway using bacterial infection systems We have
shown that Salmonella activates the PKB/Akt1 pathway to survive intracellularly.
In fact, a series of kinases in this pathway were activated for this purpose which are
almost all involved in cancer as well. We argued that testing the effects of
phosphatases on Salmonalla infection by siRNA would yield proteins that probably
target the kinases already identified in our previous study. We first identified the
phosphatases present in the human genome and then selected the corresponding
siRNAs. Using these we identified a series of phosphatases, some of which were
already defined as controlling PKB/Akt. Since esterases are homologous to
phosphatases, we also identified two esterases. Simultaneously, Huib Ovaa’s group
constructed a chemical phosphate-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.

ESTROGEN RECEPTOR AND BREAST CANCER
Estrogen Receptor a (ERa)-positive breast cancer patients are commonly treated with
tamoxifen. 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 anti-estrogens, all with compound specific properties and behavior.
The clinical benefits of clarifying pathways underlying resistance can therefore be
profound. We aim to get molecular understanding of tamoxifen and other antiestrogen
resistance mechanisms and to develop tools to use as clinical decision
markers in anti-estrogen therapy of breast cancer patients.
Structural requirements in ERa for tamoxifen resistance We found that 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 hinge, ERa can induce transcription in the
presence of its antagonist tamoxifen, a capacity which is lacking in ERb.
Modifications in ER associated with resistance to anti-estrogens We have
identified different combinations of ERa modulation by phosphorylation and/or
overexpression of its cofactors, which affect the response to specific 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 endocrine
treatment.
Overexpression of one of the three SRC-cofactors, SRC-2, predicted a poor response
in a randomized trial that evaluated adjuvant tamoxifen treatment versus nil and was
the cause of tamoxifen resistance in experimental studies.
A total profile of modifications of ER that lead to resistance to anti-estrogens is
presented in figure 1.
In contrast to ERa, phosphorylation of ERb by either Protein Kinase A or
MAPkinase, did not result in resistance to tamoxifen.
Gene signature of ERa phosphorylated at Serine 305 by Protein kinase A Since
phosphorylation of Serine 305 in ERa is causal for tamoxifen resistance, a gene
profile was determined under these conditions. Microarray experiments on U2OS
cell lines expressing ERa wt or a 305A mutant that does not become phosphorylated
at that site, show specific gene targets of a tamoxifen bound ERa phosphorylated at
Ser305 by Protein Kinase A. The results suggest an association between tamoxifen
resistance by 305P and cell movement. Furthermore, phosphorylation at Serine 305
in the presence of tamoxifen seems to affect the non-classical ER targets, rather than
ERE regulated genes. Specific targets of a tamoxifen bound ERa phosphorylated at
Serine 305 by Protein Kinase A were confirmed by both qPCR and an independent
microarray experiment.
Clinical relevance of phosphorylation of Serine 305 of ERa In collaboration with
Marleen Kok and Sabine Linn (Division of Molecular Biology) and Goran Landberg
and colleagues (University of Lund), we found that 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 a substantial fraction of ER-positive breast cancer that had poor
outcome after tamoxifen treatment.
Application of the laboratory findings of how modulation of ER and its cofactors
affects sensitivity towards anti-estrogens will lead to a tailoring of endocrine
treatment of breast cancer patients.
Group leader Rob Michalides
Rob Michalides PhD Group leader
Mariska Rondaij PhD Post-doc
Renée De Leeuw MSc PhD student
Wilbert Zwart MSc PhD student
Cristiane Bentin Toaldo BSc Technical staff
Désirée Verwoerd BSc Technical staff
Publication
33
CELL BIOLOGY II
Holm C, Kok M, Michalides R, Fles R,
Koornstra R, Wesseling J, Hauptmann M,
Neefjes J, Peterse J, Stal O, Landberg G,
Linn S. Phosphorylation of the oestrogen
receptor alpha at serine 305 and prediction of
tamoxifen resistance in breast cancer.
J Pathol 2008 (Epub ahead of print)
Figure 1: ERa resistance profile by
phosphorylation and cofactor overexpression.
Graphic representation of antiestrogen
sensitivity towards phosphorylation events on
ERa (A) in possible conjunction with SRC-1
and/or Cyclin D1 overexpression (B). The
antiestrogen inhibits the ERa (black) unless
the correct requirements are met to induce
resistance against the compound (white).

34
CELL BIOLOGY II
Group leader Huib Ovaa
Huib Ovaa PhD Group leader
Boris Rodenko PhD Post-doc
Silvia Cavalli PhD Post-doc
Farid El Oualid PhD Post-doc
Anitha Shanmugham PhD Post-doc
Celia Berkers MSc PhD student
Rieuwert Hoppes MSc PhD student
Harald Albers MSc PhD student
Henk Hilkmann Ing Technical staff
Karianne Schuurman Msc Research assistant
Annemieke de Jong MSc Research assistant
Publications
Bakker AH, Hoppes R, Linnemann C,
Toebes M, Rodenko B, Berkers CR,
Hadrup SR, van Esch WJ, Heemskerk
MH, Ovaa H, Schumacher TN.
Conditional MHC class I ligands and
peptide exchange technology for the human
MHC gene products HLA-A1, -A3, -A11,
and -B7. Proc Natl Acad Sci U S A
2008;105:3825-30
Berkers CR, de Jong A, Ovaa H, Rodenko B.
Transpeptidation and reverse proteolysis
and their consequences for immunity.
Int J Biochem Cell Biol 2009;41:66-71.
Kraus M, Malenke E, Gogel J, Muller H,
Ruckrich T, Overkleeft H, Ovaa H,
Koscielniak E, Hartmann JT, Driessen C.
Ritonavir induces endoplasmic reticulum
stress and sensitizes sarcoma cells toward
bortezomib-induced apoptosis. Mol Cancer
Ther 2008;7:1940-8
Ovaa H, van Leeuwen F. Chemical
Biology Approaches to Probe the Proteome.
Chembiochem 2008;9:2913-2919
CHEMISTRY AND BIOLOGY OF 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: MHC class I-mediated immunity: antigen processing, -loading -presentation and the T cell kill.
The proteasome is responsible for protein degradation assisted by other (endo)-proteolytic activities
affording eptitopes that can be loaded into MHC class I complexes which may result in a T cell-mediated
kill of the antigen presenting cell.
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 posttranslational
modifications, and determination of transcript levels with similar
limitations. No single ideal technique 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 should
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 cellbased
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) phosphate ester hydrolysis
(4) MHC class I antigen presentation
Figure 3: Docking molecules into the MHC class I pocket
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 mouse, 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,
4-37oC). 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.
Future directions Improvements and development of new technologies to profile
enzymatic UPS-related activities and MHC loading will allow new approaches to
understanding the ubiquitin proteasome system and antigen presentation,
unravelling 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.
Publication (continued)
35
CELL BIOLOGY II
Piva R, Ruggeri B, Williams M, Costa G,
Tamagno I, Ferrero D, Giai V, Coscia M,
Peola S, Massaia M, Pezzoni G, Allievi C,
Pescalli N, Cassin M, di Giovine S,
Nicoli P, de Feudis P, Strepponi I,
Roato I, Ferracini R, Bussolati B,
Camussi G, Jones-Bolin S, Hunter K,
Zhao H, Neri A, Palumbo A, Berkers C,
Ovaa H, Bernareggi A, Inghirami G. CEP-
18770: A novel, orally active proteasome
inhibitor with a tumor-selective
pharmacologic profile competitive with
bortezomib. Blood 2008;111:2765-75
Raijmakers R, Berkers CR, de Jong A,
Ovaa H, Heck AJ, Mohammed S.
Automated online sequential isotope
labeling for protein quantitation applied to
proteasome tissue-specific diversity. Mol Cell
Proteomics 2008;7:1755-62
Shanmugham A, Ovaa H. DUBs and
disease: activity assays for inhibitor
development. Curr Opin Drug Discov Devel
2008;11:688-96
Figure 4: MHC exchange. A. Principle of
MHC class I-exchange and exchange-based
screening assay based on fluorescence
polarization (FP). B. A modified influenza
A epitope is shown, designed to disintegrate
upon UV exposure. The UV-cleavable
2-nitrophenyl based b-peptide and cleavage
products are depicted in red.

36
CELL BIOLOGY II
Group leader Peter Peters
Peter Peters PhD Group leader
Nicole Van der Wel PhD Associate staff
scientist
Sue Godsave PhD Post-doc -
EU project-manager
Pekka Kujala PhD Post-doc
Musa Sani PhD Post-doc
Matthijn Vos PhD Post-doc
Diane Houben MSc PhD student
Jason Pierson MSc PhD student
Karin De Punder BSc Technical staff
Hans Jansen BSc Technical staff
Maaike Van Zon BSc Technical staff
Nico Ong Research assistant
Collaborators
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
Henny Zandbergen: Department of
Physic, Technical University, Delft
Helmut Gnaegi: Diatome Ltd, Biel,
Switzerland
NANOBIOLOGY
One focal point of our structural biology group is to reveal and manipulate the
macromolecular organisation of cells under normal and pathogenic conditions at the
nano-scale level. We use cryo-electron tomography of vitreous sections, currently the
only method that can obtain molecular resolution of molecular machines in cells in a
near-native situation. The tomograms contain a 3D map of the cellular proteome at
about 4 nm resolution and we are just beginning to explore its potential by placing
high effort on developing methods for our nanotechnology. The other central point of
our group is to visualise gene products in cells by electron microscopy at the highest
resolution with gold probes on cryo-sections.
Nanomachines in cells Cryo electron tomography of vitreous sections is becoming
an established technique to study the three-dimensional structure of molecular
machines in situ. Specifically we have been looking at the 80S eukaryotic ribosome
within the cell. Using high-resolution tomography of unfixed frozen hydrated cryosections
the structure and relative orientation of the ribosome, in a cellular context,
has been established by semi-automatic particle selection, volumetric alignment and
averaging. With this template a ‘molecular atlas’ was constructed, mapping the
ribosome over the entire cellular volume within a thin vitreous section of a cell (see
figure 5). This study illustrates the possibilities of this nanotechnology towards
visualizing cellular machinery in situ at macromolecular resolutions in large
eukaryotic cells. Future challenges 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.
Figure 5: Constructing a
Cellular Atlas of 80S
Ribosomes A 10 nm slice
through a reconstructed volume
displaying cellular
macromolecular machines,
specifically the 80S Ribosome.
A vacuole (V) and a
Mitochondrion (M) can also be
seen within the image. Upper
Inset. High-resolution density
map of the averaged 80S
Ribosome. Lower Inset.
A select area from the cellular
Ribosomal atlas. Scale Bar,
100 nm.
Recombinant BCG and its translocation In collaboration with the group of
Michael Brenner (Harvard, Boston) we published last year that after prolonged
infection in macrophages and dendritic cells, M. tuberculosis translocates from
phago-lysosomes to the cytosol (Cell. 2007 Jun 29;129(7):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. 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(11):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 the structure of ESX-1 secretion systems
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
(PrP Sc ) of the cellular prion protein (PrP C ). The subcellular site where PrP Sc
is formed is still unclear. In collaboration with the lab of Stan Prusiner (UCSF, San
Francisco) we have used quantitative cryo-immunogold electron microscopy to localise
different populations of PrP on hippocampal sections from prion-infected mice.
At a late subclinical stage of prion infection we detected a 6 fold overall increase in
PrP levels in the stratum oriens. The biggest increase (24 fold) occurred on vesicles
resembling early endocytic or recycling vesicles in small neurites in the neuropil,
suggesting that these vesicles are important in PrP Sc formation or pathology.
We are also 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.
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(7165):1003-7). We have now established longterm
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.
Publications
37
CELL BIOLOGY II
Godsave SF, Wille H, Kujala P, Latawiec D,
DeArmond SJ, Serban A, Prusiner SB,
Peters PJ. Cryo-Immunogold Electron
Microscopy for Prions: Toward
Identification of a Conversion Site. J
Neurosci. 2008;28:12489-12499
Hava DL, van der Wel N, Cohen N,
Dascher CC, Houben D, León L, Agarwal S,
Sugita M, van Zon M, Kent SC, Shams H,
Peters PJ, Brenner MB. Evasion of peptide,
but not lipid antigen presentation, through
pathogen-induced dendritic cell maturation.
Proc Natl Acad Sci U S A.
2008;105:11281-6
Peters PJ, Pierson J. Immunogold labeling
of thawed cryosections. Methods Cell Biol.
2008;88:131-49
Abdallah AM, Savage ND, van Zon M,
Wilson L, Vandenbroucke-Grauls CM,
van der Wel NN, Ottenhoff TH, Bitter W.
The ESX-5 secretion system of
Mycobacterium marinum modulates the
macrophage response. J Immunol.
2008;181:7166-75
Verbrugghe P, Kujala P, Waelput W,
Peters PJ, Cuvelier CA. Clusterin in human
gut-associated lymphoid tissue, tonsils, and
adenoids: localization to M cells and
follicular dendritic cells. Histochem Cell
Biol. 2008;129:311-20
Zhang L, Lee SY, Beznoussenko GV,
Peters PJ, Yang JS, Gilbert H, Brass AL,
Elledge SJ, Isaacs SN, Moss B, Mironov A
and Hsu VW. Coatomer and KDEL
Receptor: Two host proteins implicated in
early vaccinia biogenesis. PNAS (in press)
Van der Flier L, Van Gijn M, Hatzis P,
Kujala P, Haegebarth A, Stange D,
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
(Ascl2) controls intestinal stem cell fate.
Cell (in press)

38
EXPERIMENTAL THERAPY
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
Simon Joosse PhD student
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
Hasibe Iri-Kiraz Technical staff
Sten Cornelissen Technical staff
Izabela Mikolajewska Technical staff
Kim Brandwijk Technical staff
Lennart Mulder Technical staff
Publications
Broeks A, Braaf LM, Huseinovic A,
Schmidt MK, Russell NS, Van Leeuwen FE,
Hogervorst FB, Van 't Veer LJ. The
spectrum of ATM missense variants and
their contribution to contralateral breast
cancer. Breast Cancer Res Treat.
2008;107:243-8
Horlings HM, van Laar RK, Kerst JM,
Helgason HH, Wesseling J,
van der Hoeven JJ, Warmoes MO,
Floore A, Witteveen A, Lahti-Domenici J,
Glas AM, Van 't Veer LJ, de Jong D. J Clin
Gene expression profiling to identify the
histogenetic origin of metastatic
adenocarcinomas of unknown primary.
Oncol. 2008;26:4435-41
EXPERIMENTAL THERAPY
MOLECULAR PATHOLOGY AND MOLECULAR EPIDEMIOLOGY
OF BREAST AND COLORECTAL CANCER
Genome-wide genomic profiling in hereditary breast cancer (PI P Nederlof )
We have shown that breast tumors from BRCA1 and BRCA2 mutation carriers show
specific chromosomal changes, and that 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 (www.sanger.ac.uk) with routinely formalin-fixed
paraffin embedded (FFPE) tumor material. We showed that in a series of 48 breast
tumors from BRCA1/2 negative breast and ovarian cancer (HBOC) families only two
tumors showed a BRCA1-like profile, and we could confirm the inactivation of
BRCA1 in one case (promoter methylation). Currently we apply the classification
strategy to gain additional proof of significance for unclassified variants (UVs); gene
alterations for which the pathogenicity is still unclear. In collaboration with Dr. P
Devilee (Leiden University) we are collecting several breast cancer cases per family
from BRCA1/2 negative HBC families (BRCAx). Preliminary results on 60 cases
have shown that BRCAx tumors are heterogeneous but that within families the
differences are smaller. This finding will be used in a larger set of tumors to
determine different BRCAx subgroups that may allow linkage analyses within the
subgroups, in order to find additional breast cancer genes.
Molecular profiling for breast cancer classification and therapy response
Half of the recurrences of ERa+ breast tumors respond to tamoxifen while the other
half is resistant. Recently, an 81-gene tamoxifen response profile for advanced disease
has been published (Jansen et al, JCO 2005). We validated this response profile on a
more advanced whole genome platform and in an independent set of tumors,
including stable disease (initially excluded). Additionally, an even more robust
algorithm for tamoxifen response predicting is under development. Finally, these
profiles are now being tested for clinical use in the adjuvant setting. In addition, we
genotyped patients for SNPs in enzymes involved in the metabolism of tamoxifen,
the cytochrome P450 (CYP) familie. In collaboration with Dr. Els Berns (Erasmus
Medical Center) we showed that a SNP in CYP2C19 may result in a altered tamoxifen
response. The ability to accurately predict tamoxifen treatment outcome would
constitute a significant advance in the management of breast cancer.
Genetic determinants of breast cancer risk and outcome
In a retrospective nationwide cohort of Dutch breast cancer patients (BOSOM=
Breast Cancer Outcome Of Mutation carriers) as well as within a large international
collaboration (BCAC = breast cancer association consortium), we are investigating
which germline variants (pathogenic mutations in e.g. BRCA1/2 as well as SNPs)
influence breast cancer risk and outcome.
Molecular profiling of bilateral breast tumor pairs
With the increased incidence of breast cancer and the improved survival, studies on
breast cancer outcome have become an important issue. This is especially true for
young women who potentially have a long life expectancy after breast cancer, but also
a high risk of developing a contralateral breast cancer (CBC) compared to older
patients. Certain SNPs have been shown to increase the risk for the development of
breast cancer and to predispose to a certain tumor subtype (e.g basal-like), as is seen
in BRCA1 carriers. We are investigating which SNPs predispose to an increased risk
for the development of a CBC and whether part of this risk can be predicted by the
tumor subtype of the first cancer.
Validation of Adjuvant! Online
Nowadays, adjuvant treatment recommendations for early stage breast cancer
patients are based on the estimated risk of breast cancer relapse and death and the
expected benefit of adjuvant treatment. Several tools have been developed to make

estimates for the individual patient. One of these tools is Adjuvant! Online
(www.adjuvantonline.com), which calculates a 10-year survival probability based on
the patient’s age, tumor size, grade and estrogen receptor. In addition, the computer
program also calculates the likely benefit to be expected from adjuvant systemic
treatment. Adjuvant! Online is already being used by physicians. In addition, in the
MINDACT trial the clinical risk is calculated by Adjuvant! Online. To assess the
validity of Adjuvant! Online for Dutch breast cancer patients we are comparing
predicted and observed disease outcome in 2 Dutch cohorts of approximately 10000
patients in total.
Validation of the 70-gene prognosis-signature in breast cancer
subpopulations
This validation study provides evidence that the 70-gene 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
The detection of circulating tumor cells in the blood of cancer patients is a promising
tool for risk stratification. We previously developed a multi-marker mRNA QPCRbased
detection platform for the semi-quantitation of tumor cell load in the peripheral
blood of breast cancer patients. Recently we validated this assay in collaboration with
the Radiumhospitalet in Oslo, Norway (B Naume), assaying a retrospective series of
107 early-stage breast cancer patients from which blood was collected at time of
diagnosis and stored frozen (median follow-up 7.4 years). 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. The assay was
particularly powerful in lymph node negative patients (hazard ratio = 13.0 for relapsefree
survival and 6.3 for overall survival, p < 0.01) and in multivariate analyses circulating
tumor cell positivity was shown to be a significant and independent risk factor with
respect to 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
behaviour after surgery. The ability to predict liver metastasis and understanding of
their clinical behaviour 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 1. We aim to develop a gene expression profile for the
risk of LR in rectal cancer patients, allowing for selection of patients for preradiotherapy
(RT). And, by establishing additional profiles for nodal status and
circumferential margin, further refinement of the neo-adjuvant treatment can be
achieved. 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 rectal cancer patients were randomized for short-term pre-RT followed by
TME or TME alone. RNA isolation was prepared and gene expression array will be
performed on 200 samples. 2. By using PCR/sequencing on parallel extracted DNA,
we also determined PIK3CA (exons 9 and 20), K-Ras (exon 1) and BRAF V600E
mutations in 19 (7.9%), 81 (33.9%) and 5 (2.1%) rectal cancers, respectively.
Interestingly, PIK3CA mutations revealed a strong association with increased LR.
39
EXPERIMENTAL THERAPY
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.
2008 Aug 14. (Epub ahead of print)
Kok M, Linn SC, Van Laar RK, Jansen MP,
Van den Berg TM, Delahaye LJ et al.
Comparison of gene expression profiles
predicting progression in breast cancer
patients treated with tamoxifen. Breast
Cancer Res Treat 2008 Jul 27. (Epub ahead
of print)
Molloy TJ, Bosma AJ, Van 't Veer LJ.
Towards an optimized platform for the
detection, enrichment, and semiquantitation
circulating tumor cells. Breast
Cancer Res Treat. 2008;112:297-307
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; On behalf of the
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 2008 Jul 27
Reyal F, Van Vliet MH, Horlings HM,
Armstrong N, De Visser K, Kok M,
Meuleman W, Teschendorff A, Mook S,
Caldas C, Salmon R, Van de Vijver MJ,
Wessels LFA. A comprehensive analysis of
nine prognostic signatures reveals the high
predictive capacity of Proliferation, Immune
response and RNA splicing modules in
breast cancer. Breast Cancer Res.
2008;10:R93
Van 't Veer LJ, Bernards R. Enabling
personalized cancer medicine through
analysis of gene-expression patterns. Nature.
2008;452:564-70 (review)
Weigelt B, Horlings HM, Kreike B,
Hayes MM, Hauptmann M, Wessels LF,
de Jong D, Van de Vijver MJ,
Van 't Veer LJ, Peterse JL. Refinement of
breast cancer classification by molecular
characterization of histological special types.
J Pathol. 2008;216:141-50

40
EXPERIMENTAL THERAPY
Group leader Adrian Begg
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
Ben Floot Technical staff
Ingrid Hofland Technical staff
Manon Verwijs Technical staff
MECHANISMS AND PREDICTION OF TUMOR RESPONSE TO
RADIATION
We are pursuing two research lines, one on fundamental aspects of the radiation
response and the other aiming more directly at clinical translation. 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 outcome of radiotherapy, particularly using
genome wide screening methods, which has also indicated some interesting potential
targets.
Mechanisms and modulation of radiosensitivity
Possible involvement of polbDN in DSB induction or repair
We have shown that 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. More residual
double strand breaks (DSB) after irradiation were found in polbDN-expressing cells
than in vector controls, and we therefore tested polbDN’s involvement in DSB
induction and repair. Studies with a DNA-PK inhibitor indicated no involvement of
non-homologous endjoining (NHEJ) in radiosensitization by the polbDN, supporting
previous data on DNA repair mutant cell lines. Premature chromosome
condensation (PCC) assays indicated secondary double strand break formation after
the initial immediate radiation-induced breaks, which was independent of DNA-PK
activity. Interestingly, these extra breaks were not accompanied by gH2AX foci.
Irradiation induced more chromosome and chromatid type aberrations in polbDNexpressing
cells than in wildtype cells, but an increase in only chromatid type
aberrations was seen after H 2O 2, also an oxidative damage inducer. This indicates a
role for clustered damage, unique to ionizing radiation, in polbDN-induced
radiosensitization.
We further showed a replication dependence of polbDN radiosensitization, since
sensitization was less in confluent than in log phase cells, and could be modeled
assuming only S/G2 cells were sensitized. This is in contrast to polb-deficient
(knockout) cells, which were more radiosensitive only under confluent conditions or
in G1 log-phase cells. This is presumed to be partly due to the extra inhibition of long
patch repair by the polbDN, and the role and availability of the various backup
pathways.
Figure 1: Expressions of a DNA polymerase beta
dominant negative (polbDN), known to inhibit
bae excision repair, causes increased
chromosome aberrations after irradiation
(upper panel), indicating formation of
additional double strand breaks. This is
probably the cause of the observed
radiosensitization. These extra double strand
breaks do not appear to signal the formation of
gH2AX foci (lower panel).

Testing the hypothesis that BER-deficient cells are sensitive to HR inhibition
Synthetic lethality has been shown when both homologous recombination (HR) and
base excision repair (BER) are inhibited, arising from BRCA mutations and PARP
inhibition. We hypothesized that this synthetic lethality would also arise if cells with
mutations in the BER pathway, frequent in human tumors, are treated with HR
inhibitors. We are currently testing this hypothesis. 17AAG has been shown to reduce
BRCA2 and RAD51, thus affecting HR. Indeed, polDN-expressing cells (BER
deficient) were radiosensitized more by 17AAG than wildtype cells. Experiments with
XRCC1-knockout and polb-knockout cells (BER deficient) are underway, including
treatment with caffeine and other putative HR-associated inhibitors.
To look for novel HR or related inhibitors, we have set up a drug screening assay
using the in-house robotics facility, with the goal of finding drugs which
radiosensitize BER deficient more than proficient cells. This would provide tumor
specific radiosensitization for those tumors exhibiting BER defects. For this, human
tumor cells with and without expression of the polbDN were plated and irradiated in
384-well plates. To assess post-irradiation growth, a automated microscopy based cell
count assay was found to be far more sensitive than the CellTiter blue viability assay.
In order to determine survival accurately after irradiation, assay times longer than 6
days were necessary. We have begun to screen chemical libraries using this assay.
Prediction of outcome We want to understand and predict causes of failure after
radiotherapy, allowing selection of patients for alternative more effective therapies.
Last year we measured expression profiles of advanced (T3-4) head and neck cancers
(4 sites) prior to treatment, to search for genes which predict outcome in patients
treated with concurrent radiotherapy and cisplatin (collaboration with departments of
Head and Neck Surgery, Radiotherapy and Pathology). We found that a previously
published signature, reported to distinguish head and neck cancer patients at high
risk of either local or distant recurrence (Chung C et al, Cancer Res 2006), proved to
be a highly significant predictor of loco-regional recurrence after combined radiochemotherapy
in our patient series. We have now assessed the performance of this
signature in a multivariable analysis with other clinical factors on 75 tumors where
we also measured tumor volume at the time of treatment. The Chung signature
proved to be independent of tumor site, volume and T-stage, with the signature and
site being the most significant, yielding hazard ratios of around 4. This represents an
independent validation of a signature, which appears to be independent of known
clinical factors affecting outcome, further indicating its potential clinical usefulness.
We have also completed a study on a series of larynx tumors from patients treated
with radiotherapy alone and with less advanced tumors, representing a more
homogenous patient and treatment group. Each patient with a local recurrence was
matched for subsite and T-stage with one or two cured patients. This matched series
comprised 19 recurrences and 30 cures, all with T1 or T2 tumors, in which
pretreatment biopsies were taken and expression profiles measured (Illumina
platform). In an hypothesis-driven approach, we tested the predictive potential of
signatures known or suspected to affect response to radiotherapy. The putative stem
cell marker CD44 was the most significant predictor of outcome (local control), with
an acute hypoxia signature showing a strong trend. CD44 also emerged as the most
significant gene in a data-driven approach searching for any set of genes
distinguishing cures from recurrences. Signatures for intrinsic radiosensitivity and
proliferation were not significant. We are now comparing these mRNA expression
data with CD44 positivity assessed by immunohistochemistry. Further, we are
measuring expression profiles on 30 more advanced (T3) larynx tumors and some
oropharynx tumors, both treated by radiotherapy alone.
In parallel, in a search for an intrinsic radiosensitivity predictor, 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). No genes which changed expression in response to
irradiation were found which correlated with radiosensitivity. In contrast, a set of 288
genes, which did not change after irradiation (remained high or low), were found
which showed a significant correlation with intrinsic radiosensitivity. This represents
the largest such dataset on site-specific cell lines. We are further investigating and
validating the genes in this set.
Publications
41
EXPERIMENTAL THERAPY
Bartelink H, Begg AC. Prognostic and
predictive markers in oncology. Semin
Radiat Oncol. 2008;18:73-74
Vermeulen C, Verwijs-Janssen M, Begg AC,
Vens C. Cell cycle phase dependent role of
DNA polymerase beta in DNA repair and
survival after ionizing radiation. Radiother
Oncol. 2008;86:391-398
Vermeulen C, Bertocci B, Begg AC, Vens C.
Ionizing radiation sensitivity of DNA
polymerase lambda-deficient cells. Radiat
Res. 2007;168:683-688
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 2008 (in press)
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 2008 (in press)
Wouters BG, Begg AC. Irradiation induced
damage and the DNA damage response. In:
Basic Clinical Radiobiology, pp 18-35, 4th Edition 2008 (in press)
Begg AC. Molecular targeting and patient
individualization. In: Basic Clinical
Radiobiology, 4th Edition, pp 364-381, 2008
(in press)

42
EXPERIMENTAL THERAPY
Group leader Fiona Stewart
Fiona Stewart PhD Group leader
Nicola Russell 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
RADIATION-INDUCED VASCULAR DAMAGE
Improvements in detection and treatment of cancer have lead to increased numbers
of survivors at risk for developing treatment related morbidity. More than half of all
long-term survivors of cancer will have received radiotherapy as part of their
treatment. Radiotherapy has been shown to be an independent risk factor for
cardiovascular and cerebrovascular disease. 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 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.
These mice have increased cholesterol levels and develop atherosclerosis
spontaneously with age, unlike wild type mice. ApoE-/- mice given single doses (8-14
Gy) or clinically relevant fractionated irradiation (20 x 2 Gy in 4 weeks) to the neck,
developed increased atherosclerotic plaque in the carotid arteries compared with agematched
controls. The majority of lesions seen at 22-34 weeks after irradiation had an
inflammatory, thrombotic phenotype, with a thin fibrous cap. The inflammatory
phenotype was rare in unirradiated mice and lesions were protected by a thick
fibrous cap. Wild type C57Bl6 mice did not develop atherosclerotic plaque, although a
few (20%) did develop fatty streak lesions at 30 weeks after irradiation. These studies
showed that irradiation, in combination with elevated cholesterol, accelerated the
development of atherosclerosis and predisposed to the formation of a vulnerable,
inflammatory, thrombotic plaque phenotype with a thin fibrous cap.
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. Aspirin (ASA) and nitric oxide
releasing aspirin (NO-ASA) inhibited endothelial cell (EC) expression of the adhesion
molecule V-CAM and inhibited the formation of fatty streaks in these irradiated
arteries at 4 weeks after irradiation. NO-ASA, but not ASA, also inhibited the
formation of atherosclerotic lesions in unirradiated arteries at longer times (30 weeks)
but there was no significant reduction in the number or size of lesions in the irradiated
arteries. Clopidogrel and atorvastatin did not modify the development or progression
of atherosclerotic lesions in irradiated or inirradiated vessels.
In parallel clinical studies, we characterized radiation-induced vascular damage in
resection material collected from head and neck (H&N; n = 70) and breast cancer
patients (BC; n = 56) undergoing free-flap reconstructive surgery after previous
radiotherapy or surgery alone. Using imaging analysis software the ratio of the
thickness of the intima to the thickness of the media (IMR), was determined and
sections were scored for proteoglycan content and inflammatory cell content. For the
H&N cancer patients there was a 1.5-fold increase in the IMR at 4 years after
irradiation (mean 66 Gy). Inflammatory cell content was also increased in the intima
of the irradiated H&N vessels. For BC patients there was a 1.4-fold increase in IMR at
3 years after irradiation (mean 49 Gy), after correction for the IMR value of the free
flap artery, but this was not significant (p=0.072). There was a significant increase in
the proteoglycan content of the media in the irradiated mammary vessels. This study
demonstrated accelerated intimal thickening, increased proteoglycan content and
inflammatory cell content, in medium sized arteries at relatively short times after
radiation. These patients are likely to have an increased risk for development of
atherosclerosis and subsequent cerebrovascular or cardiovascular disease.
Radiation-induced telangiectasia (in collaboration with Peter ten Dijke, Leiden)
Radiation induces late vascular damage that manifests in capillaries as telangiectasia.
These are dilated, thin-walled blood vessels that may rupture thereby impairing organ
function and causing excessive bleedings. Radiation-induced telangiectasia strongly

esembles the symptoms of patients with hereditary hemorrhagic telangiectasia
(HHT). This autosomal dominant vascular disorder has been linked to mutations in
the genes encoding transforming growth factor beta (TGF-b) receptor ALK1 and the
co-receptor endoglin. Receptor haplo-insufficiency probably causes an imbalance
between endothelial cell proliferation and migration (mediated via endoglin and
ALK1 and their downstream target ID1) and vessel maturation (mediated via the
TGF-b receptor ALK5 and its downstream target PAI-1). In this study we therefore
aimed at elucidating the role of TGF-b as well as Notch signaling pathways in the
development of radiation-induced telangiectasia.
Irradiation of human microvascular endothelial cells in vitro resulted in reduced
TGF-b/ALK1 signaling, with decreased activation of the signal transducing molecules
Smad1 and Smad5 and diminished expression of the target gene ID1. TGF-b/ALK5
signalling was increased as evidenced by activation of Smad2 and Smad3 and
enhanced PAI-1 expression. Irradiation also activated the Notch signalling pathway
shown by increased expression of the Notch ligand Jagged-1 and the downstream
target gene Hey1. Functional studies showed that enhanced signalling through the
anti-angiogenic ALK5 and Notch pathway leads to decreased cell migration and tube
formation. Mechanistic and functional studies were further extended by analyzing
irradiated kidneys from mice sacrificed at different time points after irradiation.
Irradiation of wild type animals resulted in the development of telangiectasia.
Expression of PAI-1 increased progressively from 10-30 weeks, whereas Id-1
expression levels rose only transiently at 1 week after irradiation. Expression levels of
ALK1 and ALK5 steadily increased whereas activation of the signal transducing Smad
molecules changed during the course of the experiment. Further experiments
indicated that changes in activity of the respective signalling pathways might be
accomplished by altered expression of negative regulators. Endoglin levels were
found to be increased by irradiation, suggesting an attempt to repair the damaged
vasculature. As endoglin is known to have an impact on both ALK1 and ALK5
signalling, we studied its importance on irradiation-induced TGF-ß signalling and
telangiectasia development using endoglin heterozygous (Eng +/- ) mice. Eng +/- mice
developed less telangiectasia. Whether these vessels display functional differences
compared to the irradiated vasculature in wild type animals needs to be determined.
Both ALK1 and ALK5 signalling were affected in Eng +/- animals demonstrating the
importance of endoglin for both pathways in vivo. Future studies will address the
question of how endoglin influences TGF-b signalling after irradiation and whether
interfering with or promoting endoglin signalling is beneficial for irradiated cancer
patients.
Radiation induced cardiac damage (in collaboration with Christine Mummery,
Leiden) Microvascular damage and perfusion defects are major underlying causes of
heart failure after irradiation. This reflects inflammatory and thrombotic changes in
EC function, as well as apoptotic EC loss and reduced capillary density.
Revascularization of damaged heart tissue is essential to restore organ function and
prevent myocardial infarct. Endoglin is highly expressed in damaged ECs and it is
crucial for EC proliferation and vascular repair. In this project we will focus on the
role of endoglin in microvascular damage and repair in the irradiated hearts of both
wild type and endoglin deficient (Eng +/-) mice. In the first few months of this new
project we have set up the procedure for local irradiation of mouse hearts and made
base-line measurements of the heart volume. The imaging agent 99m Tc-labeled
albumin is confined to the venous system. MicroSPECT/CT imaging of its
distribution allows for a quantitative assessment of the percentage blood volume in
the heart (pre-treatment values of 11.25 ± 1.45%). Consequently, it provides an
accurate measure to study longitudinal changes in stroke volume. Alternatively,
ultrasound imaging of the blood vessel microstructure in the myocardial wall will be
performed using ultrasound and 99m Tc-sestamibi microSPECT/CT.
Publications
43
EXPERIMENTAL THERAPY
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 (in press)
Hendry JH, Akahoshi M, Wang LS,
Lipschultz SE, Stewart FA. Radiationinduced
cardiovascular injury. Radiat
Environ Biophys 2008;47:189-93
Hoving S, Heeneman S, Gijbels MJ,
te Poele JA, Russell NS, Daemen MJ,
Stewart FA. Single dose and fractionated
irradiation promote initiation and
progression of inflammatory atherosclerosis
in ApoE-/- mice. Int J Radiat Oncol Biol
Phys 2008;71:848-57
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 2008 (in press)
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 2008 (in press)
Stewart FA, Hoving, S, Kruse JJ.
Radiation-induced cardiovascular and
cerebrovascular effects: animal studies.
Radiat Res. 2007;167:350-2

44
EXPERIMENTAL THERAPY
Group leader Jan Schellens
Jan Schellens MD PhD Group leader
Jos Beijnen PhD Academic staff
Irma Meijerman PhD External staff
Serena Marchetti MD PhD Academic staff
Sander Veltkamp MSc PhD student
Roos Oostendorp MSc PhD student
Maarten Deenen MSc PhD student
Lot Devriese MD MSc PhD student
Suzanne Leijen MD PhD student
Dick Pluim Technical staff
Valerie Doodeman Technical staff
Publications
Damen CWN, Rosing H, Tibben MM,
Van Maanen MJ, Lagas JS, Schinkel AH,
Schellens JHM, Beijnen JH. A sensitive
assay for the quantitative analysis of
vinorelbine in mouse and human EDTA
plasma by high-performance liquid
chromatography coupled with electrospray
tandem mass spectrometry. J Chromatogr B
2008;868:102-09
Harmsen S, Koster A, Beijnen JH,
Schellens JH, Meijerman I. Comparison of
two immortalized human cell lines to study
nuclear receptor mediated CYP3A4 induction.
Drug Metab Dispos 2008;36:1166-71
Hoebers FJ, Pluim D, Hart AA, Verheij M,
Balm AJ, Fons G, Rasch CR, Schellens JH,
Stalpers LJ, Bartelink H, Begg AC.
Cisplatin-DNA adduct formation in
patients treated with cisplatin-based
chemoradiation: lack of correlation between
normal tissues and primary tumor. Cancer
Chemother Pharmacol 2008;61:1075-81
Marchetti S, de Vries NA, Buckle T, Pluim D,
Beijnen JH, Mazzanti R, Van Tellingen O,
Schellens JHM. Impact of the ABC-drug
transporters ABCB1, ABCG2 and ABCC2
on erlotinib disposition (Tarceva®) in in
vitro and in vivo pharmacokinetic studies
employing employing Bcrp1-/-/Mdr1a/1b-/-
(triple knockout) and wild-type mice. Mol
Cancer Ther 2008;7:2280-87
PHARMACODYNAMICS OF ANTICANCER DRUGS
Our pharmacology group has an interest in studies exploring anticancer drug
disposition, development of methods to improve oral pharmacokinetics (PK) of
anticancer drugs and development of novel pharmacodynamic (PD) methods to
support early clinical trials with anticancer drugs.
Role of ABC drug transporters in absorption and disposition of anticancer
drugs We performed a comprehensive preclinical pharmacokinetic study in wildtype,
P-gp and/or BCRP knockout mice. Our results reveal that P-gp and BCRP only
have modest effects on the absorption, distribution, metabolism and elimination of
imatinib in comparison to metabolic elimination in mice. Of these two drug
transporters, P-gp is the most dominant factor for systemic clearance and the most
important for limiting brain penetration of imatinib. Interestingly, co-administration
of the P-gp/BCRP inhibitor, elacridar, in mice leads to a 3-fold significant increase in
the systemic exposure to imatinib in the presence or absence of P-gp/BCRP. This
suggests that elacridar also inhibits other elimination pathways than P-gp and BCRP.
Previous studies have shown that the human Organic Cation Transporter 1 (OCT1) is
involved in the cellular uptake of imatinib. We show that elacridar inhibits OCT1 in
vitro. In addition, OCT1/2 has a significant effect on the pharmacokinetics of
imatinib in vivo by using OCT1/2 knockout mice. The considerable drug-drug
interaction observed with elacridar and imatinib is only partly mediated by inhibition
of OCT1/2. These uptake/elimination pathways can be important to explain the
observed variability in the clinical pharmacokinetics of imatinib.
We tested whether erlotinib hydrochloride (Tarceva, OSI-774), an orally active
epidermal growth factor receptor tyrosine kinase inhibitor, is a substrate for the ATPbinding
cassette drug transporters P-glycoprotein (P-gp; MDR1, ABCB1), breast
cancer resistance protein (BCRP; ABCG2), and multidrug resistance protein 2
(MRP2; ABCC2) in vitro and whether P-gp and BCRP affect the oral
pharmacokinetics of erlotinib hydrochloride in vivo. In vitro, erlotinib was actively
transported by P-gp and BCRP/Bcrp1. No active transport of erlotinib by MRP2 was
observed. In vivo, systemic exposure (p = 0.01) as well as bioavailability of erlotinib
after oral administration (5 mg/kg) were significantly increased in Bcrp1/
Mdr1a/1b(-/-) knockout mice (60.4%) compared with wildtype mice (40.0%; p =
0.02) (figure 1). Thus, erlotinib is transported efficiently by P-gp and BCRP/Bcrp1 in
vitro. In vivo, absence of P-gp and Bcrp1 significantly affected the oral bioavailability
of erlotinib. Possible clinical consequences for drug-drug and drug-herb interactions
in patients in the gut between P-gp/BCRP-inhibiting substrates and oral erlotinib
need to be addressed.
New insights into the pharmacology, biotransformation, and cytotoxicity of
gemcitabine and its metabolite 2’,2’-difluorodeoxyuridine We investigated the
in vitro cytotoxicity, cellular uptake, efflux, biotransformation, and nucleic acid
incorporation of dFdC and dFdU. dFdU monophosphate, diphosphate, and
triphosphate (dFdU-TP) were formed from dFdC and dFdU. dFdU-TP was
incorporated into DNA and RNA. The area under the intracellular concentration-time
curve of dFdC-TP and dFdU-TP and their extent of incorporation into DNA and RNA
inversely correlated with the IC(50) of dFdC and dFdU, respectively. The cellular
uptake and cytotoxicity of dFdU were significantly enhanced by hCNT1. dFdU
inhibited cell cycle progression and its cytotoxicity significantly increased with longer
duration of exposure. dFdU is taken up into cells with high affinity by hCNT1 and
phosphorylated to its dFdU-TP metabolite. dFdU-TP was incorporated into DNA and
RNA, which correlated with dFdU cytotoxicity. These data provide strong evidence
that dFdU can significantly contribute to the cytotoxicity of dFdC.
DPYD pharmacogenetics in patients with metastatic colorectal cancer treated
with capecitabine We are exploring the extent to which polymorphisms in DPYD
contribute to development of severe toxicity in patients treated with capecitabine.
Germline DNA was available from 576 patients with metastatic colorectal treated
with capecitabine-based first line chemotherapy participating in the CAIRO2 study.
All 23 exons of the entire DPYD gene were sequenced in 50 patients who suffered

from severe (grade 3/4) toxicity during the first two courses of treatment. The control
group consisted of 100 patients selected at random from our study population.
Polymorphisms that significantly differed between cases and cohort were assessed in
the total population of 576 patients.
A total of 29 different SNPs were detected in DPYD. The allele frequencies of the
polymorphisms DPYD*2A and A2846T differed significantly between cases and
cohort (6.0% vs 0.5% and 3.0 vs 0.0%, respectively). Assessment of these SNPs in
the total population revealed that DPYD*2A and A2846T were significantly
associated with grade 3/4 diarrhea (71% and 57%, respectively) and overall grade 3/4
toxicity (100% and 71%, respectively). Furthermore, the average cumulative
capecitabine dose per course was significantly lower compared to patients who are
wildtype for these SNPs, with the lowest average cumulative doses in course number
three being 49% and 63%, respectively.
Pharmacogenomic and pharmacokinetic safety and cost analysis in patients
treated with 5-FU / capecitabine Retrospectively, we could identify the presence of
the DPYD*2A polymorphism in 13 patients all suffering from severe toxicity with one
toxic death. Prolonged hospitalization periods were needed to recover from this
severe toxicity induced by 5-FU or capecitabine. In our prospective study 530 patients
have been genotyped for DPYD*2A. We have identified 7 patients carrying the
DPYD*2A allele, of which 4 were safely treated with capecitabine at reduced doses.
Figure 2: Systemic exposure and bioavailability of Erlotinib after oral administration were significantly
increased in Bcrp1/Mdr1a/1b(-/-) knockout mice compared with wildtype mice.
Circulating tumor cell detection in advanced solid cancer: validation of
QPCR-based detection In the development of metasases, 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
A.J. Bosma, T.J. Molloy, L.J. van ’t Veer.
Phase 1 and pharmacological study in monotherapy and in combination with
gemcitabine, cisplatin or carboplatin in advanced solid tumors 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. It potentiates the activity of cytotoxic agents in tumor cells. The protein p53
regulates cell cycle checkpoints different from those regulated by Wee1, including the
G1 checkpoint. The efficacy of MK-1775 is therefore expected to be greater in p53
deficient tumors. 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 showed that MK-1775 with
chemotherapy was well tolerated and a substantial number of patients showed stable
disease. Pharmacokinetics are linear.
45
EXPERIMENTAL THERAPY
Publications (continued)
Meijerman I, Beijnen JH, Schellens JHM.
Combined action and regulation of phase
enzymes and multidrug resistance proteins
in multidrug resistance in cancer. Cancer
Treat Rev 2008;34:505-20
Sparidans RW, Lagas JS, Schinkel AH,
Schellens JHM, Beijnen JH. Liquid
chromatography-tandem mass spectrometric
assay for diclofenac and three primary
metabolites in mouse plasma. J Chromatogr
B 2008;872:77-82
Veltkamp SA, Jansen RS, Callies S,
Pluim D, Visseren-Grul CM, Rosing H,
Kloekers-Rhoades S, Andre VAM,
Beijnen JH, Slapak CA, Schellens JHM.
Oral administration of gemcitabine in
patients with refractory tumoers: a clinical
and pharmacologic study. Clin Cancer Res
2008;14:3477-86
Veltkamp SA, Pluim D, Van Tellingen O,
Beijnen JH, Schellens JHM. Extensive
metabolism and liver accumulation of
gemcitabine following multiple oral and
intravenous administration in mice. Drug
Met Disp 2008;36:1606-15
Veltkamp SA, Witteveen EO, Capriati A,
Crea A, Animati F, Voogel-Fuchs M,
Heuvel Van den IJ, Beijnen JH, Voest EE,
Schellens JHM. Clinical and pharmacologic
study of the novel prodrug delimotecan
(MEN 4901/T-0128) in patients with solid
tumors. Clin Cancer Res 2008; 14: 7535-44
Brouwers EE, Tibben MM, Pluim D,
Rosing H, Boot H, Cats A, Schellens JHM,
Beijnen JH. Inductively coupled plasma
mass spectrometric analysis of the total
amount of platinum in DNA extracts from
peripheral blood mononuclear cells and
tissue from patients treated with cisplatin.
Anal Bioanal Chem 2008 (in press)
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
2008 (in press)
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
2008 (in press)

46
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
Murat Koseoglu PhD Post-doc
Gijs van Haaften PhD Post-doc
Maria Gomez Benito PhD Post-doc
Henning Schaefer MD PhD Post-doc
Carlos le Sage MSc PhD student
Martijn Kedde MSc PhD student
Jarno Drost MSc PhD student
Remco Nagel MSc PhD student
Marieke van Kouwenhove MSc PhD student
Arnold Bos MSc PhD student
Mariette 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 RASV12.
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.
DIVISION OF GENE REGULATION
MICRORNAS 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 the past three years we initiated studies to identify cancerous microRNAs
(miRNAs), a newly emerging gene family encoding for endogenous small RNAs.
We developed novel and unique genetic approaches to screen for cancer-causing and
cancer-preventing miRNAs. With these tools we discovered and characterized the role
of the miR-372 family in tumor growth and metastasis as well as the oncogenic role
of miR-221 in glioblastoma.
Interestingly, we noticed that the regions surrounding 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 (miR-221&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.

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 miRNAmediated
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.
Publications
47
GENE REGULATION
Kedde M, Strasser MJ, Boldajipour B,
Oude Vrielink JA, Slanchev K, le Sage C,
Nagel R, Voorhoeve PM, van Duijse J,
Orom UA, Raz R, Agami R. RNA-binding
protein Dnd1 inhibits microRNA access to
target mRNA. Cell 2007;131:1273-1286
Duursma AM, Kedde, M, Schrier M,
le Sage C, Agami R. miR-148 targets
human DNMT3b protein coding region.
RNA 2008;14: 872-877
Elkon R, Agami R. Removal of AU bias
from microarray mRNA expression data
enhances computational identification of
active microRNAs. PLoS Comput Biol
2008;4: e1000189
Huang Q, Gumireddy K, Schrier M,
le Sage C, Nagel R, Nair S, Egan DA, Li A,
Huang G, Klein-Szanto AJ, Agami R.
The microRNAs miR-373 and miR-520c
promote tumour invasion and metastasis.
Nat Cell Biol 2008;10: 202-210
Kedde M, Agami R. Interplay between
microRNAs and RNA-binding proteins
determines developmental processes. Cell
Cycle 2008; 7, 899-903
Nagel R, le Sage C, Diosdado B,
van der Waal M, Oude Vrielink JA,
Bolijn A, Meijer GA, Agami R. Regulation
of the adenomatous polyposis coli gene by
the miR-135 family in colorectal cancer.
Cancer Res 2008;68: 5795-5802
Zlotorynski E, Agami R. A PASport to
cellular proliferation. Cell 2008:134: 208-210

48
GENE REGULATION
Group leader Bas van Steensel
Bas van Steensel PhD Group leader
Katy Kolodziej PhD Post-doc
Alexey Pindyurin PhD Post-doc
Jop Kind PhD Post-doc
Guillaume Filion PhD Post-doc
Ulrich Braunschweig MSc PhD student
Daniel Peric-Hupkes MSc PhD student
Joke van Bemmel MSc PhD student
Maartje Vogel MSc PhD student
Ludo Pagie PhD Bioinformatician
Wendy Talhout BAS Technical staff
Inês de Castro Genebra de Jesus MSc
Technical staff
Publications
de Wit E, van Steensel B. Chromatin
domains in higher eukaryotes: insights from
genome-wide mapping studies. Chromosoma
2008; Oct 14. [Epub ahead of print]
de Wit E, Braunschweig U, Greil F,
Bussemaker HJ, van Steensel B. Global
chromatin domain organization of the
Drosophila genome. PLoS Genetics 2008;
4:e1000045
Guelen L, Pagie L, Brasset E, Meuleman
W, Faza MB, Talhout W, Eussen BH,
de Klein A, Wessels L, de Laat W,
van Steensel B. Domain organization of
human chromosomes revealed by mapping
of nuclear lamina interactions. Nature
2008; 453:948-951
Peric-Hupkes D, van Steensel B. Linking
cohesin to gene regulation. Cell 2008;
132:925-928
Vogel MJ. Genomic mapping of
heterochromatin components using
DamID. Thesis, University of Amsterdam;
2008.
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 major focus of investigation is currently how
the genome is organized into large, multi-gene chromatin domains that may play a
role in the coordinated control of neighboring genes. In relation to this, we
investigate the role of the nuclear envelope in the spatial organization of the genome
inside the nucleus, and its effects on gene expression.
Genomics technology for chromatin research Over the past years, we have
developed a technique (named ‘DamID’) for the genome-wide mapping of in vivo
target loci of chromatin proteins and transcription factors. This microarray-based
method allows us to generate detailed in vivo genomic binding maps of a large variety
of chromatin proteins and transcription factors. We now routinely use DamID to
construct such maps in Drosophila and mammalian cell lines. 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 also actively
pursue the development of additional novel genomics methods that will uncover new
aspects of chromatin and gene regulation.
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. It has been suggested that in these models the spreading of
heterochromatin across the rearrangement breakpoint causes the silencing of white.
However, the extent of this spreading and the precise pattern of heterochromatin
redistribution have remained unclear. We therefore constructed high-resolution
binding maps of Heterochromatin Protein 1 (HP1) on white-mottled chromosomes.
We find 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. Our data suggest that the white gene has an unusual
intrinsic affinity for heterochromatin, which may cause this gene to be more sensitive
to PEV than most other genes. These results reveal how a chromosomal
rearrangement can affect chromatin domain organization and alter the expression of
genes in the affected genomic regions.
Chromatin domain organization of the Drosophila genome Besides
heterochromatin domains, there are several other examples of multi-gene chromatin
domains. However, a global view of the chromatin organization of eukaryotic
genomes is lacking. To systematically identify multi-gene chromatin domains, we
constructed a compendium of genome-scale binding maps for a broad panel of 29
chromatin components in Drosophila melanogaster. Next, we computationally
analyzed this compendium for evidence of multi-gene chromatin domains. For this
we used a novel statistical segmentation algorithm, developed in close collaboration
with bioinformatics expert Dr Harmen Bussemaker (Columbia University, New
York). We find that at least 50% of the fly genome is organized into chromatin

domains, which often consist of dozens of genes. The domains are characterized by
various known and novel combinations of chromatin proteins. The genes in many of
the domains are coregulated during development and share biological functions.
Furthermore, fewer chromosomal rearrangements occur inside chromatin domains
than outside domains during evolution. Our results indicate that a substantial
portion of the Drosophila genome is packaged into functionally coherent, multi-gene
chromatin domains. Presently, we are extending and refining the identification of
chromatin domains by increasing the diversity, coverage and resolution of our
DamID maps. In addition, we are investigating the role of various candidate proteins
in the demarcation of specific domain types.
Chromatin Protein Discovery Project DamID maps can be used to predict the
molecular interactions and functions of chromatin proteins. In 2008 we have started
the Chromatin Protein Discovery Project, which aims to generate genome-wide
binding maps for ~100 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 will generate full-genome,
high-resolution DamID maps, which are expected to reveal many new molecular
interactions and functions of the hitherto uncharacterized proteins. A pilot run for
20 novel proteins is under way; we expect that in 2009 the mapping will be scaled up
to the full set of candidate proteins.
Nuclear lamina – genome interactions in human cells 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 have constructed for the first time a
~1kb resolution map of the interaction sites of the entire human genome with the NL
in fibroblasts. This map shows that human genome-NL interactions occur via more
than 1,300 sharply defined large domains of 0.1-10Mb in size. These laminaassociated
domains (LADs) together contain about 7,000 genes; the vast majority of
these genes are expressed at very low levels, indicating that the NL represents a
repressive chromatin environment. Furthermore, we found that the borders of LADs
are often demarcated by specific sequence elements, indicating that LAD
organization is a least in part ‘hard-coded’ in the genome. The border elements
include binding sites for the insulator protein CTCF, promoters that are oriented
away from LADs, and CpG islands, suggesting possible mechanisms of LAD
confinement. Taken together, these results demonstrate that the human genome is
divided into large, discrete domains that are units of chromosome organization
within the nucleus. We are now conducting extensive studies to reveal the functions,
dynamics, and molecular mechanisms of genome-NL interactions. This will reveal
how three-dimensional organization of DNA inside the nucleus contributes to the
regulation of gene expression.
Bioinformatics Bioinformatics tools are essential for the detailed understanding of
our genome-wide datasets (now amounting to millions of datapoints). In
collaboration with national and international experts in bioinformatics, we are
actively developing new algorithms and approaches for the systematic analysis of the
chromatin protein binding patterns that we obtain. Integration with external datasets
(gene expression profiles, genome annotation, sequence motifs) allows us to make
functional predictions that can be further tested in our laboratory. In addition, we
design our own custom oligonucleotide microrarrays.
49
GENE REGULATION
Figure 4. Model of the folding of human
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 genomic elements
that are named border elements.

50
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
Tibor van Welsem Technical staff
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 5: Telomeric silencing in budding yeast; red
(dark) and white (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. A major goal of our research is to
understand how H3-Lys79 methylation affects chromatin structure and function. We
have used a combination of genetics and biochemistry to study the mechanism of
methylation of H3-Lys79 and the consequences thereof. We found that Dot1, in
contrast to other known protein methyltransferases, acts by a non-processive
mechanism. Interestingly, this unusual mechanism of synthesis affects the function
of the methylated lysine. 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 anti-binding signal. Since human and yeast Dot1 are
structurally very similar, we expect that similar rules apply to human cells. We are
currently performing new genome-wide screens to understand how H3K79
methylation specifically affects heterochromatin function. Dot1 has also been
implicated in the DNA damage response. However, the mechanism by which H3K79
methylation affects DNA damage checkpoint activation is still unclear. Our recent
studies indicate that the role of Dot1 is modulated by other DNA damage response
pathways. We are currently investigating how Dot1 interacts with other factors to
mount a cellular response upon induction of DNA damage.

Figure 6: 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.
Histone One of the main goals of our group is to understand how chromatin
modifications can have long-term effects on gene expression. Post-translational
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. It is thought that
the post-translational modifications on histones can serve as epigenetic memory
marks during cell division. However, 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. The assay involves differential labeling of existing
and newly synthesized proteins. Using this assay we found that histones throughout
the genome are subject to extensive replication-independent exchange, suggesting
that histones and their post-translational marks are not permanent residents in
chromatin. Methods will be developed to search for proteins responsible for this
mode of histone deposition and eviction. We expect that our studies will provide
more insight into the role of histones in maintaining established patterns of gene
expression.
Publications
51
GENE REGULATION
van Welsem T, Frederiks F,
Verzijlbergen KF, Faber AW, Nelson ZW,
Egan DA, Gottschling DE, van Leeuwen F.
Synthetic lethal screens identify gene
silencing processes in yeast and implicate
the acetylated amino terminus of Sir3 in
recognition of the nucleosome core. Mol Cell
Biol 2008;28:3861-72
Frederiks F, Tzouros M, Oudgenoeg G,
van Welsem T, Fornerod M, Krijgsveld J,
van Leeuwen F. Nonprocessive methylation
by Dot1 leads to functional redundancy of
histone H3K79 methylation states. Nat
Struct Mol Biol 2008;15:550-7
Backer R, van Leeuwen F, Kraal G,
den Haan JM. CD8- dendritic cells
preferentially cross-present Saccharomyces
cerevisiae antigens. Eur J Immunol
2008;38:370-80
Ovaa H, van Leeuwen F. Chemical Biology
Approaches to Probe the Proteome.
Chembiochem 2008 (in press)

52
GENE REGULATION
Group leader Maarten Fornerod
Maarten Fornerod, PhD Group leader
Nikos Xylourgidis PhD Post-doc
Stijn Heessen PhD Post-doc
Bernike Kalverda MSc PhD student
Dieuwke Engelsma MSc PhD student
Hella van der Velde Technical staff
Publications
De Keersmaecker K, Rocnik JL, Bernad R,
Lee BH, Leeman D, Gielen O, Verachtert H,
Folens C, Munck S, Marynen P, Fornerod M*,
Gilliland DG*, Cools J*. Kinase activation
and transformation by NUP214-ABL1 is
dependent on the context of the nuclear
pore. Mol Cell 2008;31:134-42.
*co-corresponding authors
Engelsma D, Valle N, Fish A, Salome N,
Almendral JM, Fornerod M. A
supraphysiological nuclear export signal is
required for parvovirus nuclear export.
Mol Biol Cell 2008;19:2544-52
Fornerod M, Clarke PR. To the centre of
the volcano. Workshop on the mechanisms
of nucleocytoplasmic transport. EMBO Rep
2008;9:419-24
Frederiks F, et al. See page 51
Hendriksen J, Jansen M, Brown CM,
van der Velde H, van Ham M, Galjart N,
Offerhaus GJ, Fagotto F, Fornerod M.
Plasma membrane recruitment of
dephosphorylated beta-catenin upon
activation of the Wnt pathway. J Cell Sci
2008;121:1793-802
Kalverda B, Roling MD, Fornerod M.
Chromatin organization in relation to the
nuclear periphery. FEBS Lett
2008;582:2017-22
Van Vlierberghe P, van Grotel M, Tchinda J,
Lee C, Beverloo HB, van der Spek PJ,
Stubbs A, Cools J, Nagata K, Fornerod M,
et al. The recurrent SET-NUP214 fusion as
a new HOXA activation mechanism in
pediatric T-cell acute lymphoblastic
leukemia. Blood 2008;111:4668-80
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.
Nuclear transport Nucleocytoplasmic transport is accomplished by distinct classes
of soluble transport receptors that interact with transport signals and the nuclear
pore complex. We investigate how this fundamental process is used in the regulatory
circuits of the cell.
CRM1-mediated nuclear export Exportins are nuclear export receptors that bind
their cargoes cooperatively with RanGTP. They are responsible for multiple protein
and ribonucleoprotein export pathways. Present evidence suggests that most
exportins export one or a few structurally related groups of substrates. The main
exception is CRM1/Exportin1, that exports multiple functionally unrelated protein
and ribonucleoprotein cargoes, and can be thought of as the only ‘multi-purpose’
exportin identified to date.
A supraNES in parvovirus NS2 required for viral nuclear export and infectivity
We have previously identified nuclear export signals from a 15-mer random peptide
library with unusually high affinity to its receptor CRM1, which we termed
supraNESs. Characterization of these supraNESs lead to the insight that regular
NESs must have low affinity for CRM1 in order to function properly. However,
supraNESs have so far not been identified in endogenous proteins. We now identify a
supraNES in a viral protein, the NS2 protein of mouse parvovirus MVM. NS2
strongly interacts with CRM1 without the requirement of RanGTP, while addition of
RanGTP renders the complex very stable. A single mutation of a hydrophobic residue
that inactivates regular NESs, reduces the affinity of the NS2 NES for CRM1 from
supraphysiological to regular. Interestingly, an MVM mutant harbouring such
regular NES is severely hampered in capsid nuclear export and viral productivity. In
virus-infected cells, we observed a co-localization of NS2, CRM1 and mature virions,
dependent on the supraphysiological NS2 NES. These data suggest a critical role for
the NS2 supraNES in capsid nuclear export and parvovirus pathogenicity.
Kinase activation and transformation by NUP214-ABL1 is dependent on the
context of the nuclear pore Genetic alterations causing constitutive tyrosine kinase
activation are observed in a broad spectrum of cancers. Thus far these mutant
kinases have been localized to the plasma membrane or cytoplasm where they
engage proliferation and survival pathways. In collaboration with the lab of Dr. J.
Cools (Univerrsity of Leuven, Belgium, and the lab of Gary Gilliland (Harvard
University, USA), we have found that the NUP214-ABL1 fusion is unique among
these because of its requisite localization to the nuclear pore complex for its
transforming potential. We show that NUP214-ABL1 displays attenuated
transforming capacity as compared to BCR-ABL1 and that NUP214-ABL1
preferentially transforms T-cells, which is in agreement with its unique occurrence in
T-cell acute lymphoblastic leukemia. Furthermore, NUP214-ABL1 differs from BCR-
ABL1 in subcellular localization, initiation of kinase activity and signaling and lacks
phosphorylation on its activation loop. In addition to delineating a novel mechanism
for kinase activation, this study provides new insights into the spectrum of
chromosomal translocations involving nucleoporins by indicating that the nuclear
pore context itself may play a central role in transformation.

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. We have demonstrated that 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 at least in part
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 comparing CD8 T cells that did or did
not receive CD27 input in vitro or in vivo. In this way, we have identified a cytokine as
CD27 target gene that is solely responsible for the anti-apoptotic effect of CD27
during TCR-driven CD8 T cell expansion in vitro. Primary influenza virus-specific
CD8 T cells were reconstituted to express the cytokine and examined side by side
with mock-transduced cells for their accumulation after virus infection. These
experiments showed complementation of the survival defect of CD27 -/- cells in vivo,
while wild-type CD8 T cells did not benefit from provision of the cytokine gene.
Similar experiments were performed to examine the role of the anti-apoptotic
regulator Bcl-x L. Whereas Bcl-x L clearly afforded protection against apoptosis, this
effect was equal in magnitude for wild-type and CD27-deficient cells. These data tie
in with earlier studies using Bcl-x L transgenic T cells, which also pointed out that
Bcl-x L, despite being upregulated at the protein level upon CD27 signaling, is not the
key effector of its anti-apoptotic function. Gene expression profiling also identified a
chemokine as CD27 target gene in CD8 T cells which supports effector T cell
accumulation in vivo according to recent reconstitution experiments.
In a protein immunization model, we have found that CD4 T cells require CD27 to
support their own survival, but also to enable them to help CD8 T cells, in particular
to imprint memory characteristics into them. By genome-wide expression analysis of
CD4 T cells undergoing an immune response in vivo, we have found the membrane
protein MS4A4B as CD27 target gene. Strikingly, the same molecule was identified
as a unique marker for CD8 memory T cells that had received CD27-proficient CD4
T cell help. Our findings suggest that MS4A4B is instrumental in programming
memory CD8 T cells for secondary expansion. We have recently raised monoclonal
antibodies to this molecule in Armenian hamsters. These unique reagents will allow
us to examine the value of the MS4A4B as marker of helped versus helpless CD8 T
cells. Genetic interventions will be used 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. We have
made transgenic mice constitutively expressing CD70 on immature conventional DC
(CD11c-Cd70tg). Such DCs normally acquire CD70 upon maturation. In two in vivo
models, transgenic CD70 expression switched the system from tolerance induction
to induction of full CD8 T cell responsiveness, allowing the elimination of tumors
and virus, respectively (figure 1). The transgenic DC could also induce an effective
anti-tumor immune response in an adoptive transfer setting, which mimics a clinical
situation of cancer immunotherapy. We have also generated CD70-deficient mice,
which will be used as recipients to further address the potential of CD70 transgenic DC.
In DCs, transport of CD70 to the cell surface is tightly controlled. We have found that
CD70 is sorted exactly like MHC class II to the inner vesicles of late endocytic
53
IMMUNOLOGY
Division head, group leader Jannie Borst
Jannie Borst PhD Group leader
Jonathan Coquet PhD Post-doc
Sabine Middendorp PhD Post-doc
Yanling Xiao MD PhD Post-doc
Anna Keller 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
Figure 1: Transgenic CD70 expression
converts tolerance to anti-tumor immunity.
CD11c-Cd70tgCd27 -/- or control Cd27 -/mice
were injected subcutaneously with B16
tumor cells expressing an ovalbumin epitope
(OVA). Three days later, 10 5 WT or CD27 -/-
OVA-specific OT-I T cells were transferred
and one day later (day 0), mice were
challenged with OVA peptide in PBS, as
indicated. Percentages of MHC tetramer +
cells among CD8 + T cells in blood and
tumor size measured by caliper are
indicated (n=4).

54
IMMUNOLOGY
Publications
Xiao Y*, Peperzak V*, Keller AM, Borst J.
CD27 instructs CD4+ T cells to provide
help for the memory CD8+ T cell response
after protein immunization. * equal first
author J Immunol 2008;181:1071-1082.
Mousavi SF, Soroosh P, Takahashi T,
Yoshikai Y, Shen H, Lefrancois L, Borst J,
Sugamura K, Ishii N. OX40 costimulatory
signals potentiate the memory commitment
of effector CD8+ T cells. J Immunol
2008;181:5990-6001.
Keller AM, Schildknecht A, Xiao Y,
van den Broek M, Borst J. Deliberate
expression of CD70 on steady state
dendritic cells breaks CD8+ T cell tolerance
and permits effective immunity.
Immunity 2008; Dec 15.
Verbrugge I, Wissink EHJ, Rooswinkel RW,
Jongsma J, Beltraminelli N, Dupuis M,
Borst J, Verheij, M. Combining
radiotherapy with APO010 (MegaFas
Ligand) in cancer treatment, a preclinical
evaluation of feasibility. Clin Cancer Res
2008 (in press)
Verbrugge T, De Vries E, Tait SW,
Wissink EH, Walczak H, Verheij M, Borst
J. Ionizing radiation modulates the TRAIL
death inducing signaling complex, allowing
bypass of the mitochondrial apoptosis
pathway. Oncogene 2008;27:574-584
compartments, known as MIIC. We have shown that this CD70 routing serves in the
first place to coordinate CD70 cell surface deposition temporally and spatially with
antigen presentation by MHC class II. CD70 transport also impacts on the CD8 T
cell response. We found that routing of CD70 to MIIC is controlled by a chaperone
protein. In DC, genetically deficient for this chaperone, cell surface deposition of
CD70 is enhanced. This has a strong impact on CD8 T cell priming, as shown in an
in vitro cross-presentation setting with ovalbumin protein antigen. These findings
demonstrate a novel role of the chaperone in determining the threshold for CD8 T
cell priming by controlling the level of CD70 cell surface expression. This work is
carried out in close collaboration with the group of J Neefjes (Division of Cell Biology II).
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 M Verheij of the Division of Radiotherapy we are carrying out a
project aimed at exploiting death ligands to enhance the efficacy of radiotherapy. As
proof of principle, we have used human lymphoid tumor cells. In these cells,
radiation (and DNA damaging drugs) can sensitize for TRAIL receptor- and Fas/
CD95 induced apoptosis when the mitochondrial pathway is blocked and p53 is
lacking. APO010, a novel recombinant form of Fas/CD95 ligand, is currently in a
Phase I dose-escalation trial in patients with solid tumors. We have investigated the
feasibility of combination therapy with APO010 and radiotherapy in leukemic and
solid tumor cell types. This approach is attractive, since a potential combined
treatment effect would be local and limit normal tissue toxicity. We found a
significant combined effect of both treatment regimens on cell death induction, as
read out by apoptosis and clonogenic survival in vitro. In vivo, however, the
combination of systemically administered APO010 and radiotherapy did not
significantly enhance tumor growth delay, as compared to the single modality
treatments. Toxicity data indicated that further dose-escalation of APO010 was not
achievable. We suggest therefore that future studies should address the efficacy of
locally applied APO010 as radiation-enhancing agent.
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. 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 apoptosisinduction
by DNA damaging stimuli. However, its mode of regulation is distinct in
this scenario, since it is not cleaved at the conventional sites by upstream caspases.
We have developed a novel FRET-based read-out for Bid cleavage to map the relevant
sites for proteolytic activation of Bid after DNA damage and to identify the protease
involved.
In a functional genetic screen for mediators of TRAIL-induced apoptosis in MCF-7
breast cancer cells, we have identified a molecule that determines the extent of cell
surface expression of TRAIL receptor-1, but not TRAIL receptor-2. Both receptors are
partially retained in intracellular vesicles. We find that the plasma membrane pool –
rather than the intracellular pool – engages in pro-apoptotic signaling. Our data
implicate vesicular trafficking in regulating plasma membrane levels of TRAIL
receptors both before and after ligand binding, and thereby sensitivity to TRAILinduced
apoptosis.

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 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 (collaboration with Ovaa and Perrakis
labs) Peptide-(p)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 high-throughput screening, or of a collection of
clinical grade pMHC multimer reagents for cellular therapy. In collaboration with
H Ovaa (Division of Cell Biology II) we have in the past years addressed this issue, by
designing MHC class I molecules occupied with UV-sensitive ‘conditional’ peptide
ligands.
While the 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 would allow the parallel detection of a large number of 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, but is encoded by a
defined combination of two or more identifiers. Using such ‘combinatorial coding’,
the number of specificities that can be encoded increases dramatically (figure 2). In a
series of proof-of principle experiments, in which up to 25 different antigen specificities
were encoded per sample, we have demonstrated that the detection of antigen-specific T
cells from peripheral blood by combinatorial coding is highly efficient. Furthermore, to
exemplify the value of the large-scale screening of patient material made possible by
combinatorial coding, we have analyzed T cell responses against known and potential
melanoma-associated antigens in peripheral blood from melanoma patients. In parallel
work, this technology is being utilized to identify leukemia-reactive T cell responses in a
joint effort with the Heemskerk/Falkenburg group (LUMC).
Dissection of T cell immunity through retroviral barcoding 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
Figure 2: Combinatorial coding with MHC-qDots. To allow the simultaneous detection of multiple
antigen-specific T cell populations in patient samples, individual antigen-specificities are encoded by
pMHC multimers that are coupled to unique combinations of fluorochromes. Left plot depicts the binding
of pMHC multimers conjugated to two different fluorochromes to an antigen-specific T cell. Right plot
shows the maximal number of antigen-specificities that can be encoded by classical single fluorochrome
encoding and by two-dimensional encoding as a function of the number of available detection channels.
Group leader Ton Schumacher
Ton Schumacher PhD Group leader
Gavin Bendle PhD Post-doc
Bianca Heemskerk PhD Post-doc
Andrew Kaiser PhD Post-doc
Shalin Naik PhD Post-doc
Sine Reker Hadrup 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
Erwin Swart Technical staff
Mireille Toebes Technical staff
Jos Urbanus Technical staff
Publications
55
IMMUNOLOGY
Bakker AH, Hoppes R, Linnemann C,
Toebes M, Rodenko B, Berkers CR,
Reker-Hadrup S, van Esch WJE,
Heemskerk MHM, Ovaa H,
Schumacher TNM. Conditional MHC
class I ligands and MHC exchange
technology for the human MHC alleles
HLA-A1, -A3, -A11 and -B7. Proc Natl Acad
Sci USA. 2008;105:3825-30
Bos R, van Duikeren S, Morreau H,
Schumacher TN, Haanen JB,
van der Burg SH, Melief CJM, Offringa R.
Balancing between anti-tumor efficacy and
auto-immune pathology in T-cell mediated
targeting of carcinoembryonic antigen.
Cancer Res. 2008 ( in press)
Coccoris M, Swart E, van Heijst JW,
de Witte MA, Haanen JBAG, Schepers K,
Schumacher TNM. Long-term functionality
of TCR transduced T cells in vivo.
J Immunol. 2008;180:6536-43
De Witte MA, Bendle GM,
van den Boom MD, Coccoris M,
Schell TD, Tevethia SS, Mesman EM,
Song JY, Schumacher TNM. TCR gene
therapy of spontaneous prostate carcinoma
requires in vivo T cell activation. J
Immunol. 2008;181:2563-71

56
IMMUNOLOGY
Publications (continued)
De Witte MA, Jorritsma A, Kaiser A,
van den Boom MD, Dokter M, Bendle G,
Haanen JBAG, Schumacher TNM.
Requirements for effective anti-tumor
responses of TCR transduced T cells.
J Immunol. 2008;181:5128-36
De Witte MA, Jorritsma A, Swart E,
Straathof KC, de Punder K, Haanen JB,
Rooney C, Schumacher TNM. An
inducible caspase 9 safety switch can halt
cell therapy-induced autoimmune disease.
J Immunol. 2008;180:6365-73
Jorritsma A, Bins AD, Schumacher TN,
Haanen JB. Skewing the T-cell repertoire by
combined DNA vaccination, host
conditioning, and adoptive transfer. Cancer
Res. 2008;68:2455-62
Reker Hadrup S, Toebes M, Rodenko B,
Bakker AH, Egan DA, Ovaa H,
Schumacher TNM. High-Throughput
T Cell Epitope Discovery Through MHC
Peptide Exchange. In Methods in
Molecular Biology. Epitope Mapping
Protocols: Second Edition. 2008 (in press)
Schepers K, Swart E, van Heijst JW,
Gerlach C, Castrucci M, Heimerikx M,
Velds A, Kerkhoven RM, Arens R,
Schumacher TN. Dissecting T cell lineage
relationships by cellular barcoding. J Exp
Med. 2008 (in press)
Verstrepen BE, Bins AD, Rollier CS,
Mooij P, Koopman G, Sheppard NC, et
al. Improved HIV-1 specific T-cell responses
by short-interval DNA tattooing as
compared to intramuscular immunization
in non-human primates. Vaccine.
2008;26:3346-51
Van den Berg JH, Nuijen B, Beijnen JH,
Vincent A, van Tinteren H, Kluge J,
Woerdeman LAE, Hennink WE, Storm G,
Schumacher TN, Haanen JBAG.
Optimization of intradermal vaccination
by DNA tattooing in human skin. Hum
Gene Ther. 2008 (in press)
doesn’t allow the analysis of cell fate and cellular differentiation pathways.
To allow such lineage tracking we have developed an approach in which individual
T cells are tagged with a genetic barcode. For this purpose we have created an
oncoretroviral and lentiviral library containing some 5,000 different barcodes, and
the same set of barcodes has been used to generate a barcode micro-array. Infection
of cell populations by these libraries of viral vectors and subsequent micro-array
analysis can then be used to trace the progeny of individual cells. Following earlier
work in which we demonstrated how T cell populations primed at distinct sites in the
body intermingle, we have more recently used this technology to trace hematopoietic
differentiation and T cell differentiation. With respect to the latter, using a strategy
for the retroviral labelling 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. In
addition, we have utilized retroviral barcoding to determine to what extent the
magnitude of T cell responses is determined by the recruitment of antigen-specific
T cells from the naive T cell pool. These data demonstrate that under conditions in
which the magnitude of antigen-specific T cell responses varies widely, the
recruitment of antigen-specific T cell precursors remains largely unchanged and is in
fact near complete. These data demonstrate that regulation of the magnitude of T cell
responses occurs primarily at the level of T cell expansion.
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 vivo. 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 development in this line of research in the past year 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. Further experiments have shown that this
pathology can be explained by the formation of so-called mixed dimers that are
formed by the joint assembly of endogenous and exogenous TCR chains (figure 3).
These data indicate that it will be essential to restrict the formation of mixed TCR
dimers in clinical trials of TCR gene transfer. With respect to such trials, in the past
year we have initiated the production of clinical grade retrovirus encoding a
melanoma-reactive TCR for the gene modification of human T cells. 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. Finally, in a joint
effort with the Haanen lab and NKI-AVL pharmacy, a large effort is currently made to
develop the pharmaceutical process that will be utilized in this trial.
Figure 3: Off-target toxicity through TCR gene therapy. Introduction of an unmodified TCR into
peripheral T cells leads to the formation of mixed TCR dimers, consisting of endogenous and exogenous
TCR chains. As these TCR heterodimers have not been selected against reactivity with self antigens,
autoreactivity may ensue.

IMMUNOTHERAPY
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 the Schumacher 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. The
primary aim of this line of research is to evaluate specific and cytokine-based
immunotherapies, using advanced technologies for characterization of immune
responses in peripheral blood and at the tumor site. These studies include
immunomonitoring of clinical trials of cancer patients in our hospital and are
conducted in close collaboration with Christian Blank (division of Immunology and
Medical Oncology), Axel Bex (division of Surgical Oncology), Michel van den Heuvel
(division of Medical Oncology) and the clinical chemistry laboratory headed by
Willem Nooijen (division of Diagnostic Oncology).
DNA vaccination for the treatment of cancer
Phase I study in melanoma patients Induction of immunity following DNA vaccination
is generally considered a slow process. We have shown that DNA delivery to the skin
results in a highly transient pulse of antigen expression. Based on this information
we developed a novel, rapid and potent intradermal DNA vaccination method. By
short-interval intradermal DNA delivery, robust T cell responses, of a magnitude
sufficient to reject established subcutaneous tumors, are generated within 12 days.
These results were confirmed in a non-human primate model (in collaboration with
the BPRC, Rijswijk). We showed that DNA tattooing results in a 10-100-fold increase
in vaccine-specific T cells compared to intramuscular vaccination.
Together with B Nuijen (Pharmacy, Slotervaart Hospital), W Hennink and G Storm
(Department of Biopharmacy, Utrecht University) we have developed an ex vivo
human skin model to optimize DNA tattoo vaccination 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 approval by the Central
Committee on Research involving Human Subjects (CCMO) and the Dutch Ministry
for Environmental Affairs to start a first gene therapy phase I trial with DNA tattoo
vaccination. For this purpose a clinical grade DNA vaccine has been produced in the
recently built GMP DNA plasmid production unit (Amsterdam-Biotherapeutics Unit)
at the NKI-AVL/Slotervaart Hospital pharmacy department.
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 and oropharynx. Because persistence of oncogenic HPV proteins E6 and E7 is
required for carcinogenesis, these viral antigens are exquisite targets for
immunotherapeutic intervention. Indeed, therapeutic vaccinations targeting these
viral antigens have shown some promise in woman suffering from cervical cancer. In
the next years we will perform a phase I/II study in patients with HPV 16-positive
squamous cell cancer of the penis and cervix using our novel and potent intradermal
DNA vaccination strategy. We have developed highly immunogenic and safe HPV 16
E6 and E7 containing DNA vaccines that we will start producing in 2008-2009 for a
first clinical trial.
Group leader John Haanen
John Haanen MD PhD Group leader
Florry Vyth-Dreese PhD Senior staff
scientist
Bianca Heemskerk PhD Post-doc
Annelies Jorritsma PhD Post-doc
Esther Tjin PhD Post-doc
Silvia Ariotti MSc PhD student
Koen Oosterhuis MSc PhD student
Joost Van den Berg MSc PhD student
Yeung-Hyen Kim MSc PhD student
Trees Dellemijn Technical staff
Raquel Gomez MSc Technical staff
Johan Sein Technical staff
Willeke Van de Kasteele Technical staff
Martin Van der Maas Technical staff
Publications
57
IMMUNOLOGY
Abad JD, Wrzensinski C, Overwijk W,
De Witte MA, Jorritsma A, Hsu C,
Gattinoni L, Cohen CJ, Paulos CM,
Palmer DC, Haanen JB, Schumacher TN,
Rosenberg SA, Restifo NP, Morgan RA.
T-cell receptor gene therapy of established
tumors in a murine melanoma model. J
Immunother. 2008;31:1-6
Bos R, van Duikeren S, Morreau H,
Franken K, Schumacher TN, Haanen JB,
van der Burg SH, Melief CJ, Offringa R.
Balancing between antitumor efficacy and
autoimmune pathology in T-cell-mediated
targeting of carcinoembryonic antigen.
Cancer Res. 2008;68:8446-55
Coccoris M, Swart E, de Witte MA,
van Heijst JW, Haanen JB, Schepers K,
Schumacher TN. Long-term functionality
of TCR-transduced T cells in vivo. J
Immunol. 2008;180:6536-43
De Witte MA, Jorritsma A, Kaiser A,
van den Boom MD, Dokter M, Bendle
GM, Haanen JB, Schumacher TN.
Requirements for effective antitumor
responses of TCR transduced T cells. J
Immunol. 2008;181:5128-36

58
IMMUNOLOGY
Publications (continued)
De Witte MA, Jorritsma A, Swart E,
Straathof KC, de Punder K, Haanen JB,
Rooney CM, Schumacher TN. An
inducible caspase 9 safety switch can halt
cell therapy-induced autoimmune disease. J
Immunol. 2008;180:6365-73
Griffioen A, Vyth-Dreese FA. Angiostasis
as a way to improve immunotherapy.
Thrombosis and Hemostasis. 2008 (in
press)
Jorritsma A, Bins AD, Schumacher TN,
Haanen JB. Skewing the T-cell repertoire by
combined DNA vaccination, host
conditioning, and adoptive transfer. Cancer
Res. 2008;68:2455-62
Quaak SG, van den Berg JH, Toebes M,
Schumacher TN, Haanen JB, Beijnen JH,
Nuijen B. GMP production of pDERMATT
for vaccination against melanoma in a
phase I clinical trial. Eur J Pharm
Biopharm. 2008. [Epub ahead of print]
Verstrepen BE, Bins AD, Rollier CS,
Mooij P, Koopman G, Sheppard NC,
Sattentau Q, Wagner R, Wolf H,
Schumacher TN, Heeney JL, Haanen JB.
Improved HIV-1 specific T-cell responses by
short-interval DNA tattooing as compared
to intramuscular immunization in nonhuman
primates. Vaccine. 2008;26:3346-51
Figure 5: Multi parameter confocal
microscopic analysis of renal cell carcinoma
specimen. Merge graph of cryosection
stained for CD31/34 on blood vessels
(green), Ki67 proliferation marker (nuclear
label, red) and CD3 T cells (blue). Arrows
indicate T cell (1), proliferating T cell (2),
blood vessel (3), proliferating blood vessel
(4) and proliferating tumor cell (5).
Adoptive immunotherapy program TIL therapy Recently, we started a new project
concerning adoptive therapy with Tumor-infiltrating Lymphocytes (TIL). With this
approach, the Surgery Branch, NIH, Bethesda, MD, USA finds a 50% objective
response rate in heavily pretreated stage IV melanoma patients. Treatment combines
ex vivo culture of melanoma-reactive T cells from resected metastases with nonmyeloablative
chemotherapy and high dose bolus IL-2. Our goals are to show that
this treatment can be given safely in an independent cancer centre, to show in an
randomized controlled trial that this treatment improves progression-free survival
compared to standard chemotherapy and to perform a comprehensive analysis of the
T cell specificities of the melanoma-reactive TIL prior to and after adoptive transfer.
We expect to start treatment of the first patients in 2009.
T-cell receptor gene therapy In close collaboration with the Schumacher group and
several international groups, 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 will be produced by a German
GMP manufacturer. The TCR has been equipped with safeguards to prevent pairing
with endogenous TCR chains of transduced peripheral T cells to prevent potential
side effects. The process of clinical grade culturing and T cell transduction with the
1D3-MP-71 retrovirus is validated step-by-step in our laboratory and GMP facility. A
large translational gene therapy grant from ZonMW will allow us to perform a phase
I clinical study in melanoma patients in the next years.
Immunotherapy monitoring (This work is performed under supervision of Florry
Vyth-Dreese, staff scientist in the group.) The primary aim of this work is to evaluate
specific and cytokine-based immunotherapies, using advanced technologies to
characterize immune responses in peripheral blood and at the tumor site. The
Immunomonitoring Facility is implementing a full spectrum of immunomonitoring
tools and assays.
Immunotherapy in patients with renal cell carcinoma (RCC) Since the past 3 years
metastatic RCC patients have been treated by targeted therapy with tyrosine kinase-
and mTOR kinase inhibitors and anti-VEGF mAb. These therapies aim at inhibition
of angiogenesis as well as direct targeting of the tumor. In addition, they may
potentiate anti-tumor immune responses. In collaboration with others, we investigate
inhibition of angiogenesis at the peripheral blood and tumor tissue level. Clinical
tumor specimens obtained from RCC patients treated with Sunitinib, Avastin or
Interferon alpha, non small cell lung cancer (NSCLC) patients treated with Erlotinib,
or untreated patients, are examined using immunohistological methods (figure 5).
Preliminary data indicate that inhibition of angiogenesis results in enhanced tumor
infiltration by immune cells and induces apoptosis of tumor cells and endothelial
cells.
Detection of antigen-specific T lymphocytes in experimental and human tissues We have
successfully applied in situ tetramer technique for the detection of minor
Histocompatibility Antigen-specific T cells in a human ex vivo in situ skin explant
model of Graft versus Host reactivity (in collaboration with the group of E Goulmy,
Leiden University Medical Center). Recently, we published a method to detect minor
antigen specific T cells, not only in fresh, but also in cryopreserved tissues using socalled
MHC-dextramers. This will enable analysis of patient material post-surgically.
Development of a human ex vivo in situ skin model for vitiligo and melanoma In
collaboration with R Luiten (SNIP and Dermatology, Academical Medical Center,
University of Amsterdam, Amsterdam) and C Melief (Leiden University Medical
Center) a human in situ skin model has been developed to study immune and
environmental factors involved in the development of vitiligo and potential therapy of
melanoma. Using melanocyte-specific T cell clones co-cultured with normal skin
tissues, we were able to mimic the induction of vitiligo ex vivo. In addition, bulk
T cells obtained from vitiligo lesions were shown to induce severe lesions in this skin
model. Separate studies currently show that a similar model can be applied to
visualize melanoma -T cell interactions ex vivo in situ.

PROGRAMMED MUTAGENESIS
To improve immunity, B cells have the unique capacity to mutate their immunoglobulin
genes by programmed mutagenesis. Our research-focus is three-fold: i) Unravel the
molecular mechanism of programmed mutagenesis, ii) Determine the targeting
specificity of the mutation process, and iii) Understand the decision making between
repair and mutagenesis.
Binding of the mutagen AID within the chromatin of B cells In B cells of the
germinal center (GC) the antigen specific immunoglobulin (Ig) gene repertoire is
diversified by somatic hypermutation (SHM) and class switch recombination (CSR).
While SHM introduces point mutations into the Ig variable region to generate high
affinity Ig variants, CSR is a recombination process enabling isotype switching
within the Ig heavy chain constant region. Both SHM and CSR are initiated by the
activation induced cytidine deaminase (AID), which deaminates cytosine in the
variable- and switch-regions of Ig genes. Based on the translocation break points and
somatic mutations in cancer genes, aberrant targeting of AID in B cells of the GC
seems to boost the accumulation of oncogenic mutations in GC B cells. How
specifically AID is targeted to Ig loci remains unknown. To determine genome-wide
the binding specificity of AID, we applied DamID technology. We showed that
aberrant targeting of AID can occur throughout the genome. Moreover, consistent
with its single-stranded DNA binding activity, AID targets predominantly actively
transcribed regions. Having established the DamID technology for B cells, future
studies should allow us to provide a detailed, genome-wide binding profile of AID.
This profile will be critical in identifying aberrant AID target sites (ATS), which will
help to identify cancer-associated ATS causal to the development of GC- and post-GC
derived B cell lymphomas. ATS are presently used to identify molecular predictors
that favor AID binding.
Role of PCNA and TLS in programmed mutagenesis During replication, DNA
lesions can block high fidelity DNA polymerases, causing replication forks to arrest.
To increase the fitness of replicating cells in the presence of DNA lesions, i.e. to
continue replication without an a priori repair of the initial lesion, polymerase
switching is required. Monoubiquitination of the highly conserved lysine residue 164
(K164) of the proliferating cell nuclear antigen (PCNA-Ub) triggers switching from a
damage sensitive, high fidelity DNA polymerase to a damage tolerant, low fidelity
translesion synthesis (TLS) DNA polymerase. TLS polymerases can continue
replication across non Watson Crick base pairs. B cells use TLS to introduce somatic
mutations around genetic lesions caused by AID. Owing to the reduced accuracy of
TLS polymerases, TLS is the major cause of DNA-damage-induced mutations. The
finding that DNA damage-inducible ubiquitination of PCNA occurs at identical
positions in yeast and humans suggested an evolutionary conservation of this
molecular switch-board. To determine whether this concept applies to DNA damage
tolerance in mammals, we have generated recombinant mouse strains and cell lines
thereof carrying a conditional PCNA allele as well as a mutant K164R allele. While
PCNA K164R mutant B cells proliferate normally, the mutation spectrum of
hypermutated Ig genes alters dramatically. The failure to modify PCNA K164 in
homozygous mutant B cells resulted in a 10-fold reduction of transitions and
transversions at template A/T - normally accounting for 50% of all mutations
generated (figure 6b). The selective failure to mutate template A/T is compensated by
an increase in mutations at template G/C. This phenotype is similar to that found in
polymerase eta (Polh) and mismatch recognition deficient B cells. Our data indicate
the existence of two alternative mutator pathways, a major PCNA K164 dependent A/T
mutator pathway and a major PCNA K164 independent G/C mutator pathway.
PCNA-Ub, mismatch recognition (MSH2, MSH6) and Polh likely cooperate in
establishing the vast majority of mutations at template A/T during replication of Ig
genes. Consistent with the defined role of PCNA-Ub in yeast, B cells from PCNA K164R
mutant mice are highly sensitive to ultraviolet light (UV), cisplatin (CisPt) and
methyl-methanesulphonate (MMS). PCNAK164R mutant cells experience not just an
S/G2 but also a G1 arrest. We show that the increased DNA damage sensitivity is
caused by a defective recruitment of Polh.
Group leader Heinz Jacobs
59
IMMUNOLOGY
Heinz Jacobs PhD Group leader
Petra Langerak MSc PhD student
Marinus Heideman MSc PhD student
Peter Krijger MSc PhD student
Marc Hogenbirk MSc PhD student
Niek Wit MSc PhD student
Paul Van den Berk Technical Staff
Figure 6a: DNA sliding clamp usage, DNA
polymerases and mutagenesis: SHM is
dominated by the introduction of point
mutations (from: A, G, C, or T to: A, G, C,
or T). We have shown previously, that most
mutations at template A and T - which
depend on mismatch repair (MMR)
proteins Msh2 and Msh6 - require
PCNA-Ub. Equally relevant, our data
indicate that mutations at template G/C do
not depend on PCNA-Ub. PCNA-Ub
enables a polymerase switch between high
fidelity DNA polymerases (Pold/e) and the
A/T mutator Polh. Uracil, which is
generated by AID cytidine deamination
instructs a template T to generate G to A
and C to T transitions. The TLS polymerase
Rev1 contributes to the generation of C to G
and G to C transversions. The
polymerase(s) (Pol?) responsible for G to T
and C to A transversions remain to be
identified.

60
IMMUNOLOGY
Publications
Langerak P, Krijger PH, 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. 2008
Nov 12 (Epub ahead of print)
Van Maldegem F, Scheeren FA,
Jibodh RA, Bende RJ, Jacobs H,
van Noesel CJM. AID splice variants are
deaminase-activity deficient. Blood. 2008
(in press)
Our data identify a dual role for PCNA ubiquitination and Polh: Anti-mutagenic
damage tolerance of UV-induced DNA damage and mutagenic processing of AIDinduced
DNA damage in Ig genes. Polh requires PCNA-Ub to establish mutations at
template A/T. Equally essential, the G/C mutator appears independent of PCNA-Ub.
9-1-1 Complex: An alternative platform for PCNA-independent TLS? The
RAD9/RAD1/Hus1 complex (also known as 9-1-1 complex) is a heterotrimeric DNA
sliding clamp that is structurally very similar to PCNA. In yeast, 9-1-1 was shown to
interact with TLS polymerases and induce TLS independent of PCNA K164
modification. For various reasons, we consider that the 9-1-1 complex provides an
additional platform capable of recruiting specific TLS polymerases in mammalian
cells. At present, we are testing this hypothesis in mouse models carrying genetically
predefined mutations in the 9-1-1 complex.
Figure 6b: Model: A role of the 9-1-1 DNA sliding clamp in SHM Both, MMR and long patch base
excision repair (BER) generate single strand gaps at sites of DNA damage (asterisk). The ubiquitin
conjugase/ligase Rad6/Rad18 complex binds single stranded DNA and mediates ubiquitination of PCNA
and - as shown so far only in yeast - ubiquitination of an alternative DNA sliding clamp, the 9-1-1
complex. We speculate that 9-1-1 complex is required to generate most G/C transversions. This model is
currently addressed in mice carrying specific mutations in 9-1-1.
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 temporary regulator of allelic
exclusion during B cell development.

COMBATING TUMOR IMMUNE ESCAPE
The aim of our research is to identify mechanisms that tumors use to escape from
anti-tumor immune responses. The characterization of inhibitory molecules and
pathways that are involved in these escape mechanisms may help in designing novel
approaches to improve anticancer immunotherapy. One immunotherapy approach is
adoptive transfer of tumor-reactive T cells. Tumor control requires expansion and
survival of tumor-reactive T cells without exhaustion in vivo. This may best be
achieved by transferring peripheral T cells into lymphopenic hosts, since transfer is
followed by acute homeostatic proliferation (HP). We study this HP process with the
aim to optimize adoptive T cell immunotherapy.
Role of co-inhibitory molecules during tumor immune escape Sufficient T cell
activation depends on TCR ligation and positive secondary signals to prevent anergy
induction of the activated T cell. Recent work revealed that this secondary signal is
not an on-off phenomenon but a gradually modulated signal intensity orchestrated by
several co-stimulatory and co-inhibitory molecules inducibly expressed on the T cell
surface. We and others have shown that one of the ligands (PD-L1) of such a coinhibitory
molecule (PD-1) is highly expressed on tumor cells and impairs immune
responses against tumor cells in animal models as well as when testing human
T cells in vitro. We demonstrated further that susceptibility of T cells for PD-1
mediated signals inversely depends on the tumor antigen-density on tumor cells.
This indicates that a decreased tumor antigen expression combined with higher PD-
L1 expression might be the initial change towards tumor immune escape.
To show prognostic relevance of PD-L1 expression on melanomas, we currently
examine tumor samples from melanoma patients treated at the NKI-AVL. The in
vitro results and first screening of the melanomas have convinced us that interfering
with PD-L1/PD-1 interaction should be tested in early phase clinical trials in humans.
Possible technical approaches for a clinical implementation are either the blockade of
PD-L1 or PD-1 by means of monoclonal antibodies or the suppression of PD-1 T cell
surface expression via siRNA. Currently we are comparing all three approaches
concerning efficacy and availability for transfer into the clinic.
Homeostatically proliferating T cells for the treatment of cancer Transfer of
naïve peripheral T cells into lymphopenic recipients results in a slow cytokine-driven
proliferation of these T cells. During this HP, T cells acquire effector functions
(IFN-production, lytic activity) while keeping characteristics of naïve T cells resulting
in superior lymph node homing capabilities, decreased anergy induction and
superior tumor growth control compared to naïve or antigen-activated counterparts.
While the use of lymphopenia to induce HP by preconditioning the patient has been
implemented recently in pilot studies elsewhere, we focus on dissecting characteristic
of these HP T cells with the aim to induce HP in vitro, thus not requiring the
intensive pretreatment of the patient. We have identified CD62L, an adhesion
molecule, to be a pivotal molecule for homing of the transferred T cells to the
peripheral lymph node, the only compartment acute HP relies on. Characterization
of HP T cells using microarray analysis revealed several other molecules that are
exclusively expressed in/on HP T cells. The identified molecules of the HP
phenotype shall be tested for their functional relevance by means of transgenic
overexpression or siRNA-mediated silencing.
Development of an inducible spontaneous murine melanoma model Adoptive
T cell therapies have been tested so far predominantly in transplantable tumor
animals models. Such models often do not mimic the complex interaction between
the tumor cell and the tumor microenvironment and may therefore be of little
predictive value for cancer therapy. Mouse models for spontaneous melanoma better
approximate the patient situation, but due to the late onset of tumor formation are
less practical for long term immunotherapeutic experiments. To bypass this dilemma
we aim to develop an inducible mouse melanoma model with a faster tumor onset.
Group leader Christian Blank
61
IMMUNOLOGY
Christian Blank MD PhD Group leader
Kerstin Schuster PhD Post-doc
Lisa Borkner MSc PhD student
Lukas Kremmler PhD student
Jules Gadiot Technical staff

62
MOLECULAR BIOLOGY
Division head, group leader Hein Te Riele
Hein Te Riele PhD Group leader
Rob Dekker PhD Post-doc
Camiel Wielders PhD Post-doc
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
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 pathway in mutation avoidance and (2) the role of cell cycle
checkpoints in tumor suppression.
DNA MISMATCH REPAIR
Inherited defects in DNA mismatch repair (MMR) underlie the cancer syndrome
HNPCC (hereditary non-polyposis colorectal cancer). The primary function of MMR
is correction of DNA replication errors. Mismatch repair is initiated by MSH2/
MSH6 or MSH2/MSH3 protein complexes, which recognize base.base mismatches
and small loops of unpaired bases. 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.
Mismatches can also occur in heteroduplex DNA formed by exchange of homologous
but not perfectly identical DNA strands. Recognition of such mismatches by MMR
leads to dissociation of the heteroduplex and abortion of the recombination reaction.
Finally, O 6 -methylguanine lesions in DNA are processed by the MMR system into toxic
lesions causing cell death. Thus, DNA MMR has three main functions: it acts in an
anti-mutagenic and anti-recombinogenic function, and it mediates the toxicity of
methylating agents.
Oligonucleotide-directed gene modification We have recently found that MMR
imposes a strong impediment to subtle gene modification by short single-stranded
DNA oligonucleotides in mouse embryonic stem (ES) cells. As readout for
oligonucleotide-mediated gene targeting (‘oligo targeting’) we measure re-activation
of a disabled neomycin (neo) reporter gene carrying a mutation in the start codon
(ATG ➝ AAG). Single-stranded deoxyribo-oligonucleotides of ±38 residues
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 of up to
10 -5 -10 -4 (Dekker et al., NAR 2003;31:e27).
To alleviate the mutagenic effects of constitutive MMR deficiency, we render ES cells
permissive for oligo targeting by transiently suppressing MSH2 protein levels by
RNA interference. In an attempt to further increase the efficacy of oligo targeting, we
have generated a library of short-hairpin-RNA-expressing vectors suppressing the
activity of the major DNA repair pathways. Somewhat unexpectedly, none of the
knock-down vectors affected the oligo targeting efficiency. These results seem to
discard homologous recombination as a critical mediator of oligonucleotide-mediated
gene modification. We also found that the level of transcription did not affect the
efficacy of oligo targeting. Together, these results suggest that oligo targeting occurs
in the context of DNA replication.
Unclassified variants of MMR genes Besides mutations that fully disrupt gene
function, subtle mutations affecting only a single codon are frequently found in
(suspected) HNPCC. The consequences of single amino acid substitutions are often
difficult to predict. Such allelic variants are therefore termed ‘Unclassified Variants’
(UV). We have developed a three-step procedure to study the pathogenicity of
missense variants of MSH2 and MSH6 found in the human population. First, we
recreate the allelic variant in mouse ES cells by oligonucleotide-mediated codon
substitution. Secondly, we have developed a method to make ES cells homozygous
for the mutated allele. Thirdly, we study mutant ES cells for their capacity to sustain
the three main MMR functions. By this method we could thus far identify one UV as
deleterious, one as an innocent polymorphism and a third as a separation of function
mutant with reduced MMR capacity. As we use mouse ES cells, we were able to
introduce the latter mutant into the germ line of mice to study whether this variant
predisposes to cancer.

THE FANCONI ANEMIA PATHWAY
Genetic instability and cancer predisposition may also result from inherited defects
in chromosome maintenance mechanisms. An example is Fanconi anemia (FA), a
genetic disorder characterized by developmental abnormalities and a high incidence
of AML and epithelial tumors. FA is caused by bi-allelic inherited defects in either
one of 13 genes, FANCA, B, C, D1, D2, E, F, G, I, J, L, M, N. At the cellular level,
defects in FA genes cause high sensitivity to crosslinking agents as manifested by G 2
arrest, chromosomal aberrations and cell death. It has therefore been hypothesized
that chromosomal instability caused by acquired defects in FA genes may underlie
sporadic tumor development. Furthermore, such tumors may be exquisitely
susceptible to crosslinking chemotherapeutics like cisplatin.
To assess the significance of the FA genome maintenance pathway in suppression of
cancer, we have generated mouse models carrying defects in the FA genes Fancf and
Fancm. These mice are crossed into cancer prone Apc +/- mice to study whether FA
defects affect the incidence, latency and spectrum of tumorigenesis.
CELL CYCLE CHECKPOINTS
Loss of G 1/S control is a frequent, if not mandatory event in development of cancer.
However, we found that mouse embryonic fibroblasts (MEFs) devoid of proper G1/S
control still rely on mitogens and attachment to proliferate indefinitely. We made
such cells by genetically ablating the pocket proteins pRB, p107 and p130. These
proteins impede the G 1/S transition of the cell cycle by binding to and inhibiting E2F
transcription factors. Their activity is relieved upon phosphorylation by Cyclin D- and
E-dependent kinases following mitogenic signaling. Complete phosphorylation of
pocket proteins defines the so-called G 1 restriction point after which cells no longer
require mitogens to enter S-phase. Ablation of pocket proteins in MEFs (TKO MEFs)
alleviates the G 1 restriction point and allows cells to pass G1 and enter S phase in the
absence of mitogens. We are using pocket-protein-defective cells to study
mechanisms that restrict proliferation beyond the G1 restriction point.
Growth-factor independence TKO MEFs can pass through S-phase in the absence
of mitogenic signalling, but are then restricted by: (1) massive apoptosis and (2) G 2
arrest that became apparent upon expression of the apoptosis inhibitor BCL2. G2
arrest was effectuated through inhibitory interactions of the cyclin-dependent-kinase
inhibitors p21 CIP1 and p27 KIP1 with Cyclins A and B1. The arrest was reversible: readdition
of serum for 3-6 h led to mitotic entry about 15 h later, thus defining a
restriction point operating in G 2. Caffeine accelerated mitogen-induced cell cycle reentry,
which is indicative for activation of a DNA damage checkpoint. Indeed, we
found mitogen-starved G 2-arrested TKO cells to accumulate g-H2AX/RAD51 foci that
gradually disappeared upon mitogen stimulation (figure 1). Furthermore, cells
entering mitosis showed excessive chromosomal aberrations. None of these effects
were seen in TKO cells that were cultured in the presence of mitogens. Together,
these results suggest that transient mitogen starvation of TKO cells induces DNA
damage leading to chromosomal aberrations.
Anchorage independence We also showed that partial or full pocket protein
ablation was not sufficient to sustain anchorage-independent growth, even upon
expression of RAS V12 , although DKO (Rb -/- p107 -/- or Rb -/- p130 -/- ) and TKO MEFs were
immortal. Apparently, a cell cycle mechanism still operates to restrict proliferation of
these cells in soft agar. We found anchorage-deprived RAS V12 -expressing MEFs
arrested in G 1 or G 2, dependent on which pocket proteins were still active. Thus,
TKO cells arrested in G 2 while DKO cells also arrested in G 1. 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 p19 ARF .
TBX2-mediated downregulation of p53 and p21 CIP1 rescued cyclin-dependent kinase
acivities more effectively in DKO MEFs than Rb -/- MEFs, consistent with the higher
transformability of DKO MEFs.
Publications
63
MOLECULAR BIOLOGY
Foijer F, Simonis M, Van Vliet M,
Kerkhoven R, Sorger PK, Te Riele H.
Oncogenic pathways impinging on the G2
restriction point. Oncogene 2008;27:1142-54
Vormer TL, Foijer F, Wielders CLC,
Te Riele H. Anchorage-independent growth
of pocket-protein-deficient murine
fibroblasts requires bypass of G2 arrest and
can be accomplished by expression of TBX2.
Mol Cell Biol 2008;28:7263-73
Figure 1: Mitogen-starved TKO MEFs (lacking
Rb, p107 and p130) accumulate DNA damage.
(A) g-H2AX foci in TKO MEFs mitogenstarved
for 7 days. (B) Graphic representation
of g-H2AX foci accumulation in mitogenstarved
TKO MEFs. (C) Disappearance of
g-H2AX foci upon mitogen addition.

64
MOLECULAR BIOLOGY
Group leader Piet Borst
Piet Borst MD PhD Group leader
Sven Rottenberg DVM PhD Dipl. ECVP
Senior post-doc
Henri Van Luenen PhD Academic staff
Jayasree Iyer PhD Post-doc
Marina Pajic PhD Post-doc
Saara Vainio MD PhD Post-doc
Koen Van de Wetering DVM PhD Post-doc
Maaike Gonggrijp DVM PhD student
Serge Zander DVM MSc PhD student
Marcel De Haas Technical staff
Liesbeth Van Deemter Technical staff
Wouter Feddema Technical staff
Ariena Kersbergen Technical staff
Bas Ter Riet Technical staff
Publications
Borst P. Mega dose vitamin C as therapy
for human cancer? Proc Natl Acad Sci
USA 2008 (in press)
Borst P, Rottenberg S, Jonkers J. How do
real tumors become resistant to cisplatin?
Cell Cycle 2008;7:1353-9
Borst P, Sabatini R. Base J: discovery,
biosynthesis and possible functions. Ann
Rev Microbiol 2008;62:235-51
Borst P, van de Wetering K,
Schlingemann R. Does the absence of
ABCC6 (multidrug resistance protein 6) in
patients with Pseudoxanthoma elasticum
prevent the liver from providing sufficient
vitamin K to the periphery? Cell Cycle
2008;7:1575-9
De Wolf C, Jansen R, Yamaguchi H,
de Haas M, van de Wetering K,
Wijnholds J, Beijnen J, Borst P.
Contribution of the drug transporter
ABCG2 (breast cancer resistance protein)
to resistance against anticancer nucleosides.
Mol Cancer Ther 2008;7:3092-102
DNA BASE J
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). Our recent work
indicates 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. Solving the cause of death
remains a top priority. 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 (NKI-AVL) 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.
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. We use the baculovirus system to produce insect cell
membrane vesicles containing high amounts of transporter protein and suitable for
vesicular transport studies. We also use transfected polarized kidney cell monolayers
in which the transporters either route to the apical or the basolateral membrane,
allowing the study of vectorial transport through the cell layer. 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.
An interesting example is provided by resveratrol-glucuronide (R-gluc), which mainly
enters the body via MRP3. We have shown that R-gluc is a high-affinity substrate for
MRP3 and that R-mono-sulphate and R-di-sulphate are high-affinity substrates for
another ABC-transporter: BCRP (ABCG2) (figure 2). We have initiated a systematic
search for other glucuronides transported by MRPs by comparing the glucuronide
derivatives in plasma/urine of WT and KO mice using Mass Spectrometry. We have
identified several glucuronidated 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.
MRP6 and PXE Pseudoxanthoma elasticum (PXE) is an autosomal recessive disease
characterized by a progressive mineralization of connective tissue, resulting in skin,
arterial and eye disease. Classical PXE is caused by mutations in the MRP6 (ABCC6)
gene. Recent studies by Uitto et al. on Abcc6 -/- mice show that the absence of ABCC6
in the liver is crucial for PXE and confirm the ‘metabolic disease hypothesis’ for PXE,
which states that tissue calcification is due to the absence of a plasma factor secreted

from the basolateral hepatocyte membrane. We have proposed that this plasma factor
is vitamin K (precursor). We think that vitamin K (precursor) is secreted by ABCC6
from the liver as a glutathione-, glucuronide-, or sulphate conjugate and that this
supplements the vitamin K need of peripheral tissues that receive insufficient
vitamin from the diet, because dietary vitamin K is effectively extracted from blood by
the liver. Peripheral tissue vitamin K is needed for the gamma-carboxylation of
glutamate residues in proteins known to be required for counteracting calcification of
connective tissue throughout the body.
We have started an ambitious program to test this hypothesis in collaboration with
Arthur Bergen (AMC), who generated an Abcc6 -/- mouse. We have made conjugates
of menadione (Vit. K3) and are testing these in vesicular transport assays with MRP6vesicles.
DRUG RESISTANCE IN ‘SPONTANEOUS’ MOUSE TUMORS
We have started a project in collaboration with Jos Jonkers (NKI-AVL) to study
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 promotor, 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 (3-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
AstraZeneca. We are crossing 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 transporters.
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 tumor-initiating 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 (figure 3). We are currently 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.
Figure 3: Fractionation of Brca1-/-;p53-/- tumors using FACS. A. Sorting of live (propidium iodidenegative),
Lin- (non-endothelial, non-fibroblastic and non-hematopoietic) cells with CD24- and CD49fspecific
antibodies. B. Tumorigenicity of limiting dilutions of sorted fractions after orthotopic
transplantation into syngeneic animals. C. Tumor incidence summary of the graph shown in B. D.
Percentage of cell fractions in tumors before treatment and in remnants 10 days after the injection of 6mg
cisplatin (CDDP) per kg i.v.
65
MOLECULAR BIOLOGY
Publications (continued)
Rottenberg S, Jonkers J. Modeling therapy
resistance in genetically engineered mouse
cancer models. Drug Resist Updates
2008;11:51-60
Rottenberg S, Kersbergen A,
van der Burg E, Nygren AOH, Zander S,
Derksen PW, de Bruin M, Zevenhoven J,
Lau A, Boulter R, Cranston A, O’Connor
MJ, Martin NMB, Borst P, Jonkers J. High
sensitivity of BRCA1-deficient mammary
tumors to the PARP inhibitor AZD2281
alone and in combination with platinum
drugs. Proc Natl Acad Sci U S A
2008;105:17079-84
Figure 2: Transport of Resveratrol-3-sulphate
(Res-3-S) and Resveratrol-di-sulphate (Resdi-S)
by BCRP in vesicular transport
experiments. (A) Schematic overview of the
synthesis of [ 3 H]resveratrol-sulphates used in
the vesicular transport experiments
presented. [ 3 H]resveratrol was converted into
[ 3 H]Res-3-S and [ 3 H]Res-di-S using mouse
liver cystosol and the sulphate donor
3’-phosphoadenosine-5’phosphosulphate
(PAPS). Insets represent HPLC
chromatograms of the purified [ 3 H]
resveratrol-sulphates.
Time course experiments of membrane
vesicles containing human BCRP using 140
nM [ 3 H]Res-3-S (B) or [ 3 H]Res-di-S (C).
Transport was determined in the presence or
absence of 4 mM ATP as indicated.
Concentration-dependent transport of Res-
3-S and Res-di-S by BCRP is shown in (D)
and (E), respectively. Values are corrected
for transport determined in absence of ATP.
Each data point and error are the means ±
SD of an experiment performed in
triplicate.

66
MOLECULAR BIOLOGY
Group leader Jos Jonkers
Jos Jonkers PhD Group leader
Karin De Visser PhD Research associate
Peter Bouwman PhD Post-doc
Michiel De Bruin PhD Post-doc
Gilles Doumont PhD Post-doc
Marco Koudijs PhD Post-doc
Xiaoling Liu PhD Post-doc
Ewa Michalak PhD Post-doc
Petra Ter Brugge PhD Post-doc
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
Tanya Braumuller Research assistant
Eva Kregel Research assistant
Mark Pieterse Research assistant
Ewoud Speksnijder Research assistant
Eline Van der Burg Research assistant
Publications
Rottenberg S, Jonkers J. Modeling therapy
resistance in genetically engineered mouse
cancer models. Drug Resist Updat.
2008;11:51-60
Klijn C, Holstege H, de Ridder J, Liu X,
Reinders M, Jonkers J*, Wessels L*.
Identification of cancer genes using a
statistical framework for multi-experiment
analysis of non-discretized array CGH
data. Nucleic Acids Res. 2008;36:e13
(* joint corresponding authors).
Doumont G, de Visser KE, Derksen PW,
Jonkers J. Models for angiogenesis:from
fundamental mechanisms to anticancer
treatment research. Drug Discov
Today:Disease Models 2008;4:75-82
Borst P, Rottenberg S, Jonkers J. How do
real tumors become resistant to cisplatin?
Cell Cycle 2008;7:1353-1359
MOUSE MODELS OF BREAST CANCER
The focus of our research group is on the genetic dissection of human breast cancer
through the use of genetically engineered mouse models. For this, we have developed
models for p53-induced breast cancer, BRCA1- and BRCA2- associated hereditary
breast cancer, and E-cadherin-associated metastatic breast cancer. We are using these
models to (1) investigate genotype-phenotype 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.
Conditional mouse models for BRCA-associated breast cancer We have
previously generated conditional 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 share histopathological and molecular features with
BRCA1-deficient breast cancers in women: they are highly proliferative, poorly
differentiated, hormone receptor and HER2 negative mammary adenocarcinomas
with pushing borders and 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.
Tumor intervention studies in the BRCA mammary tumor models The central
role of BRCA1 and BRCA2 in DNA double-strand break (DSB) repair via homologous
recombination (HR) implies that BRCA-deficient tumors are especially sensitive to
DSB inducing chemotherapeutics. Indeed, in vitro cytotoxicity studies with BRCA2deficient
mammary tumor cell lines and in vivo tumor intervention studies in our
BRCA1 mammary tumor model showed high sensitivity of BRCA-deficient tumors to
DNA-damaging agents such as doxorubicin or platinum drugs.
In collaboration with Sven Rottenberg, Piet Borst and KuDOS Pharmaceuticals, we
have used our BRCA1/2 models to test the anti-tumor effects of PARP inhibition,
which may be selectively toxic to HR-deficient cells because it suppresses DNA
single-strand break repair. Indeed, in vitro studies showed selective toxicity of the
PARP inhibitor AZD2281 in Brca2 -/- ;p53 -/- mammary tumor cells, compared to p53 -/-
cells. Administration of AZD2281 to mice with Brca1 -/- ;p53 -/- mammary tumors
induced tumor regression without signs of toxicity, resulting in strongly increased
survival. Long-term treatment with AZD2281, however, resulted in the development
of drug resistance caused by upregulation of P-glycoprotein drug efflux pumps.
Importantly, resistance could be reversed by co-administration of AZD2281 and the
P-glycoprotein inhibitor tariquidar. Moreover, combination of AZD2281 with
platinum drugs significantly prolonged recurrence-free survival, suggesting that
AZD2281 potentiates the effect of these DNA-damaging agents. Together, these data
demonstrate in vivo efficacy of AZD2281 against BRCA-deficient breast cancer, and
illustrate how conditional mouse models of human cancer can be used for preclinical
evaluation of novel therapeutics, for investigating drug resistance mechanisms and
for testing ways to overcome therapy resistance.
Conditional mouse models for E-cadherin-deficient metastatic breast cancer
While metastatic disease is the main cause of death in breast cancer patients, the
underlying mechanisms are poorly understood. Loss of E-cadherin is strongly
associated with tumor invasion and metastasis, as well as with invasive lobular
carcinoma (ILC), which accounts for 10-15% of all breast cancers. To study the role of
E-cadherin in breast oncogenesis, we have generated a mouse model for invasive
lobular breast carcinoma (ILC) 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 several kinases that are
essential for survival of Ecad -/- ;p53 -/- cells under non-adherent culture conditions. In
vivo validation studies will be performed using pharmacological inhibitors or
inducible expression of shRNAs against these kinases in Ecad -/- ;p53 -/- mammary
tumors in mice.
Tumor intervention studies in the E-cadherin mammary tumor model We are
also using the E-cadherin mammary tumor model for preclinical testing of novel
therapeutics. For this purpose we have derived panels of clonal, luciferase-marked
mammary tumor cell lines from our mouse models. Together with the spontaneous
models, these reagents permit validation of candidate drug targets and testing of
(combinations of) anticancer drugs on four different levels: (1) in vitro, on the panels
of tumor cell lines; (2) in vivo, on tumor outgrowths from orthotopically grafted cell
lines; (3) in vivo, on tumor outgrowths from orthotopically grafted tumor fragments
(allowing parallel treatments on genetically identical tumors); (4) in vivo, on the
sporadic tumors that develop in the mouse mammary tumor models (allowing
intervention at early stages of tumor development). We are currently using this multilevel
platform to test a number of conventional and targeted anti-cancer drugs.
Array-CGH analysis of mouse mammary tumors We have performed array-based
comparative genomic hybridization (aCGH) analysis of panels of mammary tumors
derived from our conditional mouse models. To identify regions with significantly
recurrent DNA copy number aberrations (CNAs), we have developed KC-SMART
(Kernel Convolution – a Statistical Method for Aberrant Region deTection) for multiexperiment
analysis of aCGH data. KC-SMART generates a Kernel Smoothened
Estimate (KSE) of recurrent CNAs across the genome, aggregated over all tumors.
The peaks in the KSE curves are tested against randomly permutated data to identify
significantly recurrent CNAs. We have used this approach to significantly recurrent
CNAs in mouse and human breast cancer panels and in human cancer cell line
panels. We are using these data sets for (1) cross-species comparisons of CNAs in
mouse and human tumors; (2) co-occurrence analysis of CNAs in human tumor cell
lines; (3) integration of aCGH and gene expression data to identify candidate cancer
genes in recurrent CNAs.
The role of innate and adaptive immunity in breast cancer development
Studies in a transgenic mouse model for skin carcinogenesis revealed an unexpected
role for B lymphocytes and their soluble mediators in initiating chronic inflammation
during premalignancy and subsequent progression to invasive skin cancer (De Visser
et al. Cancer Cell 2005;7:411-23). We are currently investigating whether a similar link
exists between innate/adaptive immunity and breast cancer development,
progression and therapy resistance in our mouse model for E-cadherin deficient
metastatic breast cancer. Similar to human breast cancer lesions, neoplastic
mammary glands of these mice are characterized by leukocyte infiltration. To assess
the roles of innate and adaptive immune systems in breast cancer development and
progression, these mice have been crossed with various immune-deficient (Rag1 -/- ,
Il2rg -/- ) mice. In the resulting compound mutant mice, chronic inflammation,
angiogenesis, premalignant progression, cancer development and metastasis
formation are being analyzed. We will also perform adoptive transfer experiments
with bone marrow cells from mice with macrophage-specific expression of the
diphtheria toxin receptor to study the effects of diphtheria toxin-mediated
macrophage ablation on mammary tumor development and progression.
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MOLECULAR BIOLOGY
Publications (continued)
Uren AG, Kool J, Matentzoglu K,
de Ridder J, Mattison J, van Uitert M,
Lagcher W, Sie D, Tanger E, Cox T,
Reijnders M, Hubbard TJ, Rogers J,
Jonkers J, Wessels L, Adams DJ,
van Lohuizen M, Berns A. Large scale
mutagenesis in p19ARF and p53 deficient
mice identifies cancer genes and their
collaborative networks. Cell 2008;133:727-741
Evers B, Drost R, Schut E, de Bruin M,
van der Burg E, Derksen PW, Holstege H,
Liu X, van Drunen E, Beverloo HB,
Smith GC, Martin NM, Lau A,
O’Connor MJ, Jonkers J. Selective inhibition
of BRCA2-deficient mammary tumor cell
growth by AZD2281 and cisplatin. Clin
Cancer Res. 2008;14:3916-3925
Pietersen A, Evers B, Prasad AA,
Tanger E, Cornelissen-Steijger P,
Jonkers J, van Lohuizen M. Bmi1 regulates
stem cells but also proliferation and
differentiation of committed cells in
mammary gland epithelium. Curr Biol.
2008;18:1094-1099
Bouwman P, Jonkers J. Mouse models for
BRCA1 associated tumorigenesis:from
fundamental insights to preclinical utility.
Cell Cycle 2008;7:2647-2653
Rottenberg S, Jaspers JE, Kersbergen A,
van der Burg E, Nygren AO, Zander SA,
Derksen PW, de Bruin M, Zevenhoven J,
Lau A, Boulter R, Cranston A,
O’Connor MJ, Martin NM, Borst P,
Jonkers J. High sensitivity of BRCA1deficient
mammary tumors to the PARP1
inhibitor AZD2281 alone and in
combination with platinum drugs. Proc
Natl Acad Sci USA. 2008;105:17079-17084
Lehembre F, Yilmaz M, Wicki A,
Schomber T, Strittmatter K, Ziegler D,
Kren A, Went P, Derksen PW, Berns A,
Jonkers J, Gerhard Christofori G. NCAMinduced
focal adhesion assembly:a functional
switch upon loss of E-cadherin. EMBO J.
2008;27:2603-2615
Pietersen AM, Horlings HM,
Hauptmann M, Langerod A, Ajouaou A,
Cornelissen-Steijger P, Wessels LF,
Jonkers J, van de Vijver M, van Lohuizen M.
Ezh2 and Bmi1 inversely correlate with
prognosis and p53 mutation in breast
cancer. Breast Cancer Res. 2008 (in press)

68
MOLECULAR BIOLOGY
Group leader Sabine Linn
Sabine Linn MD PhD Group leader
Chiel De Bruin PhD Post-doc
Michael Knauer MD PhD Post-doc
Jolien Bueno de Mesquita MD PhD student
Marleen Kok MD PhD student
Marieke Vollebergh MD PhD student
Rutger Koornstra MD PhD student
Ingrid Van der Heijden Technical Staff
Publications
Bueno-de-Mesquita JM, Linn SC, Keijzer R,
Wesseling J, Nuyten DS, van KC, Meijers C,
de Graaf PW, Bos MM, Hart AA, Rutgers EJ,
Peterse JL, Halfwerk H, de GR, 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 2008; DOI
10.1007/s10549-008-0191-2 (e-pub ahead of
print)
Heerema-McKenney A, Wijnaendts LC,
Pulliam JF, Lopez-Terrada D,
McKenney JK, Zhu S, Montgomery K,
Mitchell J, Marinelli RJ, Hart AA,
van de Rijn M, Linn SC. Diffuse myogenin
expression by immunohistochemistry is an
independent marker of poor survival in
pediatric rhabdomyosarcoma: a tissue
microarray study of 71 primary tumors
including correlation with molecular
phenotype. Am J Surg Pathol 2008;32:1513-22
Holm C, Kok M, Michalides R, Fles R,
Koornstra R, Wesseling J, Hauptmann M,
Neefjes J, Peterse J, Stal O, Landberg G,
Linn S. Phosphorylation of the oestrogen
receptor alpha at serine 305 and prediction
of tamoxifen resistance in breast cancer. J
Pathol 2008; DOI 10.1002/path.2455
(e-pub ahead of print)
Schilder CM, Linn SC, Van Dam FS,
Schagen SB. [The effect of hormone therapy
on cognitive function in patients with breast
cancer]. Ned Tijdschr Geneeskd
2008;152:494-8
MOLECULAR DISSECTION OF BREAST AND LUNG 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 and non-small cell lung cancer (NSCLC) 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 and NSCLC patients into resistant and sensitive to a particular drug.
In addition, we use conditional mouse models for breast cancer, and derived clonal
cell lines, to study differential chemosensitivity in a controlled fashion.
A second research line focuses on the impact of prognostic molecular classifiers on
adjuvant systemic treatment advice in breast cancer.
Compliance with national guidelines, revision of guidelines and 70-gene
prognosis signature We have demonstrated that the introduction of national
guidelines for adjuvant systemic treatment of breast cancer in 2002 led to a more
uniform treatment advice in The Netherlands. The 70-gene prognosis signature did
not lead to a decrease in adjuvant systemic treatment prescription before 2008.
However, we calculated that the revision of the national guidelines in 2008 would
lead to an increased use of adjuvant systemic therapy in lymph-node negative
patients from 50% to 75%. Based on this, we calculated that use of the
70-gene prognosis signature would lead to a 10-25% decrease in the use of adjuvant
systemic treatment without compromising survival (see also Division of
Experimental Therapy).
Value of 70-gene prognosis signature in defined breast cancer subgroups In
collaboration with Division of Experimental Therapy and Agendia BV, using a pooled
database of ~1800 breast cancer patients with data on the 70-gene prognosis
signature, we have demonstrated that the 70-gene prognosis signature retained its
prognostic value in patients with tumors smaller than 20 mm (N=224).
Impact of inter-observer variation in tumor grade on adjuvant systemic
treatment advice We studied inter-observer variation in ~700 lymph-node negative
breast cancer patients. The local pathological examination was discordant with the
central review for histologic grade in 28% (kappa 0.56; grade 2 tumours 35%
discordant). If Adjuvant Online version 8.0 was applied, 8% of patients (kappa 0.83)
would have been assigned to a different clinical risk group (see also Division of
Experimental Therapy).
Development of a predictive test for tamoxifen resistance in breast cancer
Experimental studies have shown that phosphorylation of ERa at serine 118
(ERaS118-P) is required for downregulation of ERa-induced gene expression by
tamoxifen. In collaboration with the group of Rob Michalides (Division of Cell
Biology II) and the group of Göran Landberg (Lund University, Lund, Sweden and
Breakthrough Breast Cancer Research Unit, Paterson Institute for Cancer Research,
Manchester, UK) we have studied the expression pattern of ERaS118-P in 239 breast
cancer patients, who participated in a randomized trial of adjuvant tamoxifen versus
no endocrine therapy. Patients with ERaS118-P high tumors benefited from adjuvant
tamoxifen (HR=0.36, 95%CI 0.20-0.65, p=0.001) while patients with ERaS118-P low
tumors did not (HR=0.87, 95%CI 0.51-1.48, p=0.60). In multivariate analysis, the
interaction between ERaS118-P and treatment was significant (p=0.037).
We are currently collecting tumor material of approximately 1000 patients who
participated in another randomized trial of adjuvant tamoxifen versus no endocrine
therapy, in order to confirm our findings in a second, independent series.
CYP450 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

Figure 4: Relation of ERaS118-P with outcome after tamoxifen treatment. Kaplan-Meier survival analysis
according to ERaS118-P levels. Recurrence-free survival (RFS) of premenopausal patients who had been
randomly assigned to tamoxifen or no adjuvant systemic treatment. ERaS118-P Low (Allred score 0-6)
(A) and ERaS118-P High tumors (Allred score 7-8) (B) were analyzed separately.
Hazard ratios and p-values are based on univariate Cox regression analysis.
endoxifen. CYP450 analysis of estrogen receptor positive tumors has suggested that
polymorphisms of 2D6 and 2C19 are predictive of outcome after adjuvant tamoxifen
therapy in patients with early breast cancer. In collaboration with Rob van Schaik and
Els Berns (Erasmus Medical Center, Rotterdam) we are investigating the predictive
and prognostic role of these polymorphisms in relation to tamoxifen treatment and
breast cancer outcome. In addition, in collaboration with Jos Beijnen (Division of
Medical Oncology) and Hans Bonfrer (Division of Diagnostic Oncology) we are
investigating 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 (side study of EORTC 10901; accrual from
1995-1999). Breast cancer outcome of these patients will also be correlated with
CYP450 polymorphisms and serum concentrations of tamoxifen and metabolites.
Molecular mechanisms underlying sensitivity for high dose alkylating agents
Using an array comparative genomic hybridization (aCGH) classifier initially
constructed to identify BRCA1-mutated tumours we were able to identify a group of
metastatic breast cancer patients treated with platinum-based chemotherapy with a
high complete remission-rate and long progression free survival.
We found a high correlation between our BRCA1-like classifier, an expansive growth
pattern on histology and an estrogen receptor (ER) negative status. To test the
performance of this classifier in an independent patient series, we used a
randomized clinical trial comparing 5 x FEC with 4 x FEC followed by 1x carboplatinthiotepa-cyclophosphamide
(CTC) for high risk, primary operable breast cancer
[Rodenhuis et al., New Engl J Med, 2003]. We first evaluated the performance of an
expansive growth pattern versus an invasive growth pattern in de subgroup of ER
negative, HER2 negative breast cancer patients. Indeed, patients with an expansive
growth pattern had an extremely good outcome after high dose CTC. Further
analyses are ongoing. (Collaboration with Divisions of Experimental Therapy,
Molecular Biology and Medical Oncology).
Screening for drugs active against lobular breast cancer In collaboration with
the Jonkers group, we have generated and characterized a panel of 27 murine breast
cancer cell lines derived from tumors with a Trp53 -/- or Cdh1 -/- ;Trp53 -/- genotype,
modeling ductal and lobular breast carcinoma, respectively. When comparing drug
sensitivity under cluster versus low cluster plate growth conditions, differential
sensitivity was observed for src inhibitors in cell lines with a Cdh1 -/- ;Trp53 -/- genotype,
displaying greater sensitivity under low cluster growth conditions. Further analyses
are ongoing, in order to unravel the underlying mechanisms that can explain this
differential sensitivity.
Serum concentrations of EGFR ligands and sensitivity for erlotinib in NSCLC
In 2003 approximately 7200 patients were diagnosed with NSCLC in the Netherlands
of which 44% was older than 70 years. Besides relatively toxic palliative chemotherapy,
targeted therapies (i.e. EGFR-inhibitors) may be used in these patients. However,
EGFR-inhibitors have a low response rate and have side-affects. We have shown that
the EGFR ligands amphiregulin and transforming growth factor alpha, measured in
serum, can be used as predictive markers of resistance to EGFR-inhibitors.
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MOLECULAR BIOLOGY
Publications (continued)
Kok M, Linn SC, Van Laar RK, Jansen MP,
van den Berg TM, Delahaye LJ, Glas AM,
Peterse JL, Hauptmann M, Foekens JA,
Klijn JG, Wessels LF, Van’t Veer LJ,
Berns EM. Comparison of gene expression
profiles predicting progression in breast
cancer patients treated with tamoxifen.
Breast Cancer Res Treat 2008; DOI
10.1007/s10549-008-9939-y (e-pub ahead
of print)
Kok M, Linn SC, van de Vijver MJ.
Estrogen receptor phenotypes defined by gene
expression profiling. In: Fuqua SAW (ed).
Hormone receptors in breast cancer. New
York, NY: Springer, 2008 (in press)
Linn SC, Bueno-de-Mesquita JM, van de
Vijver MJ. Are gene signatures better than
traditional clinical factors? – Authors’ reply.
Lancet Oncol 2008;9:198-9
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 2008;1-10

70
MOLECULAR BIOLOGY
Group leader Alfred Schinkel
Alfred Schinkel PhD Group leader
Dilek Iusuf MSc PhD student
Jurjen Lagas MSc PhD student
Evita Van de Steeg MSc PhD student
Robert Van Waterschoot MSc PhD student
Marijn Vlaming MSc PhD student
Corina Van der Kruijssen MSc Technical staff
Anita Van Esch Technical staff
Els Wagenaar Technical staff
Publications
Marchetti S, Oostendorp RL, Pluim D,
van Eijndhoven M, van Tellingen O,
Schinkel AH, Versace R, Beijnen JH,
Mazzanti R, Schellens JH. In vitro
transport of gimatecan
(7-t-butoxyiminomethylcamptothecin) by
breast cancer resistance protein,
P-glycoprotein, and multidrug resistance
protein 2. Mol Cancer Ther 2007;6:3307-13
Van Waterschoot RA, van Herwaarden AE,
Lagas JS, Sparidans RW, Wagenaar E,
van der Kruijssen CMM, Goldstein JA,
Zeldin DC, Beijnen JH, Schinkel AH.
Midazolam metabolism in Cytochrome
P450 3A knockout mice can be attributed to
upregulated CYP2C enzymes. Mol
Pharmacol 2008;73,1029-36
Zimmermann C, van de Wetering K,
van de Steeg E, Wagenaar E, Vens C,
Schinkel AH. Species-dependent transport
and modulation properties of human and
mouse multidrug resistance protein 2
(MRP2/Mrp2, ABCC2/Abcc2). Drug
Metab Dispos 2008;36:631-40
Lagas JS, Sparidans RW,
van Waterschoot RAB, Wagenaar E,
Beijnen JH, Schinkel AH. P-glycoprotein
limits oral availability, brain penetration
and toxicity of an anionic drug, the
antibiotic salinomycin. Antimicrob Agents
Chemother. 2008;52:1034-9
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), 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.
Figure 5: Putative structure of a prototypic ABC drug
transporter
P-gp, Bcrp1, Mrp2 and Mrp3 (compound) knockout mice We found that
salinomycin, an anionic antibiotic, was transported by P-gp in vitro, and we could
subsequently demonstrate that its oral availability, brain penetration and toxicity was
markedly increased in P-gp knockout mice. This was surprising, as it was previously
thought that P-gp affected only cationic or neutral amphiphilic compounds. This
finding thus further expands the already wide spectrum of drugs that are potentially
affected by P-gp activity in vivo. We further found that the important analgesic
diclofenac was transported by BCRP and Bcrp1. Moreover, diclofenac and the
uricosuric benzbromarone could efficiently stimulate human MRP2-mediated drug
transport already at very low concentrations. Efforts to show these effects also in vivo
in mice failed, and retrospective analysis indicated that this is likely due to the fact
that mouse Mrp2 shows sometimes marked differences with human MRP2 in
stimulation by various compounds.
We generated Mrp2/Mrp3 combination knockout mice, which were viable and fertile.
The in vivo pharmacokinetics of the anticancer drug methotrexate (MTX) and its
main toxic metabolite 7OH-MTX were analyzed in these mice. This indicated an

important role of Mrp3, present in the basolateral membrane of the hepatocytes, in
transporting compounds back from the liver into the circulation, thus affecting the
urinary elimination of the drugs. This was especially evident when Mrp2, present in
the bile canalicular membrane, was absent, potentially leading to marked
accumulation of compounds in the liver. Mrp3 thus seems to function as an
important alternative clearance pathway for compounds that are potentially
accumulating in the liver. Together, these results suggest that variation in MRP2 and
MRP3 activity in patients might have profound effects on elimination and toxicity of
MTX and 7OH-MTX, and thus on the therapeutic use of MTX.
CYP3A transgenic and 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. Using
transgenic mice that specifically overexpressed human CYP3A4 in either liver or
intestine of Cyp3a knockout mice we could further show a dominant role of intestinal
CYP3A4 in oral bioavailability of docetaxel, whereas hepatic CYP3A4 dominates
clearance of i.v. docetaxel.
We subsequently characterized the Cyp3a knockout mice by studying the metabolism
of midazolam, one of the most widely used probes to assess CYP3A activity. We
expected that midazolam metabolism would be severely reduced in the absence of
Cyp3a. We used hepatic and intestinal microsomal preparations from Cyp3a knockout
and wild-type mice to assess the midazolam metabolism in vitro. In addition, in vivo
metabolite formation was determined after intravenous administration of
midazolam. Surprisingly, there was still marked midazolam metabolism in hepatic
(but not intestinal) microsomes from Cyp3a knockout mice. Accordingly, we found
comparable amounts of midazolam as well as its major metabolites in plasma after
intravenous administration in Cyp3a knockout mice when compared to wild-type
mice. These data suggest that other hepatic cytochrome P450 enzymes could take
over the midazolam metabolism in Cyp3a knockout mice. We next provided evidence
that Cyp2c enzymes, which are upregulated in the Cyp3a knockout mice, are
primarily responsible for this metabolism and that several but not all murine Cyp2c
enzymes are capable of metabolizing midazolam to its 1’-OH and/or 4-OH
derivatives. These data illustrate compensatory changes in detoxifying systems that
may occur in Cyp3a knockout mice. Studies in these mice with drugs that are partly
metabolized by Cyp2c should therefore be interpreted with caution.
We then investigated the mechanism of regulation of CYP2C55 (the most highly
upregulated Cyp2c gene in the Cyp3a knockout mice) and other detoxifying systems
in Cyp3a knockout mice further. Induction studies with prototypical inducers
demonstrated an important role for the nuclear receptors PXR and CAR in the upregulation
of CYP2C55. Subsequent diet-switch experiments revealed that foodderived
xenobiotics are primarily responsible for the increased induction of CYP2C55
as well as of several other primary detoxifying systems in Cyp3a knockout mice. Our
data suggest that CYP3A normally metabolizes food-derived activators of PXR and/or
CAR, explaining the increased levels of such activators in Cyp3a knockout mice and
subsequent up-regulation of a range of detoxifying systems. Interestingly, our studies
with tissue-specific CYP3A4 transgenic Cyp3a knockout mice revealed that not only
hepatic but also intestinal expression of CYP3A4 could reduce the hepatic expression
of detoxifying systems to near wild-type levels. Apparently, intestinal CYP3A4 can
limit the hepatic exposure to food-derived activators of nuclear receptors, thereby
regulating the expression of a range of detoxifying systems in the liver. This broad
biological effect further emphasizes the importance of intestinal CYP3A activity and
could have profound implications for the prediction of drug exposure.
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MOLECULAR BIOLOGY
Publications (continued)
Matsushima S, Maeda K, Hayashi H,
Debori Y, Schinkel AH, Schuetz JD,
Kusuhara H, Sugiyama Y. Involvement of
multiple efflux transporters in hepatic
disposition of fexofenadine. Mol Pharmacol
2008;73:1474-83
Damen CWN, Rosing H, Tibben MM,
Van Maanen MJ, Lagas JS, Schinkel AH,
Schellens JHM, Beijnen JH. A sensitive
assay for the quantitative analysis of
vinorelbine in mouse and human EDTA
plasma by high-performance liquid
chromatography coupled with electrospray
tandem mass spectrometry. J Chromatogr B
Analyt Technol Biomed Life Sci
2008;868:102-9
Enokizono J, Kusuhara H, Ose A,
Schinkel AH, Sugiyama Y. Quantitative
investigation of the role of breast cancer
resistance protein (Bcrp/Abcg2) in limiting
brain and testis penetration of xenobiotic
compounds. Drug Metab Disp
2008;36:995-1002
Sparidans RW, Lagas JS, Schinkel AH,
Schellens JHM, Beijnen JH. Liquid
chromatography-tandem mass spectrometric
assay for diclofenac and three primary
metabolites in mouse plasma. J Chromatogr
B Analyt Technol Biomed Life Sci
2008;872:77-82
Vlaming MLH, Pala Z, van Esch AM,
Wagenaar E, van Tellingen O,
de Waart DR, Oude Elferink RPJ,
van de Wetering K, Schinkel AH. Impact
of Mrp2 (Abcc2) and Mrp3 (Abcc3) on the
in vivo elimination of methotrexate and its
main metabolite 7-hydroxymethotrexate.
Clin Cancer Res 2008;14:8152-60
Lagas JS, van der Kruijssen CMM,
Beijnen JH, Schinkel AH. Transport of
diclofenac by BCRP (ABCG2) and
stimulation of MRP2- (ABCC2-) mediated
drug transport by diclofenac and
benzbromarone. Drug Metab Disp
2009;37:129-36
Van Waterschoot AB, Rooswinkel RW,
Wagenaar E, van der Kruijssen CMM,
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

72
MOLECULAR BIOLOGY
Group leader Lodewyk Wessels
Lodewyk Wessels PhD Group leader
Nicola Armstrong PhD Academic staff
Michael Hauptmann PhD Academic staff
Christiaan Klijn MSc PhD student
Wouter Meuleman MSc PhD student
Jeroen De Ridder MSc PhD student
Martin Van Vliet MSc PhD student
Jorma De Ronde PhD student
Bram Gerritsen Bioinformatician
Publications
Van Uitert M, Meuleman W, Wessels LFA.
Biclustering Sparse Binary Genomic Data.
Journal of Computational Biology
2008;15:1329-45
Reyal F, Van Vliet MH, Armstrong NJ,
Horlings HM, de Visser KE, Kok MJT,
Teschendorff AE, Mook S, Van’t Veer L,
Caldas C, Salmon RJ, Van de Vijver MJ,
Wessels LFA. A comprehensive analysis of
prognostic signatures reveals the high
predictive capacity of Proliferation,
Immune response and RNA splicing
modules in breast cancer. Breast Cancer Res
2008;10:R93
Van Vliet MH, Reyal F, Horlings HM,
van de Vijver MJ, Reinders MJT,
Wessels LFA. Pooling breast cancer
datasets has a synergetic effect on
classification performance and improves
signature stability. BMC Genomics
2008;9:375
Knijnenburg TA, Wessels LFA and
Reinders MJT Combinatorial influence of
environmental parameters on transcription
factor activity. Bioinformatics.
2008;24:i172-81
Klijn C, Holstege H, de Ridder J, Liu X,
Reinders MJT, Jonkers J., Wessels LFA.
Identification of cancer genes using a
statistical framework for multi-experiment
analysis of non-discretized array CGH
data. Nucleic Acids Res 2008;36:e13
BIOINFORMATICS AND STATISTICS
Our group focuses on developing novel computational approaches which exploit a
wide variety of (cancer) biological and epidemiological questions. These include, but
are not limited to, stratifying tumors into groups with distinct and homogeneous
outcome and therapy response, elucidation the function of genes – and especially
pathways – involved in tumorigenesis and understanding molecular regulatory
mechanisms, in general. Below a number of exemplary projects are presented 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 significant occurrences of multiple
independent insertions within one tumor. This approach employs a predefined
significance level while controlling the family-wise error. It operates at multiple
biologically relevant scales, providing insight in the behavior of co-occurrences across
these scales. This framework was augmented by an approach which reprioritizes the
co-occurrences to compensate for co-occurrences with frequently hit targets. An
approach was also developed to detect co-occurrences between single gene inserts
and inserts associated with a family of genes. This provides a powerful technique to
detect gene families where the family members can substitute for each other during
oncogenesis. The results were validated by comparing these to co-occurrences of
genes obtained through literature mining of published abstracts and expert
evaluation. We have extended this approach to capture more complex interactions
between insertion loci. More specifically, we detect simple logic circuits modeling
combinations of co-occurring and mutually exclusive insertions that accurately
predict the expression pattern of downstream targets.
Detecting cancer genes in aCGH data derived from tumor panels We have
developed a new approach which inputs non-discretized array comparative genomic
hybridization (aCGH) data to identify regions that are significantly aberrant across an
entire tumor set. In an analysis of 89 human breast tumors, our method showed
enrichment for known cancer genes in the detected regions and identified
aberrations that are strongly associated with breast cancer subtypes and clinical
parameters. Furthermore, we identified 18 recurrent aberrant regions in a new
dataset of 19 p53-deficient mouse mammary tumors. These regions, combined with
gene expression microarray data, point to known cancer genes and novel candidate
cancer genes. This framework for aberrant region detection has been extended to also
capture significantly co-occurring and mutually exclusive aberrations. Application of
this approach to a large cell line panel revealed a number of known as well as
interesting new oncogenic interactions.
Functional analysis of expression signatures on a large breast cancer
compendium In order to better understand the relative lack of overlap between
breast cancer prognostic signatures that are seemingly similar in terms of predictive
capacity, we performed a comprehensive analysis of the performance of nine gene
expression signatures on seven different breast cancer datasets. To minimize interdataset
variability, we only employed a collection of 947 breast cancer samples
hybridized on the Affymetrix platform, collected the raw data, and pre-processed and
normalized these samples jointly. For 44% of these patients all nine classifiers are
concordant. In the group where all signatures predict a good outcome, only seven
percent of the tumors recur. In the group where all signatures predict a poor
outcome, 35% of the tumors recur, indicating a large degree of heterogeneity in this
group that is not captured by gene expression. The percentage of ‘poor outcome’
events relative to the number of ‘poor outcome’ calls is correlated, indicating a benefit
when combining the separate classifiers. To better characterize the functional
processes associated with the largely non-overlapping signatures, we created an
enlarged signature for each of them by including genes that are highly correlated
with the genes in the signature. Identification of functional gene sets that are overrepresented
in the intersection of the enlarged signatures revealed a common core of
ten different functional modules. The identified modules include: Immune response,

KRAS, proliferation, RNA splicing, Rb pathway, sterol biosynthesis, extra-cellular
matrix constituent, focal adhesion, negative regulation of proliferation and apoptosis.
All modules are associated with survival in an independent test set. The combination
of the RNA splicing and immune response modules resulted in a high prognostic
performance on an independent validation set.
Classifier signature stability Pooling the abovementioned collection of 947 breast
cancer samples from seven studies would limit the effect of any sampling biases
from the individual datasets, and alleviate small sample size problems. We showed
that pooling datasets and deriving a classifier from the pooled data leads to more
accurate classifiers. More specifically, when starting with six breast cancer datasets,
and pooling pairs of datasets, we observed a synergetic effect on the classification
performance in 73% of the cases. By increasing the number of datasets pooled (from
three to six), we observed a significant association between the number of datasets
that are pooled, the validation performance and the number of genes selected.
Functional enrichment of the signatures derived from pooled datasets indicates high
significance for the proliferation categories. Microtubule-associated pathways only
emerge when pooling more than five datasets, while a plethora of gene sets that are
only significant in signatures derived from single datasets, were no longer significant
in pooled variants, thus identifying those as likely false positives.
Computational Analysis of Lamina Associated Domains (LADs) LADs are very
large (0.1-10Mb), sharply defined segments of the human genome that are associated
with the lamina. These domains provide insight in how the genome is organized
within the cell nucleus. On a more general level it is possible that LADs encode
mechanisms involved in, e.g., higher level gene regulation and/or higher order
packaging of the DNA. LADs are demarcated by specific sequence elements,
indicating that the folding of DNA is in part hard-wired in the genome. Some of the
more prevalent elements identified at the borders of LADs include an abundance of
CpG-islands, particular transcription factor binding site motifs and promoters of
specifically oriented genes. The sequence elements found so far are, unfortunately,
insufficient to fully characterize LADs. Initial experiments show that LAD structures
change across cell types. We aim to employ machine learning approaches to integrate
various data sources (lamin binding profiles, expression data, genomic features and
chromatin features) to formulate testable hypotheses regarding mechanistic and
functional properties of LADs.
Flexible statistical modeling of exposure-time-response relationships An
adequate depiction of exposure-time-response relationships is important in assessing
public health implications of an occupational or environmental exposure. Recent
advances have focused on flexible modeling of the overall shape of latency. However,
methods are needed to allow for varying shapes of latency under different exposure
profiles. We developed a tensor product spline model for describing exposureresponse
relationships for protracted time-dependent exposure histories in
epidemiologic studies. The methods use flexible multi-dimensional techniques to
jointly model age, latency and exposure-response effects. In analyzing lung cancer
mortality and exposure to radioactive radon gas based on data from the Colorado
Plateau Uranium Miners cohort, a model that allows for varying exposure-dependent
latency shapes is found to be superior to models that only allowed for an overall
latency curve. Specifically, the model suggests that, at low exposure levels risk
increased at short latencies followed by a slow decline for longer latency periods.
On the other hand, risk was higher but did not change much by latency for higher
exposure levels. The proposed methodology has the advantage of allowing for latency
functions that vary by exposure levels and, conversely, exposure-response
relationships that are influenced by the latency structure.
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Publications (continued)
Meuleman W, Engwegen JYMN,
Gast M-CW, Beijnen JH, Reinders MJT
and Wessels LFA. Comparison of
normalisation methods for Surface-
Enhanced Laser Desorption and Ionisation
Time-Of-Flight Mass Spectrometry data
BMC Bioinformatics 2008;9:88
Weigelt B, Horlings HM, Kreike B,
Hayes MM, Hauptmann M, Wessels LFA,
de Jong D, van de Vijver MJ,
van ‘t Veer LJ, and Peterse JL. Refinement
of breast cancer classification by molecular
characterisation of histological special types.
J Pathol 2008;216:141-50
Berhane K, Hauptmann M, Langholz B.
Using tensor product splines in modeling
exposure-time-response relationships:
application to the Colorado Plateau
uranium miners cohort. Stat Med
2008;27:5484-96
Sigurdson AJ, Ha M, Hauptmann M,
Bhatti P, Sram R, Beskid O, Tawn EJ,
Whitehouse CA, Lindholm C, Nakano M,
Kodama Y, Nakamura N, Vorobstova I,
Oestreicher U, Stephan G, Yong LC,
Bauchinger M, Schmid E, Chung HW,
Darroudi F, Roy L, Voisin P, Barquinero JF,
Livingston G, Blakey D, Hayata I, Zhang W,
Wang C, Bennett LM, Littlefield LG,
Edwards AA, Kleinerman RA, Tucker JD.
International study of factors affecting
human chromosome translocations. Mutat
Res 2008;652:112-21
Hooning MJ, Aleman BMP, Hauptmann M,
Baaijens MH, Klijn JG, Noyon R, Stovall M,
Van Leeuwen FE. Roles of radiotherapy
and chemotherapy in the development of
contralateral breast cancer. J Clin Oncol
(in press)
Holm C, Kok M, Michalides R, Fles R,
Koornstra RH, Van ‘t Veer LJ, Neefjes J,
Peterse JL, Hauptmann M, Stal O,
Landberg G, Linn SC. Phosphorylation of
the estrogen receptor alpha at serine 305
and prediction of tamoxifen response in
breast cancer. J Pathol (in press)

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MOLECULAR BIOLOGY
Group leader Rob Wolthuis
Rob Wolthuis Group Leader
Linda Clijsters PhD student
Wouter Van Zon PhD student
Erik Voets PhD student
Janneke Ogink Technical staff
Bas Ter Riet Technical staff
Publications
Wolthuis R, Clay-Farrace L, van Zon W,
Yekezare M, Koop L, Ogink J, Medema R
and Pines J. (2008) Cdc20 and Cks Direct
the Spindle Checkpoint-Independent
Destruction of Cyclin A. Molecular Cell
2008;30:290-302
van Leuken RJ, Clijsters L, Wolthuis RMF
(2008) To cell cycle, swing the APC/C.
BBA Reviews in Cancer 2008;1786:49-59
van Leuken RJ, Luna-Vargas MP,
Sixma TK, Wolthuis RMF and Medema RH
Usp39 is essential for mitotic spindle
checkpoint integrity and controls
mRNA-levels of Aurora B. Cell Cycle
2008;17:2710-19
Lindqvist A, van Zon W,
Karlsson Rosenthal C, Wolthuis RMF.
(2007) Cyclin B1-Cdk1 activation continues
after centrosome separation to control
mitotic progression. PLoS Biology
2007;5:e123
REGULATION OF MITOSIS IN NORMAL AND CANCER CELLS
In our group we aim to understand how mitosis works in both normal and cancer
cells, by using a combinatorial approach of biochemistry, molecular genetics in
human cells, and dedicated live-cell fluorescence imaging. We are focussing on two
problems that cells face at the end of the cell cycle: a) how to enter mitosis from G2
phase and b) how to successfully exit mitosis and divide. Equally important is how
cells respond to insults or mistakes in G2 or mitosis.
Mitosis: decisive finish of the cell cycle Cells duplicate their chromosomes and
increase their size during the S and G2 phases. Mitosis forms the spectacular end of
the cell cycle when sister chromatids are irreversibly separated and two new cells are
formed. Cells should delay mitotic entry under conditions that are unfavourable for
safe and successful division, such as after DNA damage, or when cells are too small.
The decision to enter mitosis is controlled by signalling pathways called the G2
checkpoints that direct the activation of the principal mitotic kinase, Cyclin B1-Cdk1.
Once cells are in mitosis, they need to ensure that all the duplicated chromosomes
are properly attached to the mitotic spindle. This is safe-guarded by the Mitotic
Spindle Checkpoint, which works by inhibiting another key enzyme of our interest, the
Anaphase-Promoting Complex or Cyclososme (APC/C). Once active, the APC/C triggers
destruction of regulatory proteins, to initiate sister chromatid separation and mitotic
exit. Progression through mitosis needs to be swift to prevent DNA damage as a
result of the hyper-condensed state of the chromosomes, as well to avoid cellular
damage caused by transcriptional and translational silencing during mitosis. Rapid
dynamics are thus critical to mitosis. This also makes mitosis the most exciting stage
of the cell cycle, yet the most difficult to study by biochemical means.
Getting into Mitosis: look before you leap The transition from G2 to mitosis is
catalyzed by activation of Cyclin B1-Cdk1. Because Cdk1 levels remain fairly constant
throughout the cell cycle, and are in excess over Cyclin, all Cyclin B1 that is newly
synthesized in G2 phase binds Cdk1. At this stage Cyclin B1-bound Cdk1 is kept
inactive by phosphorylation of Thr14 and Tyr15, resulting in slow accumulation of
inactive Cyclin B1-Cdk1 complexes. In Cyclin B1 immunoprecipitates of synchronized
cells, Cdk1 activity rises very gradually in time, reaching no more than 25% of its
maximum activity at the end of G2. The Cdc25 phosphatases are activated by the
slowly rising Cyclin B1-Cdk1 activity, which enhances Cyclin B1-Cdk1 activation.
In contrast, the inactivating kinases Wee1 and Myt1 are inactivated by rising Cyclin
B1-Cdk1 activity. The result is a double positive-feedback loop leading to steep
activation of Cyclin B1-Cdk1. Only at the very end of G2 this auto-catalyzing feed-back
loop is initiated and cells become committed to mitosis. The protein levels,
intracellular localisations and intrinsic activities of the Wee1/Myt1 inhibitory kinases
and Cdc25 activating phosphatases which control Cyclin B1-Cdk1 are intensely
regulated. We aim to create further insights into the mechanisms that trigger abrupt
activation in late G2 and the parameters of an ‘activation threshold’ for mitosis
(figure 7). Recently, in collaboration with Arne Lindqvist and Christina Karlsson-
Rosenthal, we used a novel assay to monitor the activation of Cyclin B1-Cdk1 in single
cells. We discovered that human Cyclin B1-Cdk1 activity most likely has a ‘bi-stable’
response curve in vivo. Apparently, Cyclin B1-Cdk1 activation develops in a non-linear
fashion after the activation threshold. Similar models are required to understand the
threshold for mitotic entry and progression, either under normal conditions or after
cellular insults in G2. By determining these conditions in both normal and cancer
cells, we aim to create useful models for Cyclin B1-Cdk1 control and understand the
effects of interfering with Cyclin B1-Cdk1 activity as an approach for cancer therapy.

Figure 6: Requirement of Cdk1 for Mitotic Entry and Mitotic Progression in Human Cells. (A) Cdk1
knock-down cells were synchronized in G2/M 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. (B) Mitotic cells were lysed (lanes 1 and 2) or released from nocodazole and incubated in fresh
medium for 3 h, recollected, and lysed (lanes 3 and 4). Differences in mitotic phosphorylation shift of APC3
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 co-expressed Cdk1-YFP with a silent
mutation in the RNAi targeting region (lane 2). (D) Metaphase duration in Cdk1 RNAi cells (right) or
Cdk1 RNAi cells rescued by co-expression of non–RNAi-sensitive Cdk1-YFP (left). (E) Time-lapse
microscopy analysis of mitotic progression after entry with normal or impaired Cdk1 levels. Bottom panels
are consecutive images of tubulin-YFP in a pS-control cell in mitosis; top panels show delayed chromosome
alignment (frames 2 and 3) and stalled metaphase (frames 4–6) after Cdk1 shRNA. From: Lindqvist A,
van Zon W, Karlsson Rosenthal C, Wolthuis RMF Cyclin B1-Cdk1 activation continues after centrosome
separation to control mitotic progression. PLoS Biology 2007;5:e123.
Mitotic Exit: coordination of events by protein destruction Mitotic entry
requires accumulation of regulatory proteins, such as the mitotic Cyclins. In contrast,
these proteins must be destroyed at distinct, sequential time points in mitosis. A
multi-subunit ubiquitin ligase –the APC/C- ubiquitinates these mitotic proteins at
highly defined mitotic time points, which initiates their proteolysis by the 26S
proteasome. Sequential degradation of for instance Cyclin A, Cyclin B1 and Securin
coordinates cell division with segregation of sister chromatids. Therefore, precise
regulation of protein destruction by the APC/C is essential to guarantee cell viability
and genomic integrity.
The question remains how the APC/C recognizes a critical substrate at the right
time. To address this important issue in human cells, we are using a combination of
gene silencing, biochemistry, in vitro ubiquitination assays, and live cell imaging to
identify regulation of the APC/C activation (figure 7). Our recent work revealed
unexpected roles for the potentially oncogenic Cks protein family in directing Cdc20dependent
destruction of Cyclin A, and in coordinating the concurrent destruction of
Cyclin B1 and Securin. We are using these new findings to search for roles of
individual APC/C subunits, phosphorylation events as well as the intracellular
localization of APC/C-complexes, in controlling mitosis. Furthermore, we think that
depending on the way the APC/C is inhibited, distinct molecular programs may be
activated which affect the cellular responses to a prolonged arrest in mitosis.
The next research goal is to understand how oncogenic mutations may impinge on
critical steps in mitotic entry, mitotic progression and mitotic exit, but also how they
determine the way cells respond to errors in mitosis, or to anti-mitotic drugs. It is
anticipated that answers to these questions could create exciting opportunities to
improve anti-cancer therapies.
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Figure 7: Measuring APC/C activity by
time-lapse fluorescence microscopy of Cyclin
B1 destruction. A plasmid encoding for
fluorescent Cyclin B1 was injected into G2phase
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 ubiquitindependent
protein destruction in live cells
undergoing division and was first
established in the laboratory of Jon Pines,
Gurdon Institute, Cambridge, UK.

76
MOLECULAR CARCINOGENESIS
Division head, group leader René Bernards
René Bernards PhD Group leader
Katrien Berns PhD Post-doc
Annette Dirac PhD Post-doc
Michael Hölzel MD PhD Post-doc
Sidong Huang PhD Post-doc
Rianne Oosterkamp MD Clinical fellow
Miguel Rubio PhD Post-doc
Linda Smit PhD Post-doc
Mirjam Epping MSc PhD student
Ernst-Jan Geutjes MSc PhD student
Guus Heynen MSc PhD student
Roderik Kortlever MSc PhD student
Jasper Mullenders Msc PhD student
Miranda van Dongen Technical staff
Marielle Hijmans MSc Technical staff
Mandy Madiredjo Technical staff
Publications
Bernards R. Cancer: Entangled pathways.
Nature 2008;455:479-80
Van ‘t Veer LJ, Bernards R. Enabling
personalized cancer medicine through
analysis of gene-expression patterns. Nature
2008;452:564-70
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 research
2008;68:9221-30
Epping MT, Bernards R. Molecular basis of
the anti-cancer effects of histone deacetylase
inhibitors. The international journal of
biochemistry & cell biology 2009;41:16-20
Epping MT, Hart AA, Glas AM,
Krijgsman O, Bernards R. PRAME
expression and clinical outcome of breast
cancer. British journal of cancer
2008;99:398-403
DIVISION OF MOLECULAR
CARCINOGENESIS
FUNCTIONAL GENOMICS
My group uses functional genomics technologies to find novel cancer-relevant genes
and to identify mechanisms of resistance to cancer drugs. We use both highthroughput
gain-of-function genetic screens and RNA interference-based loss-offunction
genetic screens to achieve these goals.
Identification of mechanisms of drug resistance Unresponsiveness to therapy is
a recurring problem in the treatment of cancer. It is therefore important to identify
the molecular pathways that contribute to unresponsiveness to cancer therapeutics.
We use both gain-of-function and loss-of-function genetic screens to identify genes
that contribute to drug resistance, in particular to new classes of targeted
therapeutics. For the loss-of-function genetic screens, we use our vast collection of
RNA interference vectors, which at present consists of 55,000 shRNA vectors that
together target over 23,000 mouse and human genes for suppression. We use this
vector set in conjunction with an efficient way to screen shRNA libraries: siRNA bar
code screening (in collaboration with Roderick Beijersbergen, division of Molecular
Carcinogenesis, see below). In the past year, we have focused on the identification of
genes whose suppression contributes to resistance to retinoic acid (RA)-based
therapies. Retinoic acid is a key morphogen in early vertebrate development and
tissue regeneration, which induces growth arrest, differentiation and apoptosis. In
the clinic, retinoids are increasingly being used for the treatment of cancer, including
leukemias and neuroblastoma. However, the molecular mechanisms of resistance to
retinoid-based therapies remain obscure.
To address this, we performed a large-scale RNA interference screen with a
30,000-vector mouse shRNA library to identify genes whose suppression confers
resistance to RA-mediated differentiation in mouse F9 teratocarcinoma cells. To date,
we have validated two genes whose suppression confers resistance to RA-induced
differentiation and growth arrest in these F9 cells: ZNF423 and CtBP2 (figure 1). For
ZNF423, a large zinc finger protein, we have shown that it binds directly to the RAR/
RXR receptor heterodimer in chromatin and that this binding is required for receptor
activation by RA. ZNF423 knockdown also prevents differentiation of neuroblastoma
cell lines in vitro. Conversely, ectopic expression of ZNF423 enhances RAdifferentiation
in human neuroblastoma cells. Significantly, we found that ZNF423 is
a marker of poor outcome in two independent 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. We have recently performed an additional loss of
function genetic screen in human neuroblastoma to find additional genes whose
suppression confers resistance to RA. Unexpectedly, we found here that suppression
of the tumor suppressor gene NF1 causes resistance to RA-induced differentiation.
This suggests that RAS signaling can modify responses of neuroblastoma cells to
retinoids. We are currently asking if NF1 is mutated in human neuroblastoma and
how loss of NF1 contributes to RA resistance.
We are also using a similar approach to identify mechanisms of resistance to drugs
that act downstream of HER2 in breast cancer: inhibitors of PIK3CA and mTOR.
Drugs that inhibit these kinases are currently in early stage clinical trials. Uncovering
mechanisms of resistance against these drugs may help identify those patients that
benefit most from these drugs. We have identified and validated one gene whose
suppression can cause resistance to the mTOR inhibitor rapamycin in breast cancer
cell lines. The mechanism leading to resistance to rapamycin is under investigation.

Figure 1: Znf423 is required for RA-induced differentiation in mouse ES cells.
Down-regulation of Znf423 by RNAi in mouse Embryo Stem (ES) cells confers resistance to RA-induced
differentiation. ES cells expressing two different shRNAs against Znf423 or GFP (control) were grown in
the absence or presence of exogenous RA (1 µM) for 1 week, after which cells were fixed, stained for
alkaline phosphatase (AP) and photographed. The alkaline phosphatase stain represents a marker of the
undifferentiated state of ES cells, demonstrating that knockdown of Znf423 blocks differentiation.
Functional studies on de-ubiquitinating enzymes My laboratory has established a
collection of shRNA vectors that target deubiquitinating enzymes (DUBs) for
suppression. Currently, we have a set to some 100 DUB shRNA vectors and a similar
set of siRNA reagents. We have used these RNA interference reagents to identify
novel DUBs in cancer relevant pathways. In a screen aimed at identifying DUBs that
can remove ubiquitin moieties from the androgen receptor, we identified USP26.
Remarkably, mutations in USP26 have been reported in patients suffering from male
infertility syndromes, underscoring the potential role of USP26 in androgen receptor
signaling.
Using a single-well siRNA screening approach, we have identified DUBs whose
suppression either inhibits or enhances TNF-alpha mediated apoptosis. We are
currently studying how these DUBs affect signaling through their respective
pathways.
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MOLECULAR CARCINOGENESIS
Publications (continued)
Herold S, Hock A, Herkert B, Berns K,
Mullenders J, Beijersbergen R, Bernards R,
Eilers M. Miz1 and HectH9 regulate the
stability of the checkpoint protein, TopBP1.
Embo J 2008;27:2851-61
Kortlever RM, Nijwening JH, Bernards R.
Transforming growth factor-beta requires its
target plasminogen activator inhibitor-1 for
cytostatic activity. J Biol Chem
2008;283:24308-13
Kortlever RM, Brummelkamp TR,
van Meeteren LA, Moolenaar WH,
Bernards R. Suppression of the p53dependent
replicative senescence response by
lysophosphatidic acid signaling. Mol Cancer
Res 2008;6:1452-60
Stemke-Hale K, Gonzalez-Angulo AM,
Lluch A, Neve RM, Kuo WL, Davies M,
Carey M, Hu Z, Guan Y, Sahin A,
Symmans WF, Pusztai L, Nolden LK,
Horlings H, Berns K, Hung MC,
van de Vijver MJ, Valero V, Gray JW,
Bernards R, Mills GB, Hennessy BT. An
integrative genomic and proteomic analysis
of PIK3CA, PTEN, and AKT mutations in
breast cancer. Cancer research
2008;68:6084-91
Wittner BS, Sgroi DC, Ryan PD,
Bruinsma TJ, Glas AM, Male A, Dahiya S,
Habin K, Bernards R, Haber DA,
Van ‘t Veer LJ, Ramaswamy S. Analysis of
the MammaPrint breast cancer assay in a
predominantly postmenopausal cohort. Clin
Cancer Res 2008;14:2988-93
Bernards R. Reaction to American Society
of Clinical Oncology 2007 update of
recommendations for the use of tumor
markers in breast cancer. J Clin Oncol
2008;26:2057-8
Rutgers EJ, Pusztai L, Bernards R. Are
short-term or long-term recurrence rates
more important in breast cancer screening?
Annals of internal medicine 2008;149:357
Eichhorn PJ, Creyghton MP, Bernards R.
Protein phosphatase 2A regulatory subunits
and cancer. Biochim Biophys Acta 2008 (in
press)
Bernards R. RNAi delivers insight into liver
cancer. Cell 2008;135:793-5

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MOLECULAR CARCINOGENESIS
Group leader Roderick Beijersbergen
Roderick Beijersbergen PhD Group leader
David Egan 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
THE RNAI STRATEGY IN TARGET DISCOVERY
The research in our lab continues to evolve around the identification of novel drug
targets in cancer using large scale cell-based screening technologies. The goal of our
work is to identify essential components in disease-related pathways that can be
explored as drug targets in cancer therapy and to identify potential mechanisms of
resistance and to achieve a more complete understanding of drug action, thereby
enabling more rational decisions on drug application.
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 have begun to 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 background of a tumor specific alteration and
represent ‘genotype specific’ drugs. 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. 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 more than 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
(figure 2). We are in the process of extensive validation and elucidation of the
mechanisms underlying these phenotypes.
Figure 2: Synthetic lethal interaction with loss of p53
Scatter plot representing the results of inactivation of 8000 genes by siRNA in BJ cell in the presence
(X-axis) or absence (Y-axis) of p53 expression and a low dose of cis-platin. Each dot represents 1 gene
targeted by 4 individual siRNAs and indicates the relative effect on cell viability in each of the 2 different
genotypes. Depicted in dark grey is a panel of control siRNAs, depicted in light grey are potential synthetic
lethal interactions with loss-of-p53. In addition to siRNAs that specifically decrease viability in p53 null
cells, we have also identified siRNAs causing enhanced proliferation only in the presence of p53 expression.
Bar code screens The bar code screening technology has been developed to monitor
the effect of the inactivation of gene expression in large complex populations of cells
(figure 3). In this way the effect of gene inactivation on the phenotype of individual
cells can be followed. The design of the NKI shRNA libraries allows for the
identification of individual shRNA vectors in a large pool of shRNA vector containing
cells. Each hairpin vector contains a unique 19-mer sequence (“barcode”) designed to
target one specific cellular gene. This 19-mer sequence can be used to determine the
relative abundance of a shRNA vector by hybridization to DNA micro-arrays

containing the 19-mer barcode sequences. By using this technique, large complex
populations of shRNA expressing cells can be monitored for the abundance of each
individual shRNA expressing cell (see figure 3) reflecting the effect of specific gene
inactivation on cellular behavior. The bar code screening technology facilitates the
identification of genes that contribute to drug resistance in cancer cells, in particular
to new classes of targeted therapeutics. An example of this strength is the recent
finding that PI3 kinase activation contributes to resistance to lapatinib treatment. At
this moment several novel cancer drugs, in early stages of clinical development, are
used with our bar code screening technology to identify genes that either enhance the
drug’s effect or result in resistance to drug treatment.
Figure 3: Bar code screen technology
Large populations of shRNA expressing cells are analyzed in parallel using molecular barcode screens.
Each shRNA vector is tagged with a unique ‘barcode’ sequence (the 19-mer targeting sequence) that is used
to monitor the representation of each vector in a complex population by hybridizing to an oligonucleotide
microarray containing the 19-mer barcode sequences. When a population of cells is subjected to a selective
pressure, there will be selection for those cells that express a shRNA that modulates the cellular response. As
a result the representation of individual shRNA constructs is expected to change. This change in the
relative abundance of individual shRNA vectors can be determined by comparison of a control population
versus the population exposed to selection. The signal intensity of shRNAs that confer resistance to the
selection will increase compared to the control population.
siRNA screens and High Content Imaging Over the last year we have expanded
our capabilities to perform large scale single well siRNA screens. An example of
these efforts is a screen for the identification of novel components of the TGFb
signaling pathway using a TGFb responsive reporter controlling the expression of
luciferase. We have identified several siRNAs that either increase or decrease TGFb
signaling. These hits are used for follow-up experiments to elucidate their
mechanism of action.
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MOLECULAR CARCINOGENESIS
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 research
2008;68:9221-30
Herold S, Hock A, Herkert B, Berns K,
Mullenders J, Beijersbergen R, Bernards R,
Eilers M. Miz1 and HectH9 regulate the
stability of the checkpoint protein, TopBP1.
Embo J 2008;27:2851-61
OttoT, Horn S, Brockmann S, Eilers U,
Schüttrumpf L, Popov N, Kenney A,
Schulte JH, Beijersbergen RL,
Christiansen H, Berwanger B, Eilers M.
Stabilization of N-Myc is a critical function
of Aurora-A in human neuroblastoma.
Cancer Cell (in press)

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MOLECULAR GENETICS
Division head, group leader Maarten Van Lohuizen
Maarten Van Lohuizen PhD Group leader
Elisabetta Citterio PhD Senior Post-doc
Jean Bernard Beaudry PhD Post-doc
Jaap Kool PhD Post-doc
Karim Nacerdine PhD Post-doc
Inka Pawlitzky PhD Post-doc
Alexandra Pietersen PhD Post-doc
Anke Sparmann PhD Post-doc
Bas Tolhuis PhD Post-doc
Bart Westerman PhD Post-doc
Sophia Bruggeman MSc PhD student
Panthea Taghavi MSc PhD student
Joep Vissers MSc PhD student
Marleen Blom Technical staff
Paulien Cornelissen Technical staff
Danielle Hulsman Technical staff
Ellen Tanger Technical staff
Hans Teunissen Technical staff
Els Verhoeven Technical staff
Huub van Vugt Technical staff
Julian Puppe MD Visiting scientist
Publications
Terranova R, Yokobayashi S, Stadler MB,
Otte AP, Van Lohuizen M, Orkin SH,
Peters AH. Polycomb group proteins Ezh2
and Rnf2 direct genomic contraction and
imprinted repression in early mouse
embryos. Dev Cell. 2008:15:668-79
Taghavi P, Verhoeven E, Jacobs JJ,
Lambooij JP, Stortelers C, Tanger E,
Moolenaar WH, Van Lohuizen M. In vitro
genetic screen identifies a cooperative role
for LPA signaling and c-Myc in cell
transformation. Oncogene 2008:27:6806-16
Pietersen AM, Evers B, Prasad AA,
Tanger E, Cornelissen-Steijger P, Jonkers J,
Van Lohuizen M. Bmi1 regulates stem cells
and proliferation and differentiation of
committed cells in mammary epithelium.
Curr Biol. 2008:18:1094-9
Boutsma E, Noback S, Van Lohuizen M.
The Polycomb protein and E3 ubiquitin
ligase Ring1B harbors an IRES in its highly
conserved 5’ UTR. PLoS ONE 2008:3:e2322
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
Polycomb-group (Pc-G) protein complexes, and the effects of deregulation of Pc-G
genes on development, Cell cycle control, cancer formation and stem cell
maintenance. In addition, we are performing large-scale genetic screens in primary
cells and in cancer-predisposed mice to identify cancer-relevant networks of
oncogenes and tumor-suppressor genes. Model organisms comprise Mouse and
Drosophila.
Functional characterization of Pc-G protein complexes Repressive Pc-G proteins
and the counteracting Trithorax-group (Trx-G) of nucleosome remodeling factors are
involved in the 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 Gretel
Buchwald and Titia Sixma, Division of Biochemistry). This enzymatic activity is part
of the core PRC1 complex and conditional Ring1b loss-of-function 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,
transcriptional repressors as well as key stem cell signaling pathways such as Wnt,
Hh, Notch and several new distinct functional categories. Currently we are
investigating the possibility that these domains may interact in 3D nuclear space
using optimized in vivo 4C (chromatin conformation capture on Chip).
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
primary embryo fibroblasts (MEFs) and neural precursors in the cerebellum of Bmi1
knockouts 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 well-established 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

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.
differentiation upon loss of Bmi1 in mammary epithelial precursors is in part
mediated via INK4a/ARF. Importantly, these studies revealed a dual role for Bmi1/
Pc-G: controlling both proliferation and differentiation. A key characteristic of cancer
cells is their unlimited self-renewal. In this respect, cancer cells resemble stem cells,
and accumulating evidence suggests that many forms of cancer may indeed contain
cells carrying stem cell markers. In studying the proliferation defects and progressive
loss of cells 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 recently
demonstrated a critical role for Bmi1 in maintenance of brain tumor formation
(figure 2). Interestingly, Bmi1 acts in this tumor setting in an Ink4a/ARFindependent
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 nonsmall
cell lung cancer, breast 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 David Adams and Allen Bradley from
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.
These screens yielded over 10.000 insertion sites implicating over 300 loci in
tumorigenesis and uncovered new pathway-specific oncogenes and candidate tumorsuppressors.
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 using bone marrow
or fetal liver hemapoietic cell-transplatation systems.
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.
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MOLECULAR GENETICS
Publications (continued)
Van der Stoop P, Boutsma EA, Hulsman D,
Noback S, Heimerikx M, Kerkhoven RM,
Voncken JW, Wessels LF, Van Lohuizen M.
Ubiquitin E3 ligase Ring1b/Rnf2 of
polycomb repressive complex 1 contributes to
stable maintenance of mouse embryonic
stem cells. PLoS ONE. 2008:3:e2235
Uren AG, Kool J, Matentzoglu K,
De Ridder J, Mattison J, Van Uitert M,
Lagcher W, Sie D, Tanger E, Cox T,
Reinders M, Hubbard TJ, Rogers J,
Jonkers J, Wessels L, Adams DJ,
Van Lohuizen M, Berns A.
Large-scale mutagenesis in p19(ARF)- and
p53-deficient mice identifies cancer genes
and their collaborative networks. Cell
2008:133:727-41
Vissers JH, Nicassio F, van Lohuizen M,
Di Fiore PP, Citterio E. The many faces of
ubiquitinated histone H2A: insights from
the DUBs. Cell Div. 2008:3:8
Vékony H, Raaphorst FM, Otte AP,
Van Lohuizen M, Leemans CR,
Van der Waal I, Bloemena E. High
expression of Polycomb group protein EZH2
predicts poor survival in salivary gland
adenoid cystic carcinoma. J Clin Pathol.
2008:61:744-9
Puschendorf M, Terranova R, Boutsma E,
Mao X, Isono K, Brykczynska U, Kolb C,
Otte AP, Koseki H, Orkin SH,
Van Lohuizen M, Peters AH. PRC1 and
Suv39h specify parental asymmetry at
constitutive heterochromatin in early mouse
embryos. Nat Genet. 2008:40:411-20
Pietersen AM, Van Lohuizen M. Stem cell
regulation by polycomb repressors:
postponing commitment. Curr Opin Cell
Biol. 2008:20:201-7
Miyazaki M, Miyazaki K, Itoi M, Katoh Y,
Guo Y, Kanno R, Katoh-Fukui Y, Honda H,
Amagai T, Van Lohuizen M, Kawamoto H,
Kanno M. Thymocyte proliferation
induced by pre-T cell receptor signaling is
maintained through polycomb gene product
Bmi-1-mediated Cdkn2a repression.
Immunity 2008:28:231-45

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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
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: Mapping interaction networks
between Common Insertion Sites.
Co-occurrence between the top 25 CISs. CIS
names and CIS rank are indicated on
vertical axis, numbers on horizontal axis
are CIS rank. The horizontal axis
represents CISs that are assumed to be the
predisposing, more clonal event and the
vertical axis represents CISs that are
presumed to be subsequent, subclonal events.
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
genotype-phenotype 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. Some of the gene families identified in our
models are being studied in more depth.
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
indistinguishable from that of human SCLC. The tumors also metastasize to the
same sites. Array CGH showed frequent amplification of L-Myc further supporting
their resemblance with the human counterpart. These tumors are heterogeneous and
consist of different cell types. Cloning of the different cells from a single tumor
showed very different marker profiles. Both cells with neuroendocrine and progenitor
markers were found. Interestingly, these phenotypically highly diverse cell lines
shared some highly distinct genetic aberrations indicating that they were derived
from a common progenitor. Some known recurrent lesions were found that
specifically marked the tumor cells with neuroendocrine markers, such as L-myc
amplification. Forced overexpression of L-myc in the non-neuroendocrine cells did
not substantially change their phenotype. However, K-ras introduction into
neuroendocrine cells profoundly altered these cells. They now more closely
resembled NSCLC. This is of interest since this has also been reported for relapses of
SCLC. This raises the question about the cell type in which tumorigenesis is initiated
and that can differentiate to seemingly quite different lineages. Therefore, 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 I and II cells, and neuroendocrine
cells to drive Cre expression upon Adeno-Cre infection we will establish the marker
profile of cells in lung that give rise to SCLC and NSCLC. Preliminary results indicate
that distinct subsets of cells serve as a compartment from which these tumors derive.
This presents a proof of concept for the approach and therefore, we are now
generating a larger set of Adeno-Cre viruses to more precisely define the target cells
that give rise to the different types of lung cancer.
Mesotheliomas Inactivation of Nf2 and Ink4a has been reported in a subset of
human mesotheliomas. This has served as a basis to design a mesothelioma mouse
model. We have inactivated Nf2 in combination with either Ink4a/p19Arf, p53, or p53/
Ink4a loss by Adeno-Cre infection of mesothelia of the thoracic and intraperitoneal
cavity. Concomitant loss of Ink4a/p19Arf or p53 strongly accelerated mesothelioma
development. We found that p53 deficiency or proficiency does not result in a distinct
resistance profile towards cytotoxic drugs. Initially we have performed the studies
with cell lines cloned from these tumors. However, these cell lines appear irreversibly

altered, as measured by expression profiling, likely as a result of in vitro propagation.
Therefore, 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. We are making an inventory of the activated signaling pathways in the
individual tumors to gain better insight in combination therapies that might
specifically target the signaling pathways activated these tumors.
Ink4 proteins We have shown that disrupting Ink4a, Ink4b and p19Arf together, a
situation often seen in human tumors, results in very aggressive tumor development,
not seen in Ink4a/p19Arf knockout mice. It appears that Ink4b is a critical backup
for Ink4a. The backup function of Ink4b for Ink4a raises the question whether Ink4c
and Ink4d might fulfill similar backup functions. Therefore we have begun to cross
the Ink4c and Ink4d knockout alleles into the conditional Inka-/-;Ink4b-/-;p19Arflox/
lox mice. Although in mouse embryo fibroblasts a constitutive-active Cdk4 mutant
shows a similar phenotype as the Ink4a-/-;Ink4b-/- alleles in a Ras transformation
assay, it is evident from studies of others that in vivo Ink4c and Ink4d have tumor
suppressor functions in specific tissues. The knockout of different combinations of
all four genes should teach us to what extent this tumor suppressor family plays a
role in tumor suppression in additional tissues. In addition, this might reveal
unexpected physiological functions of these proteins.
Identification of new oncogenic pathways Proviral insertional mutagenesis
screens marks genes instrumental in tumorigenesis. In collaboration with the groups
of Maarten van Lohuizen, Lodewyk Wessels, Jos Jonkers, and John Hilkens we have
performed a large-scale proviral insertional mutagenesis study in tumor suppressor
knockout mice. In collaboration with the group of David Adams of the Sanger Centre
we have sequenced the insertion sites of approximately 1000 lymphoid tumors
induced in wild-type and genetically-predisposed genetic backgrounds. The largest
subgroup study was performed in p53 and p19Arf mutant mice. p53 and p19Arf are
tumor suppressors frequently mutated in human tumors. In a high throughput
screen in mice for mutations collaborating with either p53 or p19Arf deficiency,
we identified 10.806 retroviral insertion sites, implicating over 300 loci in
tumorigenesis. This dataset reveals 20 genes that are specifically mutated in either
p19Arf -deficient, p53-deficient or wild-type mice (including Flt3, mmu-mir-106a-363,
Smg6 and Ccnd3), as well as networks of significant collaborative and mutually
exclusive interactions between cancer genes (see figure 4). Furthermore, we found
candidate tumor suppressor genes, as well as distinct clusters of insertions within
genes like Flt3 and Notch1 that induce mutants with different spectra of genetic
interactions. Cross species comparative analysis with aCGH data of human cancer
cell lines revealed known and candidate oncogenes (Mmp13, Slamf6, Rreb1) and
tumor suppressors (Wwox, Arfrp2). This dataset should prove to be a rich resource
for the study of genetic interactions that underlie tumorigenesis.
Figure 4: Common Insertion site interaction network representing the co-occurrence or mutual exclusivity
of the 20 most significant CISs. Co-occurring CISs are connected by uninterupted lines (thin line, 0.001<
p-value < 0.05; heavy line, p-value < 0.001), mutual exclusive CISs are connected with broken lines (thin
line, 0.001< p-value < 0.05; heavy line, p-value < 0.001).
Publications
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MOLECULAR GENETICS
Berns A. Kras and Hras – What is the
difference. Nature Genetics 2008;40:1149-50
Boyle P, Anderson BO, Andersson LC,
Ariyaratne Y, Auleleyb GR, Barbacid M,
Bartelink H, Baselga J, Behbehani K,
Belardelli F, et al. Need for global action for
cancer control. Ann Oncol 2008;19:1519-
1521
Lehembre F, Yilmaz M, Wicki A,
Schomber T, Strittmatter K, Ziegler D,
Kren A, Went P, Derksen PW, Berns A, et
al. NCAM-induced focal adhesion assembly:
a functional switch upon loss of E-cadherin.
EMBO J. 2008;27:2603-15
Uren AG, Kool J, ,Matentzogly K,
de Ridder J, Mattison J, van Uitert M,
Lagcher W, Sie D, Tanger E, Cox T,
Reinders M, Hubbard TJ, Rogers J,
Jonkers J, Wessels L, Adams DJ,
van Lohuizen M, Berns A. Large-scale
mutagenesis in p19 ARF and p53 deficient
mice identifies cancer genes and their
collaborative networks. Cell 2008;133:727-41
Berns A. A tRNA with oncogenic capacity.
Cell 2008; 133:29-30
Jongsma J, van Montfort E, Vooijs M,
Zevenhoven J, Krimpenfort P,
Van der Valk M, Van de Vijver M, Berns A.
A conditionele mouse model for malignant
mesothelioma. Cancer Cell 2008;13:261-271
Van Amerongen R, Berns A. Targeted
anticancer therapies: mouse models help
uncover the mechanisms of tumor escape.
Cancer Cell 2008;13:5-7

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MOLECULAR GENETICS
Group leader Daniel Peeper
Daniel Peeper PhD Group leader
Gireesh Anirudhan PhD Post-doc
Christophe Desmet PhD Post-doc
Tristan Gallenne PhD Post-doc
Alexandre Prieur PhD Post-doc
Cornelia Hömig PhD Post-doc
Christelle Lenain MD PhD Post-doc
Katrin Meissl PhD Post-doc
Celia Vogel MSc Post-doc
Thomas Geiger MSc PhD student
Joanna Kaplon MSc PhD student
Thomas Kuilman Msc PhD student
Chrysiis Michaloglou BSc PhD student
Marjon Smit MSc PhD student
Liesbeth Vredeveld MSc PhD student
Sirith Douma MSc Technical staff
Aranzazu Rosado Technical staff
Publications
Kuilman T, Peeper DS. Senescence-
Messaging Secretome: SMS-ing cellular
stress. Nature Reviews Cancer (in press).
Smit MA, Peeper DS. Deregulating EMT
and senescence: double impact by a single
twist. Cancer Cell 2008;14:5-7
Kuilman T, Michaloglou C, Vredeveld LC,
Douma S, van Doorn R, Desmet CJ,
Aarden LA, Mooi WJ, Peeper DS.
Oncogene-induced senescence relayed by an
interleukin-dependent inflammatory
network. Cell 2008;133:1019-31
Prieur A, Peeper DS. Cellular senescence in
vivo: a barrier to tumorigenesis. Curr Opin
Cell Biol. 2008;20:150-5
Schlisio S, Kenchappa RS, Vredeveld LC,
George RE, Stewart R, Greulich H, Shahriari K,
Nguyen NV, Pigny P, Dahia PL, Pomeroy SL,
Maris JM, Look AT, Meyerson M, Peeper DS,
Carter BD, Kaelin WG Jr. The kinesin KIF1Bbeta
acts downstream from EglN3 to induce apoptosis
and is a potential 1p36 tumor suppressor. Genes
Dev. 2008;22:884-93
Michaloglou C, Vredeveld LC, Mooi WJ,
Peeper DS. BRAF(E600) in benign and malignant
human tumours. Oncogene. 2008;27:877-95
RESOLVING CANCER CELL NETWORKS AND IDENTIFYING
CANCER DRUG TARGETS
The Peeper laboratory is dedicated to resolving mechanisms that protect mammalian
cells against oncogenic transformation, aiming to identify novel cancer drug targets.
Non-malignant cells require multiple genetic mutations to transform into tumor
cells. With both classical biochemical and genetic approaches, as well as with
functional screens (based on both cDNA expression and RNA interference genomewide
libraries), we have been focusing on two of these events: bypass of oncogeneinduced
growth arrest (‘premature senescence’, which limits the proliferative capacity
of cells) and suppression of ‘anoikis’ (apoptosis resulting from lack of adhesion,
suppression of which may contribute to metastasis). In addition, we are studying the
role of senescence as an in-vivo mechanism protecting against tumorigenesis. We
anticipate that these approaches, together, will lead to the identification of a network
of gene products, deregulation of which allows for tumorigenesis and metastasis. In
turn, these proteins may represent novel therapeutic drug targets.
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 upon tumor and dissemination. In an attempt to identify
metastasis-associated 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 caspaseassociated
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
suppressed the metastatic potential of the tumor cells as well. 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 particular 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 Most normal mammalian cells have a finite lifespan, thought to
constitute a protective mechanism against unlimited proliferation. This phenomenon
(‘senescence’) is driven by telomere attrition, which triggers the induction of tumor
suppressors, including p16INK4a. In cultured cells, senescence can be elicited also
prematurely, by oncogenes. However, whether such Oncogene-Induced Senescence
(OIS) represents a physiological process has long been debated. Human nevi
(‘moles’), benign tumors of melanocytes, frequently harbor oncogenic mutations in
BRAF (predominantly V600E). Nonetheless, nevi typically remain growth-arrested

for decades and only rarely progress into malignancy (melanoma). We previously
found that nevi have many hallmarks of senescent cells, and proposed that they are
restricted from progression to malignant melanoma by virtue of their ability to
undergo OIS.
Currently, we are using various functional genomic approaches to identify novel OIS
pathways whose deregulation contributes to melanoma. 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, we postulated that
putative tumor suppressors, similar to p16 INK4A , are transcriptionally upregulated
during the senescence response. Indeed, 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. L. 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/EBPb 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 (figure 5).
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Figure 5: Potential benefits and threats of
association of secreted factors with
senescence. The use of SMS factors for
senescence has potential ‘bright’ and ‘dark’
sides. a-c | Several potential advantages
(‘bright’ side) could explain the evolutionary
pressure for the SMS. a | SMS factors could
constitute a ‘danger signal’, which sensitizes
normal neighbouring cells (pink) to senesce.
For example, this may reduce the chance
that damaged cells at risk for neoplastic
transformation would fail to enter
senescence. b | Another possible benefit is
that also (normal) stromal cells could
receive the ‘danger signal’ and stop
supporting growth of the incipient neoplastic
cell, thereby helping to implement a full
senescence response in this cell. Alternatively,
they could start secreting proliferationinhibitory
(SMS) factors. c | A third
advantage is the possible attraction of the
innate immune system, which can dispose of
the senescent cell. d-e | In later stages of
tumourigenesis, tumour cells can misuse
SMS signalling (‘dark’ side). d | By secreting
SMS factors, cancer cells can induce
senescence in stromal cells, such as
fibroblasts. These in turn secrete factors that
contribute to tumour growth. This model of
two-way communication between tumour
cells and stromal cells may explain why
tumour cells commonly express several SMS
factors. e | Stress and/or aging can lead to
an increase in the number of senescent cells,
thereby potentially stimulating expansion of
premalignant cells as a function of the SMS.

86
MOLECULAR GENETICS
Group leader John Hilkens
John Hilkens PhD Group leader
Annabel Zwaagstra MSc PhD student
Mandy Boer Technical staff
Figure 6: Irs4 overexpression in HC11
mammary epithelial cells induces anchorage
independent growth. HC11 mammary
epithelial cells retrovirally transduced with
Irs4 or with an empty control vector
(pMSCV) were grown in soft agar. Colonies
were counted after 3 weeks in culture.
GENES INVOLVED IN BREAST CANCER PROGRESSION
AND METASTASIS
Cancer is the result of sequential accumulation of multiple genetic changes in
normal somatic cells affecting oncogenes and tumor suppressor genes. However,
only a limited number of these genes have been identified. Complete knowledge of
the cancer causing genes and the pathways they control 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 and unravel the
collaborating genetic pathways leading to breast cancer and breast cancer progression
using mouse mammary tumor virus (MMTV) induced insertional mutagenesis in
mouse models.
Identification of mammary cancer genes in various mammary tumor models
by MMTV tagging MMTV proviral insertion in the genome of murine mammary
epithelial cells can activate flanking proto-oncogenes leading to mammary tumor
induction. We have searched for common MMTV insertion sites (CISs, which
indicate the presence of a nearby oncogene) in primary tumors from MMTV infected
BALB/c mice, BALB/c mice conditionally deficient for p53 in the mammary gland
and transgenic FVB mice that overexpress Erbb2 in the mammary gland. We
identified over 50 CISs in a screen of almost 400 mammary tumors from these
strains. Approximately 50% of the CIS associated genes are novel candidate cancer
genes not previously implicated in mammary or other cancers. The number of
tumors with proviral insertions in specific CISs differed strongly in each strain.
A detailed expression analysis of the most frequently tagged genes revealed that
oncogenic (i.e. those genes that are tagged by MMTV) Wnt and Fgf genes were
activated in almost all wild type BALB/c tumors, less frequently in P53 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 most
frequently MMTV tagged genes in the tumors from wild type and conditionally
deficient P53 mice. Rspo3, but not Rspo2, was upregulated in all Erbb2 tumors but
rarely MMTV tagged. Most likely, Rspo3 can also be epigenetically upregulated or is
downstream Erbb2. Eras is frequently tagged in the Erbb2 transgenic mice.
Expression analysis showed that the gene was significantly more frequently
expressed in MMTV induced tumors from the Erbb2 transgenic mice than in MMTV
induced tumors from the FVB background strain, suggesting that Eras maybe
specifically cooperating with Erbb2 in FVB mice.
Irs4 is a novel mammary tumor oncogene We identified the Irs4 locus as a novel
CIS in all three tumor models. Subsequently, we have validated Irs4 as an oncogene
by showing that overexpression of this gene in NMuMG normal mammary epithelial
cells rendered these cells highly tumorigenic in vivo. In addition to Irs4 that is
activated in approximately 20% of the mammary tumors, also Igf2 is targeted by
MMTV in approximately 10% of the tumors, indicating that the insulin-like growth
factor pathway is a major oncogenic pathway in mouse mammary tumorigenesis.
Interestingly, most mammary tumors showing overexpression of Irs4 frequently also
expressed high levels of Irs1 and Irs2 suggesting that the downstream oncogenic
activity of Irs4 is different from that of its family members in mammary tumor cells.
In vitro studies showed that Irs4 overexpression in HC11 mammary epithelial cells
induces anchorage independent growth and protects NMuMG cells against apoptosis
induced by serum deprivation. Both properties are hallmarks for tumorigenesis. Irs4
overexpression did not stimulate cell proliferation but reduced insulin requirement
for growth of NMuMG mammary epithelial cells. Unexpectedly, Irs4 overexpression
did not have a major effect on the Akt and MAP kinase pathways using MAPK and
AKT phosphorylation as read outs, suggesting that Irs4 affects different pathways
than Irs1 and Irs2.

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. E.g., histone modifications, including
acetylation, are central to the transcriptional control of cancer-relevant pathways
governing processes of cell proliferation, apoptosis, differentiation and DNA-repair.
A growing list of non-histone targets is also subject to regulation through reversible
acetylation (e.g. p53) via the opposing enzymatic actions of the same set of acetylases
(HATs) and deacetylases (HDACs) that act on histones. Histone deacetylases
enzymes involved in removing acetyl groups from lysine residues, are promising
drug targets in cancer therapy. Despite evidence of clinical efficacy, the molecular
mechanisms underlying the selective anti-tumorigenic 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. Hdac2 -/- mice are born with a two-fold lower Mendelian frequency
indicating that some Hdac2 -/- mice die during embryonic development. Also, Hdac2deficient
mice display a 25% reduction in total bodyweight. Although Hdac2 -/- MEFs
displayed no obvious phenotype, we observed a significant increase in Hdac1
expression, suggesting a compensatory function of Hdac1 in the absence of Hdac2.
Indeed, inactivation of Hdac1 in Hdac2 -/- MEFs, resulted in a G1 cell cycle arrest
accompanied by senescence-associated b-galactosidase activity (SA-b-gal). Whereas
the expression of several cell cycle inhibitors was not altered in cells lacking Hdac1
and Hdac2, p21 Cip was significantly upregulated in Hdac2-null MEFs and even more
in cells lacking both HDACs. Using p21 Cip shRNA vectors in Hdac1/2 deficient MEFs
we observed an almost complete rescue of the cell cycle arrest, indicating that Hdac1
and Hdac2 collectively suppress p21 Cip in order to maintain cell cycle progression. In
contrast to the results obtained in MEFs we did not observe a reversal of the Hdac2defiency
linked growth-defect in mice that lacked both Hdac2 and p21 Cip , suggesting
that the growth-defect observed in Hdac2 -/- mice is p21 Cip -independent.
Identification of novel HDAC1- and HDAC2-associated transcriptional nodes
and cis-regulatory elements. Inhibition of class I HDAC mediated transcription is
believed to be crucial for the efficacy of HDACi in tumor treatment. The
identification of HDAC1 and HDAC2 target genes will therefore be of importance to
identify pathways targeted by HDACi. Since it is likely that HDAC1 and HDAC2
regulate many genes and the effects of HDAC inhibition can be contributed to the
collaborative action of various genes we established a system to identify HDAC1- and
HDAC2-associated transcriptional nodes through analysis of HDAC1 and HDAC2
transcriptomes using bioinformatics. By determining over-represented cis-regulatory
elements in the promoters of validated HDAC1 and HDAC2 target genes we will
identify DNA binding proteins that recruit HDAC1 and HDAC2 to accomplish their
transcriptional function.
87
MOLECULAR GENETICS
Junior group leader Jan-Hermen Dannenberg
Jan-Hermen Dannenberg PhD Junior
group leader
Inès Bejaoui MSc PhD student
Eva Yanover Technical Staff
Figure 7: Simulatenous ablation of Hdac1
and Hdac2 results in a cell cycle arrest (left
panel, bottom left) accompanied by
senescence-associated b-galactosidase
activity (SA-b-gal) (panel below). p21 Cip
knockdown results in the rescue of this cell
cycle arrest (upper panel, bottom right)

88
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
Publication
Taghavi P, Verhoeven E, Jacobs JJL,
Lambooij JP, Stortelers C, Tanger E,
Moolenaar WH and van Lohuizen M.
In Vitro Genetic Screen Identifies a
Cooperative Role for LPA Signaling and
c-Myc in Cell Transformation. Oncogene
2008;27:6806-6816
Figure 8: Severe fusion of mouse
chromosomes upon loss of telomere capping
due to inactivation of TRF2 (bright white
signals mark telomeric sequences present at
the ends of each chromosome)
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 a 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 different 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. Although TRF2
inhibition, oncogenic stress and DNA double-strand breaks (DSBs) all induce
phenotypically similar senescence responses, we found that on gene expression level
the telomere damage response is very different from oncogene-induced senescence
and highly similar to the response to DSBs. Extensive analysis of the signaling pathways
activated upon TRF2 inhibition is ongoing and has helped us identify a signaling
pathway not known before to directly respond to telomere dysfunction. We are currently
investigating this pathway for its contribution to telomere-induced senescence.
Telomere-induced senescence involves activation of the p53 pathway and, as we and
others have shown, also of the p16INK4a/RB pathway. By rendering telomeres
dysfunctional by partially removing TRF2 from telomeres we were previously able to
demonstrate that, despite its relatively late activation, p16INK4a significantly
contributes to the senescence response triggered by such dysfunctional telomeres.
P16INK4a most likely acts as a backup to p53 in maintaining human cells growth
arrested upon loss of telomere function. We are now investigating the mechanism by
which p16INK4a is induced upon telomere damage.
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 know 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 are
involved in telomere-induced genome instability we have developed an RNAi loss-offunction
genetic screen in mouse cells in which we can instantly uncap telomeres by
inactivating TRF2 to induce severe telomere fusion.
An increasing number of micro-RNAs (miRNA) are being found to regulate an
increasing number of pathways and changes in miRNA levels have been associated
with several cancers. MiRNAs might also affect aging and cancer by affecting the
expression levels of genes involved in telomere maintenance. To identify such
miRNAs we have initiated two types of screens in which we use a collection of
miRNAs constructed by the group of Reuven Agami (Division of Gene Regulation).

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 may contribute to carcinogenesis.
The short generation time (3 months), large progeny size (females typically lay 200
eggs at a time), and availability of readily accessible transparent embryos combine to
make the zebrafish an ideal system for dissecting developmental pathways and
cellular mechanisms that when deregulated underlie malignant transformation.
Analysis of cell polarity and energy metabolism during development and
cancer Mutations in the serine-threonine kinase LKB1 lead to a gastrointestinal
hamartomatous polyposis disorder with increased predisposition to malignancies of
epithelial origin, in particular of the gastro-intestinal tract in humans. LKB1 activates
AMPK under low energy conditions and that leads to growth suppression through
several pathways including inhibition of the mTOR pathway. Additionally, activation
of LKB1 induces complete polarization of human intestinal epithelial cells. To gain
insight into how LKB1 mediates its effects during development and cancer, we have
identified two mutations in the single zebrafish LKB1 ortholog by TILLING. Both
mutations are stop codons in the kinase domain and fortuitously the Y246 mutation
has also been identified in Peutz-Jeghers patients and has been shown to abolish the
catalytic activity of LKB1.
Lkb1 mutant zebrafish die at the larval stage owing to severe problems in maintaining
intestinal architecture. Mutant larvae are indistinguishable from wt siblings the first
5 days of development. Soon after yolk absorption, we observe an abrupt collapse of
the intestinal villi and flattening of the epithelium. Perhaps surprisingly, cell
polarization of intestinal epithelial cells is not affected as localization and levels of
actin and aPKC on the apical membrane are normal. Interestingly, lkb1 mutant
intestines resemble those of starved wild-type larvae at around day 12 pf. Consistent
with this finding we observe premature depletion of liver glycogen. We are currently
investigating whether also at the molecular level the lkb1 mutants exhibit accelerated
metabolism. We are also comparing transcriptional profiles of wt and lkb1
mutants with the aim to unravel the pathways regulated by LKB1 and a possible
interconnection between maintenance of cell polarity, physiology and energy status
as well as implications for cancer. Preliminary analysis of adult aged lkb1/+ fish,
indicates that they are susceptible to bile duct adenocarcinomas, establishing that
LKB1 acts as a tumor suppressor also in fish.
The developmental roles of the Polycomb group proteins of transcriptional
repressors (Collaboration with Maarten Van Lohuizen) Polycomb-mediated
transcriptional repression has critical roles in embryonic development, maintenance
of stem cell fate and cancer. Important targets of repression include developmental
regulators, the Homeobox gene cluster and the Ink4a/ARF tumor suppressor locus.
We aim to uncover the developmental pathways regulated by Polycomb repression.
To this end, we perform morpholino (MO)-mediated knock-down of function and
characterize the resulting phenotype. In particular, we are investigating the effects of
transient inactivation of the E3 ubiquitin ligase, Ring1b. Targeted inactivation of
Ring1b in mice leads to early embryonic lethality precluding analysis of the role of
Ring1b in embryonic development. Fortuitously, maternal deposition of Ring1b
mRNA in zebrafish allows them to complete gastrulation. Transient inactivation of
Ring1b in zebrafish results in heart abnormalities, aberrant heart looping and
diminished blood circulation. We have established that microinjection of three
independent MOs leads to the same phenotype, results in dramatic reduction of
protein levels and reduced ubiquitination of H2A. We are currently characterizing
the phenotype further. In addition, we are generating a stable knock-out line by
employing zinc finger nucleases methodology.
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
Publications
89
MOLECULAR GENETICS
Goessling W, North TE, Lord AM, Ceol C,
Lee S, Weidinger G, Bourque C,
Strijbosch R, Haramis AP, Puder M,
Clevers H, Moon RT, Zon LI. APC mutant
zebrafish uncover a changing temporal
requirement for wnt signaling in liver
development. Dev Biol. 2008;320:161-74
Hawkins TA, Haramis AP, Etard C,
Prodromou C, Vaughan CK, Ashworth R,
Ray S, Behra M, Holder N, Talbot WS,
Pearl LH, Strähle U, Wilson SW. The
ATPase-dependent chaperoning activity of
Hsp90a regulates thick filament formation
and integration during skeletal muscle
myofibrillogenesis. Development
2008;135:1147-56
Figure 9: Vibratome section of a zebrafish
intestine at day 7 post-fertilization stained
with an antibody against aPKC (red).
aPKC is localized at the apical surface of
intestinal epithelial cells. The nuclei are
stained for DAPI (blue).

90
PSYCHOSOCIAL RESEARCH
Division head, group leader Neil Aaronson
Neil Aaronson PhD Group leader
Hester Oldenburg MD PhD Academic staff
Marc Van Beurden MD PhD Academic staff
Emiel Rutgers MD PhD Academic staff
Senno Verhoef MD Academic staff
Saskia Duijts PhD Post-doc
Eric Vermeulen PhD Post-doc
Kirsten Douma 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
Marijke Wevers MSc PhD student
Chad Gundy MSc Senior statistical analyst
Miranda Gerritsma Resarch assistant
Tanja Nagtegaal Research assistant
Marianne Kuenen Research assistant
Publications
Buijs C, Rodenhuis S, Bontenbal M, Beex LVAM,
van der Wal E, Smit W, Nooij MA, Voest E,
Hupperets P, ten Vergert EM, van Tinteren H,
Willemse PHB, Mourits MJE, Aaronson NK,
Post WJ, de Vries EGE. Prospective study of
long-term impact of adjuvant high-dose and
conventional chemotherapy on health-related
quality of life. J Clin Oncol 2007;25:5403-9
Gehring K, Sitskoorn MM, Aaronson NK,
Taphoorn MJB. Interventions for cognitive
deficits in adults with brain tumours.
Lancet Neurol 2008;7:548-60
Gundy CM, Aaronson NK. The influence
of proxy perspective on the evaluation of
health-related quality of life: an empirical
study. Med Care 2008;46:209-16
Hilarius DL, Kloeg PH, Gundy CM,
Aaronson NK. The use of health-related quality
of life assessments in daily clinical oncology
practice: A community hospital-based
intervention study. Cancer 2008;113:628-637
Hoopman R, Terwee CB, Aaronson NK.
Translated COOP/WONCA charts found
appropriate for use among Turkish and
Moroccan ethnic minority cancer patients.
J Clin Epidemiol 2008;61:1036-48
DIVISION OF PSYCHOSOCIAL
RESEARCH AND EPIDEMIOLOGY
PSYCHOSOCIAL ONCOLOGY
The subdivision of psychosocial research is pursuing three major research lines:
(1) health-related quality of life (HRQL) assessment in clinical research and clinical
practice (group Neil Aaronson); (2) symptom perception and management (group
Sanne Schagen and group Neil Aaronson); and (3) psychosocial issues in cancer
clinical genetics (group Eveline Bleiker and group Neil Aaronson).
Development and validation of a prostate cancer-specific health-related
quality of life (HRQL) questionnaire for use in international clinical trials
This multinational study evaluated the psychometrics of the EORTC QLQ-PR25, a
questionnaire assessing the HRQL of prostate cancer patients. The QLQ-PR25 and
the QLQ-C30 were administered to 642 prostate cancer patients from 13 countries
treated with curative or palliative intent. The QLQ-PR25 assesses urinary, bowel, and
sexual symptoms and functioning, and the side-effects of hormonal treatment. 509
patients were available for the final analysis. Multi-trait scaling analyses confirmed
the hypothesized scale structure of the QLQ-PR25. Internal consistency reliability
was good (coefficient a = 0.70-0.86) for the urinary symptoms and sexual function
scales, but lower for the bowel function and side-effects of hormonal treatment scales
(a < 0.70). The module discriminated clearly between clinically distinct patient
subgroups, and was responsive to changes in health status over time. In general, the
QLQ-PR25 demonstrates acceptable psychometric properties and clinical validity.
Some caution should be used in interpreting the bowel function and side-effects of
hormonal therapy scales; results can be reported at the individual item and scale level.
Cognitive rehabilitation of glioma patients: A prospective, randomized study
This multicenter, randomized trial, carried out in collaboration with the University
Medical Center Utrecht (M Sitskoorn, K Gehring) and the VU Medical Center (M.
Taphoorn), evaluated the effectiveness of a cognitive rehabilitation program (CRP) on
the neuropsychological (NP) test performance, cognitive complaints, and HRQL of
patients with brain cancer. In total, 140 adult patients with low-grade and anaplastic
gliomas, favorable prognostic factors, and both subjective cognitive symptoms and
objective cognitive deficits were recruited from 11 hospitals. Patients were
randomized to either an intervention group or a waiting-list control group. The
intervention incorporated both computer-based attention retraining and
compensatory skills training of attention, memory and executive functioning. All
patients completed a battery of NP tests, and self-report questionnaires at baseline,
immediately following completion of the CRP, and at 6 month follow-up.
At immediate post-treatment, statistically significant intervention effects were
observed for measures of subjective cognitive functioning and its perceived burden,
but not for the objective NP outcomes or for any of the other self-report measures.
At 6 month follow-up, the CRP group performed significantly better than the control
group on NP tests of attention and verbal memory. Group differences in subjective
outcomes were no longer statistically significant at 6 months.
These results indicate that the CRP has a salutary effect on short-term cognitive
complaints and on longer-term cognitive performance. Additional research is needed
to identify which elements of the intervention are most effective, and to enhance its
effect on other health outcomes.
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 a usual care control group.
Primary outcomes include musculoskeletal performance (knee extension and grip

strength), 6-minute walk test and 15-meter walking speed, and self-reported physical
functioning. Secondary outcomes include self-reported HRQL and fatigue, selfreported
and objectively assessed physical walking activity, whole body composition
and hematological values. Patient recruitment has recently been completed (N = 128).
To date, 107 patients have completed the study, including 6-month follow-up. The
overall adherence to the exercise program is high (87% of the 24 physical exercise
session attended; range = 21% to 100%). Loss to follow-up during the course of the
study is approximately 15%, primarily due to disease recurrence. It is anticipated that
the final follow-up will be completed by the end of 2009.
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. In total, 325 consenting women, younger than 50 years of age, with
histologically confirmed primary breast cancer, will be randomized to one of the three
intervention groups or to a usual care, ‘waiting list’ control group. All participating
women will be 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 accrual
started in January 2008. To date, 1835 women have been identified as being
potentially eligible for study participation, of whom 1046 returned the initial brief
screening questionnaire. Of these 1046 women, 431 met the eligiblity criteria. To
date, 268 women have been randomly allocated to the one of the 4 study groups.
Patient recruitment, intervention, and data collection (including follow-up) will
continue until approximately mid-2010.
Behavioral and psychosocial effects of rapid genetic counseling and testing in
newly diagnosed breast cancer patients: A multicenter, randomized trial This
multicenter, randomized trial, being carried out in collaboration with the University
Medical Center Utrecht (Margreet Ausems), is investigating the uptake of rapid
genetic counseling and testing (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 (see below).
During an 18 month period, 255 eligible, consenting women from 13 hospitals in the
Amsterdam and Utrecht regions of the Netherlands will be randomized either to a
RGCT group or a “usual care” (UC) group at a ratio of 2:1. Women in the RGCT
group will be referred for genetic counseling within days after diagnosis, and those
who follow through on this referral will receive genetic counseling within 1 week of
diagnosis. If a DNA-test is indicated, the results can be generated within 3 to 6 weeks.
Women in the UC condition will receive standard advice and care. In some cases,
patients may be referred by the treating surgeon or self-refer to genetic counseling.
However, in current practice, this occurs rarely during the pre-surgery period.
The study endpoints include: (1) uptake of RGCT; (2) choice of clinical management
strategy, including the uptake of direct bilateral mastectomy (BLM) or of delayed
preventive contralateral mastectomy (PCM); (3) cancer risk perception, and cancerrelated
distress; (4) knowledge of genetic aspects of breast cancer; (5) decisional
satisfaction; and (6) health-related quality of life (HRQL); and (6) women’s experience
of and satisfaction with RGCT. Standardized questionnaires will be administered to
all patients at study entry, and at 6 and 12 months to assess all psychosocial
outcomes. A subset of women will be interviewed to obtain supplementary,
qualitative data about the RGCT experience.
In 2008, the study protocol and all study materials were finalized, and ethical
approval for the study was obtained from the NKI-AVL institutional review board.
The protocol is currently under review by the UMCU and other participating
hospitals. The first patient from the NKI-AVL was recently randomized.
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PSYCHOSOCIAL RESEARCH
Publications (continued)
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 (in press)
Langendijk JA, Doornaert P, Aaronson NK,
Verdonck-de Leeuw IM, Leemans CR,
Slotman BJ. The impact of late treatmentrelated
toxicity on quality of life (EORTC
QLQ-C30) among patients with head and
neck cancer treated with radiotherapy.
J Clin Oncol 2008;26:3770-3776
Mols F, Aquarius AE, Essink-Bot ML,
Aaronson NK, Kil PJM, van de Poll-
Franse LV. Does Diabetes Mellitus as
comorbid condition affect health-related
quality of life in prostate cancer survivors?
Results of a population-based observational
study. BJU International, 2008 June 3;
(Epub ahead of print)
Scott NW, Fayers PM, Aaronson NK,
Bottomley A, de Graeff A, Groenvold M,
Koller M, Petersen MA, Sprangers MAG.
The relationship between overall quality of
life and its subdimensions in cancer patients
was influenced by culture: analysis, of an
international database. J Clin Epidemiol
2008;61;868-74
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 2008
Sept. 5: (Epub ahead of print)
Van Andel G, Bottomley A, Fosså SD,
Efficace F, Coens C, Guerif S, Kynaston H,
Gontero P, Thalmann G, Akdas A,
D’Haese S, Aaronson NK. An
international field study of the EORTC
QLQ-PR25: A questionnaire for assessing
the health-related quality of life of patients
with prostate cancer. Eur J Cancer
2008;44:2418-2424
Van Seters M, van Beurden M,
ten Kate FJW, Ewing PC, Eijkemans MJC,
Kagie MJ, Meijer CJM, Aaronson NK,
Burger MPM, Helmerhorst THM.
Effectiveness of imiquimod 5% cream in
women with multifocal high-grade vulvar
intrapithelial neoplasia (VIN): results of a
randomized controlled trial. New Engl
J Med 2008;358:1465-73

92
PSYCHOSOCIAL RESEARCH
Group leader Sanne Schagen
Sanne Schagen PhD Group leader
Frits Van Dam PhD Academic staff
Willem Boogerd MD PhD Academic staff
Dieta Brandsma MD PhD Academic staff
Sabine Linn MD PhD Academic staff
Michiel De Ruiter PhD Post-doc
Christien Schilder MSc PhD student
Vincent Koppelmans MSc PhD student
Riejanne Seigers MSc PhD student
Chad Gundy MSc Senior statistical analyst
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 (in press)
Schilder CMT, Eggens PC, Seynaeve C,
Linn SC, Boogerd W, Gundy C, Beex LV ,
van Dam FS, Schagen SB.
Neuropsychological effects of tamoxifen and
exemestane after AC-chemotherapy in
postmenopausal women with early breast
cancer: cross-sectional findings from the
neuropsychological TEAM-side study. Acta
Oncol 2008;5:1-10
Kreukels BP, Van Dam FS, Ridderinkhof RK,
Boogerd W, Schagen SB. Persistent
neurocognitive problems after adjuvant
chemotherapy for breast cancer. Clin Breast
Cancer 2008;8:80-7
Schilder CMT, Linn SC, Van Dam, FSAM,
Schagen SB. De invloed van hormonale
therapie op het cognitief functioneren van
patiënten met het mammacarcinoom. Ned
Tijdschr Geneeskd 2008;152:494-8
Kreukels BP, Hamburger HL, de Ruiter MB,
van Dam FS, Ridderinkhof KR, Boogerd W,
Schagen SB. ERP amplitude and latency
in breast cancer survivors treated with
adjuvant chemotherapy. Clin Neurophysiol.
2008;119:533-41
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.
Effects of endocrine treatment on cognitive function This prospective
multicenter study is examining the neuropsychological sequelae of exemestane and
tamoxifen in postmenopausal breast cancer patients compared to healthy controls.
Patients were recruited from the TEAM trial, an open label, randomized multicenter
study of exemestane treatment (25 mg once daily) or 2-3 years of adjuvant tamoxifen
(20 mg once daily) followed by 3-2 years exemestane treatment (25 mg once daily).
Patients were assessed at T1, before the start of endocrine treatment, and at T2 after
12 months of treatment. At T2, after adjustment for T1 performance, exemestane
users (n=99) did not perform significantly worse than healthy controls (n=120) on
any of 8 cognitive domains. Tamoxifen users (n=80) performed worse than healthy
controls on verbal memory (p=.003) and executive functioning (p=.005), and worse
than exemestane users on information processing speed (p=.02).These findings
underscore the need to include cognitive effects of endocrine treatment in long-term
safety studies and stress the need for more knowledge about differential effects on
cognitive function of these treatments.
Nature and mechanism of cognitive deficits following chemotherapy:
an (f )MRI study (NKI-AMC) In this ‘seed money’ project, 18 breast cancer patients
treated with high-dose CTC chemotherapy (CT) and 15 breast cancer patients not
treated with chemotherapy (non-CT) were scanned while performing neuropsychological
tests at the 3 Tesla MRI facility of the Academic Medical Centre in
Amsterdam, combining structural, functional and biochemical imaging. Results
indicate that, 10 years after treatment, CT patients performed significantly worse
than non-CT patients on several cognitive tests while being scanned. These
impairments where accompanied by pronounced hypoactivation of bilateral posterial
parietal cortex, indicating dysfunction of the dorsal attention network during memory
encoding and executive functioning. 1H-MR spectroscopy of cerebral white matter, in
addition, indicated significantly lower levels of N-acetyl aspartate, a marker of
neuronal viability, suggesting decreased axonal integrity in CT patients relative to
non-CT patients. Together, these data demonstrate detrimental effects of CT on brain
function and biochemistry almost ten years after treatment.
CTC Control
Figure 1: Hypoactivation of posterior parietal cortex in breast cancer patients almost ten years after being
treated with adjuvant high-dose chemotherapy (left) compared to patients not treated with chemotherapy
(right) during an fMRI memory encoding task.

Animal studies exploring the underlying mechanisms of cognitive impairment
(NKI-RUG) Previous experiments by our group revealed both short- and long-term,
dose-dependent adverse effects of methotrexate (MTX) on learning behavior and
hippocampal cell proliferation in rats. We have now investigated the potential effect
of single high-dose (250 mg/kg) MTX on neuroinflammation and brain energy
supply in rats using PET and immunohistochemistry. PET data showed no
significant differences in neuroinflammation between control and MTX animals,
while immunohistochemistry indicated clearly more activated microglia in MTX
compared to control animals, one and three weeks after treatment. PET Glucose
metabolism was decreased one week after treatment in MTX animals.
Immunohistochemistry results showed that MTX animals had significantly less
blood vessels compared to controls at both time points. Subsequent experiments will
focus on the causes of microglia activation and reduced brain vascularization, the role
of multidrug resistance pumps on the effect of MTX, and the potential reversing of
the negative effect of MTX on learning.
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. 70 breast cancer survivors are
currently included in the study.
The influence of priming chemotherapy related cognitive problems on
self-reported complaints and performance (NKI-VU University)
We previously showed that pre-existing knowledge of chemotherapy-associated
cognitive problems and priming the “chemo-brain” schema can increase the
reporting of cognitive complaints by facilitating the accessibility of this concept. In a
pilot project, we examined whether increased accessibility of a schema (in this case
an age-impairs-cognition schema), also has a negative impact on cognitive test
performance in healthy women (N=100). We found that increased accessibility
increased self-reported cognitive complaints and also lowered performance on
objective NP tests, showing that information influences participants’ mindsets which,
in turn, may induce expectancy-congruent self-perceptions and performance.
93
PSYCHOSOCIAL RESEARCH
Publications (continued)
Vardy J, Wefel JS, Ahles T, Tannock IF,
Schagen SB. Cancer and cancer-therapy
related cognitive dysfunction: an
international perspective from the Venice
cognitive workshop. Ann Oncol,
2008;19:623-9
Schagen SB, Boogerd W, Muller MJ,
Huinink WT, Moonen L, Meinhardt W,
Van Dam FS. Cognitive complaints and
cognitive impairment following BEP
chemotherapy in patients with testicular
cancer. Acta Oncol. 2008;47:63-70
Seigers R, Schagen SB, Beerling W,
Boogerd W, Van Tellingen O, Van Dam FS,
Koolhaas JM, Buwalda B. Long-lasting
suppression of hippocampal cell
proliferation and impaired cognitive
performance by methotrexate in the rat.
Behav Brain Res. 2008;186:168-75
Schilder CTM, Schagen SB,
FSAM van Dam. Effect of hormones and
hormonal treatment on cognition. In.
Cognition and Cancer. Meyers & Perry eds.
Cambridge University Press 2008

94
PSYCHOSOCIAL RESEARCH
Group leader Eveline Bleiker
Eveline Bleiker PhD Group leader
Senno Verhoef MD Academic staff
Irma Kluijt MD Academic staff
Daniela Hahn MSc Academic staff
Leonie Woerdeman MD Academic staff
Kirsten Douma MSc PhD student
Chantal Lammens MSc PhD student
Willem Eijzenga MSc PhD student
Chad Gundy MSc Senior statistical analyst
Tanja Nagtegaal Research assistent
Publications
Bleiker EMA, Hendriks JHCL, Otten JDM,
Verbeek ALM, Ploeg HM van der.
Personality factors and breast cancer risk: a
13-year follow-up. JNCI 2008;100:213-218
Bleiker EMA, Hahn DEE, Smets, EMA.
Familiaire Tumoren Hoofdstuk. In:
Psychologische patiëntenzorg in de
oncologie, 2008 Editors JCJM de Haes, LM
Gualthérie van Weezel, R Sanderman,
HBM van de Wiel (Ed.) (in press)
Bleiker EMA, Hahn DEE. Beslissen bij
erfelijkheidsonderzoek voor kanker: kansen
en keuzen. De Psycholoog, 2008;43:12-19
Douma KFL, Aaronson NK, Vasen HFA,
Bleiker EMA. Psychosocial issues in genetic
testing for familial adenomatous polyposis:
a review of the literature. Psycho-Oncology
2008;17:737-745
Figure 2: compliance with screening by
endoscopy, in 75 individuals from FAP
families at high risk of developing colorectal
cancer: % of individuals that reports pain
and embarrassment with and without a
sedative.
PSYCHOSOCIAL ISSUES IN CANCER GENETICS
The research line is being conducted in close collaboration with the family cancer
clinic. It comprises a number of studies which are focused on two psychosocial
themes in genetic counseling for cancer: 1) early detection of psychosocial problems
and the development of psycho-educational interventions; and 2) the uptake and
long-term psychosocial impact of risk-reducing behavior.
The long term psychosocial impact of genetic testing among familial
adenomatous polyposis (FAP) families This study began in 2004, in collaboration
with the Netherlands Foundation for the Detection of Hereditary Tumors. The aim of
the study is to evaluate the long-term (>1 yr) psychosocial impact of the diagnosis of
FAP among individuals from high-risk families. In total 525 FAP-family members
(response 64%) and 131 partners of individuals with a FAP-diagnosis have completed
the questionnaire, and 60 respondents have been interviewed. Data collection has
been completed and analyses are ongoing. Preliminary analyses indicate that: 1) 20%
of individuals from FAP-families have moderate to severe levels of distress; 2) only a
quarter 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%); 4) based on
medical record data, 20% of the at-risk group is less than fully compliant with
surveillance advice; and 5) under-compliance is associated significantly with
perceived self-efficacy, use of sedatives during surveillance, pain after surveillance
and low perceived benefits of surveillance.
Psychosocial aspects of genetic testing in families at high risk of multiple
tumors at various sites and ages The aim of this study is to investigate the uptake
of genetic testing for Li-Fraumeni Syndrome (LFS) and Von Hippel-Lindau Disease
(VHL), evaluate the psychosocial consequences of (not) undergoing genetic testing,
and to assess compliance with recommended surveillance programs. Data collection
has been completed. In total, 243 individuals (78%) participated by completing a selfreport
questionnaire. A subset of 94 respondents also participated in a semi-structured
interview. First analyses of the questionnaire date indicate that: 1) 39% report moderate
to high levels of distress; 2) of these, 33% had received psychosocial support; 3) of
those who had not received psychosocial support, 28% indicated they would have
liked to; 4) 92% of the high risk VHL family members who had received screening
advice self-reported that they had been fully compliant with the advice given.
Surveillance for early pancreatic neoplasia in high-risk individuals This multicenter
study is evaluating the feasibility and effectiveness of surveillance for early
pancreatic neoplasia in high-risk individuals. The purpose of the psychosocial arm of
this study is to assess experiences and compliance with the offered surveillance
program for pancreatic cancer (MRI and EUS), to evaluate the psychosocial impact of
the pancreatic surveillance program, and to investigate the communication of the
pancreatic risk and its preventive options to first, second and third degree relatives.
Approval was obtained from the Review Board of the AMC and Erasmus MC.
Experiences with nipple banking This study is being carried out in collaboration
with the Department of Plastic Surgery (Leonie Woerdeman, Kalam Ahmed) and the
Department of Psychosocial Counseling and Support (Daniela Hahn). During the
past three years, about 300 women who had had a mastectomy directly followed by
reconstruction, were offered nipple banking. Of these, about 100 women opted for
nipple banking, about 160 women opted for a nipple tattoo, and about 40 women did
not have any nipple reconstruction. In 2008, two focus groups were conducted,
including 11 women who had undergone nipple banking. These groups provided
information for the development of a research proposal and a questionnaire to be used
in a cross-sectional study in which experiences with three types of nipple ‘reconstruction’
are compared. All 300 women will receive a psychological questionnaire, including
questions about their decision about their nipple reconstruction, and their experiences
with the option chosen. A prospective study is also currently being planned.

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 1) whether
hormonal/life-style factors modify cancer risk in BRCA1/2 families, 2) the agespecific
cumulative risks of breast, ovarian and other cancers based on full pedigree
information.
We estimated the risks of breast and ovarian cancer in BRCA1/2 carriers, using the
pedigree information of 19,668 family members in 758 families. Data were analyzed
with a modified segregation analysis model adjusting for ascertainment. The
increased risk of breast cancer for BRCA1- and BRCA2 carriers, as compared to the
general population, was strongly age-dependent for BRCA1, but not for BRCA2.
Younger BRCA1 carriers showed highest hazard ratios (HRs). No trend was found for
ovarian cancer. The cumulative risk (CR) of developing breast cancer before age 70
was 45% for BRCA1, and 31% for BRCA2, while the CR of ovarian cancer was 31% for
BRCA1 and 7% for BRCA2. Among BRCA1 families, younger birth cohorts had
much higher risks (65% for breast cancer and 45% for ovarian cancer for women
born >1940), while no birth cohort effects were found for BRCA2. In addition, a
higher number of family members with breast cancer strongly increased the lifetime
risk among BRCA1 carriers, but not among BRCA2 carriers. The birth cohort and
family history effects may partly explain the differences in estimated lifetime risks as
found in the literature.
We examined the association between physical activity and breast cancer risk among
1,026 BRCA1/2 carriers in a retrospective cohort analysis. Survival bias was examined
by comparing the results of the analysis of the entire cohort with those of a pseudoincident
cohort, diagnosed/censored within ten years preceding completion of the
questionnaire. The entire cohort analysis showed a non-significant breast cancer risk
increase of ever sports activity lifetime (HR=1.21), while the pseudo-incident cohort
analysis showed a decreased risk (HR=0.84), which suggested that survival bias was
present. Among active women in the pseudo-incident cohort we observed a risk
reduction for medium and high levels of intensity lifetime sports activity as
compared to low levels (HR=0.59 and HR=0.77, respectively). For frequency and
duration differences were smaller. Among women active in sports before age 30,
dose-response associations of increasing levels of intensity, frequency, or duration
with lower breast cancer risk were observed, with the strongest trends found for
intensity and frequency. Ever versus never having participated in sports activity after
age 30 was inversely associated with breast cancer risk (HR=0.59), but no doseresponse
trends were found. These results suggest that BRCA1/2 mutation carriers
can reduce their risk of breast cancer by engaging in sports activities, preferably
starting at a young age.
We started to coordinate the data collection of the International BRCA1/2 Carrier
Cohort Study (IBCCS), in which three national studies (UK, NL, F) participate, as
well as 11 (multi)center-based studies. In addition, we started to coordinate the risk
factor data collection for the Consortium of Investigators of Modifiers of BRCA1/2
(CIMBA), an international collaborative study on more than 10,000 BRCA1/2
carriers.
In our prospective cohort of 12,091 DES daughters (DESnet project), followed from
December 1992 till June 2008 cancer incidence was assessed through linkage with
the Dutch pathology database (PALGA) and the Netherlands Cancer Registry (NCR).
Median age at the end of follow-up was still relatively young, 44.0 years. A total of
340 incident cancers occurred. No overall increased risk of cancer was found
(standardized incidence ratio (SIR)=1.0). The risk of clear cell adenocarcinoma of the
vagina and cervix (CCA) was strongly increased (SIR=24.2), with the increased risk
also present at ages above 40 years. Overall breast cancer risk was not increased, nor
in women older than age 40. Melanoma risk diagnosed before age 40 was 60%
Group leader Flora Van Leeuwen
Group leader Matti Rookus
95
EPIDEMIOLOGY
Flora Van Leeuwen PhD Group leader
Matti Rookus PhD Group leader
Annegien Broeks PhD Academic staff
Berthe Aleman MD Academic staff
Petra Nederlof PhD Academic staff
Nicola Russell PhD Academic staff
Emiel Rutgers MD PhD Academic staff
Laura Van ’t Veer PhD Academic staff
Senno Verhoef PhD Academic staff
Marjanka Schmidt PhD Senior post-doc
Marieke De Bruin PhD Post-doc
Peggy Manders PhD Post-doc
Ina Mulder PhD Post-doc
Eric Vermeulen PhD Post-doc
Dorien Voskuil PhD Post-doc
Sandra Van den Belt-Dusebout MSc PhD student
Naomi Boekel MSc PhD student
Richard Brohet MSc PhD student
Mathilde Cardous-Ubbink MSc PhD student
Tamara Marees MSc PhD student
Anouk Pijpe MSc PhD student
Janneke Verloop MSc PhD student
Willem Klokman MD MSc Senior statistical analyst
Thea Mooij MSc Statistical analyst
Wieke Heideman MSc Research assistant
Esther Janssen Research assistant
Kiki Jeanson MSc Research assistant
Marianne Kuenen Research assistant
Renée Mulder MSc Research assistant
Gabey Ouwens Research assistant
Renate Udo Research assistant
Marie-José Blom MSc Data manager
Eduard Ivanov MSc Data manager

96
EPIDEMIOLOGY
Publications
AC Antoniou, MA Rookus, N Andrieu,
RM Brohet, J Chang-Claude, S Peock,
M Cook, DG Evans, R Eeles, EMBRACE,
C Nogues, L Faivre, P Gesta, GENEPSO7 ,
FE van Leeuwen, MGEM Ausems,
GEO-HEBON, A Osorio, T Caldes,
J Simard, J Lubinski, AM Gerdes, E Olah,
C Fürhauser, H Olsson, B Arver, P
Radice, DF Easton and DE Goldgar.
Reproductive and hormonal factors, and
ovarian cancer risk for BRCA1 and BRCA2
mutation carriers: results from the
International BRCA1/2 Carrier Cohort
Study. Cancer Epidemiol Biomarkers Prev.
(in press).
Broeks, A., Braaf, L.M., Huseinovic, A.,
Schmidt, M.K., Russel, N.S.,
Van Leeuwen, F.E., Hogervorst, F.B., and
‘t Veer L.J. The spectrum of ATM missense
variants and their contribution to
contralateral breast cancer. Breast Cancer
Res. Treat. 2008; 107: 243-248.
Ceelen M, van Weissenbruch MM,
Vermeiden JPW, van Leeuwen FE,
Delemarre-van de Waal HA. Increased
blood pressure in children born after in vitro
fertilization. J Clin Endocrinol Metab
2008: 93: 1682 – 1688.
Ceelen M, van Weissenbruch MM,
Roos JC, Vermeiden JP, van Leeuwen FE,
Delemarre-van de Waal HA. Body
composition in children and adolescents
born after in vitro fertilization or
spontaneous conception. J Clin Endocrinol
Metab 2007;92:3417-3423
De Bruin ML, Huisbrink J, Kuenen MA,
Ouwens GM, Hauptmann M,
van ’t Veer MB, Aleman BMP,
van Leeuwen FE. Treatment-related risk
factors for premature menopause
following Hodgkin’s lymphoma. Blood
2008; 111: 101-108
De Kok IMCM, Habbema JDF,
MJE Mourits, Coebergh JWW,
Van Leeuwen FE. Onvoldoende gronden
voor opname van vaccinatie tegen Humaan
papillomavirus in het Rijksvaccinatieprogramma.
Ned Tijdschr Geneeskd;
2008;152:2001-2006
increased, but the risk increase was no longer present at higher ages. No increased
risks were found for other types of cancer. Thus, we found no increased risks for
cancer except for CCA at all ages and melanoma before age 40. The increased risk of
breast cancer at higher ages, as found in an US cohort of DES daughters, was not
confirmed so far.
Our nation-wide cohort of 25,152 women treated for subfertility was recently linked
with the Dutch pathology database (PALGA), to assess the risk of breast cancer.
The cohort consists of 19,145 women who received ovarian stimulation for in vitro
fertilization (IVF) between 1983 and 1995 and 6,007 subfertile control women not
treated with IVF. In total, we observed 378 first invasive breast cancers. Compared to
the general population, the risk of breast cancer was not increased in the full cohort,
nor in the IVF and control group separately (Standardized Incidence Ratio
(SIR)=0.9). The risk of breast cancer among IVF-treated women was similar to that
of subfertile controls (HR=1.08), adjusting for confounders like parity, age at
menarche, family history of breast cancer and BMI. Ongoing analyses focus on doseresponse
relations and differences in breast cancer risk according to treatment period
and cause of subfertility. In 2008, we started with the data-collection for the
expansion of the IVF cohort (n = 13,000) through on-line questionnaires
(collaboration with CW Burger, Erasmus MC Rotterdam).
In part of our cohort of breast cancer patients aged

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 for Hodgkin’s lymphoma (n=3,400), testicular cancer (n=2,707)
and breast cancer (n=50,000) over a period of up to 30 years after primary treatment.
After our previous findings of increased risk for cardiovascular diseases among
patients treated for Hodgkin lymphoma, we further investigated the excess risk for
cerebrovascular disease in this group of patients. Among 2201 5-year survivors,
treated for Hodgkin lymphoma before age 51 (median follow-up time of 17.5 years),
we observed 96 patients developing cerebrovascular disease (55 strokes, 31 transient
ischemic attacks (TIA), 10 patients with TIA and stroke; median age 52 years). Most
ischemic events were of large-artery atherosclerotic (36%) or cardioembolic subtype
(24%). The 30-year cumulative incidence accounting for death as a competing risk
for ischemic stroke or TIA 30 years after Hodgkin’s lymphoma treatment was 7%
(figure 1). Compared to the general population Hodgkin lymphoma survivors
experience a 2-fold increased risk for stroke and a 3-fold increased risk for TIA.
Radiation to the neck and mediastinum was an independent risk factor for ischemic
cerebrovascular disease (HR=2.5). Treatment with chemotherapy was not associated
with an increased risk. Hypertension, diabetes mellitus and hypercholesterolemia
were associated with the occurrence of ischemic cerebrovascular disease, whereas
smoking and overweight were not. Patients treated for Hodgkin’s lymphoma
experience a substantially increased risk of stroke and TIA, associated with radiation
to the neck and mediastinum. Physicians should consider appropriate risk-reducing
strategies.
With regard to second cancer risk among survivors of Hodgkin lymphoma we
focused this year on pleural malignancies and more detailed analyses of breast cancer
risk. Malignant mesothelioma is a relatively uncommon malignancy. Although the
pathogenesis is primarily related to asbestos, malignant mesothelioma may be
associated with radiation exposure. Recently, increased risks for mesothelioma
following radiation for lymphoma have been reported. Because these findings are
based on small numbers of patients, confirmation is needed. We examined
malignant mesothelioma risk in 2567 5-year survivors of Hodgkin lymphoma.
Overall, the risk was almost 26-fold increased compared to the general population
leading to an absolute excess risk of 3.5 cases per 10,000 patients per year. Histology
and survival of the mesothelioma cases following Hodgkin lymphoma were
comparable to mesothelioma cases from the general population, but the proportions
of males and asbestos-exposed individuals were lower than expected. The evidence
for radiotherapy as a cause of mesothelioma independent of exposure to asbestos is
expanding, and the diagnosis ‘mesothelioma’ should be kept in mind whenever
related symptoms arise in previously irradiated patients.
Figure 3. The cumulative incidence of ischemic stroke and TIA following Hodgkin’s lymphona treatment,
accounting for death as a competing risk.
Publications (continued)
97
EPIDEMIOLOGY
Hoogendoorn WE, Hollema H,
van Boven HH, Bergman E,
de Leeuw-Mantel G, Platteel I, Fles R,
Nederlof PM, Mourits MJE,
van Leeuwen FE, and the Comprehensive
Cancer Centers’ TAMARISK-group.
Prognosis of uterine corpus cancer after
tamoxifen treatment for breast cancer.
Breast Cancer Res Treat 2008;112:99-108
De Jong D, Vasmel WLE, De Boer JP,
Verhave G, Barbé E, Caparie MK,
van Leeuwen FE. Risk of anaplastic large
cell lymphoma in women with breast
implants. JAMA 2008;300/17: 2030
Gronwald J, Pijpe A, Byrski T, Huzarski T,
Stawicka M, Cybulski C, van Leeuwen Fe,
Lubínski J, Narod SA. Early radiation
exposures and BRCA1-associated breast
cancer in young women from Poland.
Breast Cancer Res Treat 2008;112:581-4
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 (in press)
Hooning MJ, Aleman B, Baaijens MHA,
Hauptmann M, Klijn JGM, Noyon R,
Stovall M, van Leeuwen FE. Roles of
radiotherapy and chemotherapy in the
development of contralateral breast cancer.
J Clin Oncol 2008;26:5561-8
JM Korse, JMG Bonfrer, M van Beurden,
RHM Verheijen, MA Rookus. Estradiol
and testosterone levels are lower after an
oophorectomy than after a natural
menopause. Tumor Biology (in press)
Marees T, Moll AC, Imhof SM,
de Boer MR, Ringens PJ, van Leeuwen FE.
Risk of Second Malignancies in Survivors of
Retinoblastoma: More Than 40 Years of
Follow-Up. J Natl Cancer Inst. 2009 Dec 9
Schaapveld M, Visser O, Louwman MJ,
Willemse PHB, de Vries EGE,
van der Graaf WT, Otter R, Coebergh JW,
van Leeuwen FE. The impact of adjuvant
therapy on controlateral breast cancer risk
and the prognostic significance of
controlateral breast cancer: a populationbased
study in the Netherlands. Breast
Cancer Res Treat 2008;110:189-197

98
EPIDEMIOLOGY
Publications (continued)
Schaapveld M, Visser O, Louwman MJ,
de Vries EGE, Willemse PHB, Otter R,
van der Graaf WTA, Coebergh JWW,
van Leeuwen FE. Risk of new primary nonbreast
cancers following breast cancer
treatment: A Dutch population-based study.
J Clin Oncol 2008;8:1239-1246
Travis LB, Hodgson D, Allen J,
Van Leeuwen FE. Second cancers. In:
DeVita VT Jr, Hellman S, Rosenberg SA
(eds). Cancer: principles and practice of
oncology (ed 8). Philadelphia, PA:
Lippincott 2008:2718-2742
Van Leeuwen FE, Swerdlow AJ, Travis LB.
Second cancers after treatment of Hodgkin
lymphoma. In: Hoppe RT, Mauch PM,
Armitage JO, Diehl V, Weiss LM. (Eds.).
Hodgkin Lymphoma, second edition.
Philadelphia: Lippincott Williams &
Wilkins. 2007;347-370
Vermeulen E, Schmidt MK, Aaronson NK,
Kuenen M, van Leeuwen FE. Obtaining
‘fresh’ consent for genetic research with
biological samples archived ten years ago.
EJC (in press)
Voskuil DW, Vrieling A, Korse CM,
Beijnen JH, Bonfrer JM, van Doorn J,
Kaas R, Oldenburg HS, Russell NS,
Rutgers EJ, Verhoef S, van Leeuwen FE,
van’t Veer LJ, Rookus MA. Effects of
lycopene on the insulin-like growth factor
(IGF) system in premenopausal breast
cancer survivors and women at high
familial breast cancer risk. Nutr. Cancer
2008;60:342 – 353
Vrieling A, Voskuil DW, Bonfrer JM,
Korse CM, van Doorn J, Cats A,
Depla AC, Timmer R, Witteman BJ,
van Leeuwen FE, Van’t Veer LJ,
Rookus MA, Kampman E. Lycopene
supplementation elevates circulating
insulin-like growth factor binding protein-1
and -2 concentrations in persons at greater
risk of colorectal cancer. Am J Clin Nutr.
2007;86:1456-1462
Vrieling A, Rookus MA, Kampman E,
Bonfrer JMG, Bosma A, Cats A,
van Doorn J, Korse CM, Witteman BJM,
van Leeuwen FE, van ‘t Veer LJ, Voskuil DW.
No effect of red clover-derived isoflavone
intervention on the Insulin-like Growth Factor
(IGF) system; a randomized cross-over trial in
women. Cancer Epidemiol Biomarkers Prev.
Oct. 2008;17:2585-2593
The strongly elevated risk of breast cancer following treatment for Hodgkin
lymphoma has become a major concern for female survivors of this disease. Within
our Dutch Hodgkin lymphoma survivors cohort, we examined the risk of breast
cancer up to more than 30 years after treatment of Hodgkin lymphoma, focusing on
the effect of radiation fields. 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 30-year cumulative incidence of breast
cancer accounting for death as a competing risk was 19%. 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.
To further study the effects of radiotherapy, chemotherapy, reproductive and genetic
factors on the risk of breast cancer after Hodgkin lymphoma, we are performing a
nationwide case-control study (collaboration with Division Experimental Therapy,
A. Broeks, L. van ‘t.Veer). So far we have identified 138 case patients, who were
individually matched to 398 controls. All women still alive receive a questionnaire on
lifestyle factors and hormone use and are asked to provide a blood sample for genetic
analyses.
In collaboration with division of Experimental Therapy we also examined tumor
characteristics and gene expression profiles of 22 breast cancers following
radiotherapy for Hodgkin lymphoma from our larger series, and compared these
with age-matched sporadic breast cancer without prior radiation exposures. The
results point to a distinct molecular signature of radiation-associated breast cancer.
We also assessed the risk of secondary non-breast cancers in a recently treated
population-based cohort of breast cancer patients focused on the association with
treatment and prognostic implications. The cohort comprised 58,068 Dutch patients
diagnosed with invasive breast cancer between 1989 and 2003. After a median
follow-up of 5.4 years, 2,578 second non-breast cancers had occurred. Compared with
the Dutch female population at large, in this cohort, the SIR of second non-breast
cancers was significantly increased (SIR=1.22). The absolute excess risk was 13.6 per
10,000 person-years. SIRs were elevated for cancers of the esophagus, stomach,
colon, rectum, lung, uterus, ovary, kidney, and bladder cancers, and for soft tissue
sarcomas, melanoma, non-Hodgkin’s lymphoma, and acute myeloid leukemia. The
10-year cumulative incidence of second non-breast cancers (adjusting for competing
risks) was 5.4%. Among patients younger than 50 years, radiotherapy was associated
with an increased lung cancer risk (HR=2.31) and chemotherapy with decreased risk
for all second non-breast cancers (HR=0.78) and for colon and lung cancer. Among
patients age 50 years and older, radiotherapy was associated with raised risk of soft
tissue sarcoma (HR=3.43); chemotherapy with increased risks of melanoma, uterine
cancer, and acute myeloid leukemia; and hormonal therapy with uterine cancer
(HR=1.78). A second non-breast cancer significantly affected the patient’s prognosis
(HR=3.98 for overall mortality).

EARLY STAGE TECHNOLOGY ASSESSMENT
Constructive Technology Assessment of the introduction of a 70-gene micro
array prognostic test in breast cancer treatment From 2003 until end 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. As the
diffusion of this technology is in an early stage and the course of development not
easy to predict, an evaluation approach is chosen that takes the technology dynamics
into account, constructive technology assessment (CTA).
The project proceeds well: a paper on the overall results on CTA was accepted, an
internal guideline on patient rights concerning banked tissue finalised and a scenario
session was conducted together with the breast group international (BIG). The
results of these scenarios will be used for the Cost Effectiveness Analysis of which
the preliminary (promising) results will be send to the Dutch Health Insurance
Board by end of 2008.
In 2009 finalising the CEA modelling, a CEA of the 70-gene test in France and a
comparison between cost-effectiveness in France and the Netherlands are foreseen.
Building on the experiences of early stage technology assessment, funds were
granted to conduct scenario analysis and cost effectiveness modelling on the most
promising results of the CTMM project focusing on the use of markers in the
treatment of breast cancer.
In preparation of the establishment of a surgical center for minimal invasive
interventions, a preparatory study is being conducted into the appropriate
methodology for early stage technology assessment for surgical innovations, using
the operation robot as a case.
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.
In order to define optimal performance levels a series of international benchmarking
projects has been performed on comparing performance of 3 Comprehensive Cancer
Centers, 3 ambulatory chemotherapy treatment centers (ACT), 6 fundamental research
organisations and in 2008 a study on 4 Radiotherapy departments was concluded.
Applying operations research methods have resulted in improvements in efficiency
of the AVL ACT of 25-30% and after after acceptance of a paper on this topic, a
multicenter proposal will be drafted.
Using the experience of earlier OR simulation techniques (such as a verification of
the efficiency of the operation room planning and scheduling), in 2008 a project was
concluded on the use of simulation to attain a high degree of open access in the
radiology department; reduction of the length of the diagnostic track, including CT,
form three to one week and reducing the number of hospital visits was proven to be
possible and resulted in implementation proposals.
In cooperation with the UT PhD student Peter van Berkel started halftime on
mathematic analysis of care pathways within the oncologic hospital setting and
measuring the effect of increased focus on efficient capacity use.
Rehabilitation, physical activity and cancer Survivorship care and rehabilitation
are important elements of a cancer center’s program.In 2008 we have focused on
strengthening and expanding the infrastructure in which studies in this area can be
conducted. A multidisciplinary rehab program was started for breast cancer survivors
receiving adjuvant treatment in cooperation concerning Herstel and and an
agreement was reached to start a formal head and neck rehab program, approved by
health insurance companies. A retrospective needs-assessment study was performed
under 109 breast cancer survivors. Between 20 and 30% indicated need for care or
rehabilitation in both physical and psychological health domains.
Apart from cooperation in the drafting of a meanwhile funded RCT on physical
exercise during chemotherapy, WvH was requested to act as scientific coordinator of
a program proposal within Alpe d’Huzes focussing on ‘patient empowerment, return
to work, tele-monitoring and implementation of relevant findings and programs all
related to physical exercise and supported by innovative IT’. It is foreseen that this
large action research program will be submitted early 2009.
99
PSYCHOSOCIAL RESEARCH
Group leader Wim Van Harten
Wim Van Harten MD PhD Group leader
Valesca Retel MSc Research staff
Jolien Bueno de Mesquita MD Research staff
Marian Hummel Academic staff
Wineke Van Lent Research staff
Peter Van Berkel Research staff
Rozan Gilles Research assistant
Miranda Van Duijn MSc Academic staff
Rutger Dahmen MD Academic staff
Inge Haenen Research assistant
Joost Deetman Research assistant
Gijs Hesselink Research assistant
Ingrid Nota Research assistant
Publications
Retèl VP, Hummel JM, Van Harten WH.
Early phase Technology Assessment of
Nanotechnology in Oncology, Tumori, 94:
284-290, 2008 Genova
Van der Ploeg HP, Streppel KR,
van der Beek AJ, Van der Woude LH,
Vollenbroek-Hutten MMR, Van Harten WH,
Van Mechelen W. Underlying Mechanisms of
Improving Physical Activity Behavior after
Rehabilitation. Int. J. Behav. Med.
2008;15:101-108
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. Int. J. Techn. Ass. in Health Care.
2009;25:73-83
Klopper AHJ, Meerding NM,
Van Harten WH, Wilderom CPM.
Measuring Stereotypical Images between
Medical Doctors and Managers in Hospitals.
J. Health Org. and Manag (in press)
Van Lent WAM, Goedbloed N,
Van Harten WH. Improving the efficiency
of a chemotherapy day unit; applying a
business approach to oncology. Eur. J.
Cancer (in press)

100
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 Graduate
student
Tessa Buckle Technical staff
Tiny Korse Technical staff
Dorothé Linders Technical staff
Marian Buning - Kager Technical staff
Arifa Fazalalikhan Technical staff in training
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
Jaap Teunissen MD Registrar
Saskia Baank Technical staff
Martine Bakker Technical staff
Carolien Beers - Bauhuis Technical staff
Natascha Bruin Technical staff
Petra Doodeman 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
Douwe Atsma Technical staff
Lucie Boerrigter- Barendsen Technical staff
Michael Knauer MD Academic staff
Jolien Bueno de Mesquita Graduate student
DIVISION OF
DIAGNOSTIC ONCOLOGY
DEPARTMENT OF CLINICAL CHEMISTRY
Pharmacological studies in mice
Nienke van den Brink-de Vries, Tessa Buckle, Roos Oostendorp, Arifa Fazalalikhan,
Olaf van Tellingen
High-grade gliomas, in particular glioblastoma multiforme (GBM), are among the
deadliest and most devastating of human cancers. Current treatment involves a
combination of surgery, radiotherapy and temozolomide chemotherapy but this
offers only a very modest survival benefit. A major part of our work is dedicated to
define the role of the BBB in the chemo-resistance of GBM with special emphasis on
the role of drug transporting proteins. The expression of several ABC transporters
(e.g. ABCB1, ABCG2, ABCC1-5) has been reported and we are investigating their
functional importance using knockout mouse models. Because of overlapping
substrate specificities of these transporters the use of compound knockouts is
essential. Recently, we have been able to create Abcb1a/b;abcg2;abcc4 and
abcg2,abcc4; abcc5 compound knockout strains that will be used for our
pharmacokinetics studies. We have now also created abcb1a/b;Abcg2 knockout nude
mice and have used these to demonstrate that the efficacy of temozolomide against
intracranial tumors is higher than in wildtype nude mice, which is in line with the
50% higher brain penetration of this agent in abcb1a/b;abcg2 knockout mice. We
have also created new cell lines which over-express EGFRvIII and that will be
characterized and used to determine the efficacy of agents that target the EGFR-PI3K-
AKT-mTor pathway. Many of these agents are substrates of ABC transporters.
The second part of our research involves the development of murine brain tumor
models using Cre/LoxP conditional mice. This platform is suited to generate
spontaneous high-grade gliomas in adult mice. Grade III and IV tumors arise in
conditional Kras V12 ;Ink4a/Arf, Kras V12 ;Ink4a/Arf;P53 and Kras V12 ;Ink4a/Arf;Pten
mice after stereotactic intracranial injection of a self-deleting lentivirus mediating
astrocyte-specific expression of Cre (GFAP-Cre lentivirus). Unfortunately, we have not
been successful in creating tumors by injecting a mixture of the GFAP-Cre virus and
a lentivirus containing EGFRvIII into conditional Ink4a/Arf and Ink4a/Arf;Pten
mice. Most likely, the expression of EGFRvIII in infected cells is too low. We expect
that our models will be very useful to supporting the selection of the most promising
combinations of molecularly-targeted agents and cytotoxic drugs for clinical trial.
Besides protecting the brain, ABC transporters (e.g. ABCB1, ABCG2) are considered
to be markers of stem cells and are thought to protect them from xenotoxic stress.
We have now undertaken in vivo studies to establish the protective role of ABC
transporters in bone marrow progenitor cells using the chemotherapeutic agent
topotecan TPT), which is a substrate of several ABC transporters. Wildtype (WT)
mice were sub-lethally irradiated (9 gy) and, subsequently, received donor bone
marrow from WT or ABC transporter knockout mice the next day. After a recovery
period of 6 – 8 weeks, these chimeric mice were exposed to TPT and hematological
parameters (WBC, Hb, RBC) were assessed prior (baseline), during and after
treatment. Thus, each animal served as it own reference. Following the exposure to
0.5 mg TPT/kg/day x5 for three consecutive weeks, only the abcg2 and abcb1a/b;abcg2
chimeras fell ill with Hb levels declining to less than 40% of the baseline values.
WBC values also declined during the first 2 wks but stabilized thereafter, suggesting
that this regimen did not deplete the most primitive stem cell population. 51-Cr
labeling confirmed that the more marked decline of Hb was not due to a higher loss
of mature RBC’s in abcg2 chimeras. Since we had previously found that Abcc1 was
involved in the protection of the WBC lineage, we also checked abcg2;abcb1a/b;abcc1
chimeras but Hb and WBC values were similar as in abcg2 and abcg2;abcb1a/b
chimeras. In conclusion, when abcg2 is absent, TPT causes significant toxicity in the
erythroid lineage, but does not cause a depletion of the more primitive bone marrow
stem cell compartment. Further experiments involving abcc4 are ongoing, to address
recent reports that this ABC transporter is also expressed in bone marrow cells.

Neuroendocrine tumours
Tiny Korse, Hans Bonfrer, Otto Visser, Babs Taal
In collaboration with the Division of Medical Oncology and Department of
Cardiology of the Slotervaart Hospital, we investigated the role of NT-proBNP as
diagnostic marker for carcinoid heart disease and as prognostic marker in patients
with neuroendocrine tumours. From 102 NET-patients serum samples were obtained
and cardiac ultrasound studies was performed from 1999 thru 2007. The criterion
for CHD was tricuspidal regurgitation (TR) stage III/IV. We found that NT-proBNP is
a promising diagnostic marker for CHD. Elevated NT-proBNP in addition to elevated
CgA levels showed worse survival than elevated CgA only. In association with the
Comprehensive Cancer Centre we evaluate epidemiological data of neuroendocrine
tumours from 1990 until 2006. The total number of patients is 9126.
The Department of Clinical Chemistry is also involved in a study of the Division of
Molecular Biology (Marieke Vollebergh, Sabine Linn group). Amphiregulin, TGFa,
IGF-1 and IGFBP-3 were measured in serum and pleura effusion of patients with
non-small cell lung cancer to investigate their predictive value for Erlotinib response.
In collaboration with the Department of Internal Medicine of the AMC, we measured
S100B and NSE in elderly patients with hip fracture to investigate the possible
associations with different subtypes of delirium. We found that S100B levels were
higher during delirium from 1 day before up until 8 days after surgery. No significant
difference in S100B or NSE levels was seen between the hyperactive, hypoactive and
mixed subtype of delirium.
Biomarker discovery using MALDI-MS based proteomics tools We are involved
in a pilot study of the OncoProteomics Laboratory of the VUmc. In this pilot we will
compare exoprotease activities in serum samples of diffent storage time. These
exoprotease activities are measured by using MALDI-TOF mass spectrometric
analysis of the degradation peptides of exogenous labeled substrates. Each labeled
degradation peptide is quantitated by an internal standard peptide.
DEPARTMENT OF NUCLEAR MEDICINE
Clinical Nuclear Medicine
Joke Baars, Axel Bex, Jose Belderbos, Jan Paul de Boer, Sjaak Burgers, Paul Baas, Kenneth Gilhuijs,
Niels Graafland, Michel van den Heuvel, Simon Horenblas, Ingrid Kappers, Houke Klomp, Bin Kroon,
Charlotte Lange, Joost Leijte, Wim Meinhardt, Omgo Nieweg, Hester Oldenburg, Iris van der Ploeg,
Henk van der Poel, Emiel Rutgers, Marieke Straver, Marcel Verheij, Marie-Jeanne Vrancken-Peters,
Babs Taal, Jelle Teertstra, Ly Tran, Michel Wouters, Tjeerd Aukema, Philippe Baars, Cornelis Hoefnagel,
Marina Kartachova, Saar Muller, Ferida Sivro, Michiel Sinaasappel, Renato Valdés Olmos,
Lenka Vermeeren, Wouter Vogel
The extension of the hybrid imaging activities has led to new studies using
SPECT-CT and PET-CT. The majority of current studies are based on the evaluation
of the sentinel node procedure and the use of 18F-FDG PET-CT. New studies using
PET tracers for imaging of hypoxia, apoptosis, and other tumor cell processes are
ongoing. The pioneer activities with a portable gamma camera for real time
intraoperative radioguided imaging will be complemented with a mini PET ring
device for dedicated breast molecular imaging. Finally, radioinmunotherapy with
131I-rituximab in non-Hodgkin’s lymphoma is in progress.
SPECT-CT for sentinel node identification SPECT-CT, which was introduced at
the NKI-AVL in 2006, evolved from two-dimensional to three-dimensional display
(figure 1) and was further incorporated for the anatomical localization of sentinel
nodes in various malignancies.
In 30 out of 85 melanoma patients (35%) with conventional lymphoscintigrams that
were difficult to interpret or showed unusual lymphatic drainage patterns or did not
depict a sentinel node SPECT-CT led to significant more information for the surgeon;
this resulted in a different incision in 17 patients, an incision at another site in 8 and
101
DIAGNOSTIC ONCOLOGY
Stella Mook MD Graduate 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
THE NETHERLANDS CANCER INSTITUTE
FAMILY CANCER CLINIC
Senno Verhoef MD PhD Head
Frans Hogervorst PhD Academic staff, head
Diagnostic Laboratory
Laura van ’t Veer PhD Academic staff
Irma Kluijt MD Academic staff
Priscilla Axwijk MD Academic staff
Eveline Bleiker Academic staff
Daniela Hahn Academic staff
Petra Nederlof PhD Academic staff
Efraim Rosenberg 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
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
Tanja Alderliesten PhD Post-doc
Fijs van Leeuwen PhD Post-doc
Christian Siedschlag PhD Post-doc
Anita Paape Technical staff
Kenneth Pengel Technical staff
Lukas Batteau Technical staff
Patrick Chin Post-doc
Tessa Buckle Technical staff
Anne van Leeuwen Graduate student
Alexander Schmitz Graduate student
Annemarie Schmitz Graduate student
Jincey Sriram Post-doc

102
DIAGNOSTIC ONCOLOGY
Publications
Ackerstaff AH, Balm AJ, Rasch CR,
de Boer JP, Wiggenraad R, Rietveld DH,
Gregor RT, Kröger R, Hilgers FJ. First-year
quality of life assessment of an intra-arterial
(RADPLAT) versus intravenous
chemoradiation phase III trial. Head Neck.
2008 Oct 28 (epub ahead of print)
Antoniou AC, Spurdle AB, Sinilnikova OM,
Healey S, Pooley KA, Schmutzler RK,
Versmold B, Engel C, Meindl A, Arnold N,
Hofmann W, Sutter C, Niederacher D,
Deissler H, Caldes T, Kämpjärvi K,
Nevanlinna H, Simard J, Beesley J, Chen X;
Kathleen Cuningham Consortium for
Research into Familial Breast Cancer,
Neuhausen SL, Rebbeck TR, Wagner T,
Lynch HT, Isaacs C, Weitzel J, Ganz PA,
Daly MB, Tomlinson G, Olopade OI, Blum JL,
Couch FJ, Peterlongo P, Manoukian S,
Barile M, Radice P, Szabo CI, Pereira LH,
Greene MH, Rennert G, Lejbkowicz F,
Barnett-Griness O, Andrulis IL, Ozcelik H;
OCGN, Gerdes AM, Caligo MA, Laitman Y,
Kaufman B, Milgrom R, Friedman E;
Swedish BRCA1 and BRCA2 study
collaborators, Domchek SM, Nathanson KL,
Osorio A, Llort G, Milne RL, Benítez J,
Hamann U, Hogervorst FB, Manders P,
Ligtenberg MJ, van den Ouweland AM;
DNA-HEBON collaborators, Peock S, Cook M,
Platte R, Evans DG, Eeles R, Pichert G,
Chu C, Eccles D, Davidson R, Douglas F;
EMBRACE, Godwin AK, Barjhoux L,
Mazoyer S, Sobol H, Bourdon V, Eisinger F,
Chompret A, Capoulade C, Bressac-de
Paillerets B, Lenoir GM, Gauthier-Villars M,
Houdayer C, Stoppa-Lyonnet D; GEMO,
Chenevix-Trench G, Easton DF; CIMBA.
Common breast cancer-predisposition alleles
are associated with breast cancer risk in
BRCA1 and BRCA2 mutation carriers.
Am J Hum Genet. 2008;82:937-48
Booman M, Szuhai K, Rosenwald A,
Hartmann E, Kluin-Nelemans H, de Jong D,
Schuuring E, Kluin P. Genomic alterations
and gene expression in primary diffuse large
B-cell lymphomas of immune-privileged sites:
the importance of apoptosis and
immunomodulatory pathways. J Pathol.
2008;216:209-17
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 Aug 14
Figure 1: Three-dimensional anatomical display of fused SPECT-CT images using volume rendering
showing drainage from the tumor injection site (T) to sentinel nodes (arrows) in oral cavity malignancy
(A), melanoma of the trunk (B), breast cancer (C) and penile cancer (D).
an extra incision in 5. In another 19 patients sentinel nodes were more clearly
visualized by SPECT-CT than by conventional lymphoscintigraphy, but the surgical
approach remained unchanged.
In 134 breast cancer patients with unusual lymphatic drainage or a pattern that was
difficult to interpret or non-visualization on conventional lymphoscintigraphy
SPECT-CT was additionally performed. Fused SPECT-CT images showed additional
sentinel nodes in 15 patients (11%) and enabled an accurate incision in 102 patients
(76%), an extra incision in 6, and avoided an incision in two patients with spots due
to skin contamination of the tracer.
Besides two-dimensional display fused SPECT-CT images can be displayed using
volume rendering which enables depiction of sentinel nodes in relation to
surrounding muscle and bone structures in a 3D image. This modality was evaluated
as an additional tool in 23 patients with melanoma and 7 with breast cancer. In 23
patients (77%) a more precise anatomical location was accomplished leading to an
adjustment of the surgical incision or exploration route in 15 (50%).
Both two- and three-dimensional fused SPECT-CT were used to analyze the drainage
of the groin in 50 melanoma patients according to the Daseler anatomical
classification of the inguinal areas. Sentinel nodes were depicted in the superior
lateral zone (10%), superior medial (13%), inferior medial (42%), central (26%), and
external iliac (8%). Majority of second-tier nodes were located in the superior lateral
zone (9%), inferior medial (5%), central (13%), external iliac (54%), common iliac
(2%) and obturator fossa (6%). Tumor-positive sentinel nodes were found in the
inferior medial (83%) and central (17%) zones. No lymphatic drainage was seen in
the inferior lateral zone which suggests that this zone could be excluded from a
complete dissection.
SPECT-CT was also used to visualize tumor-blockage and rerouting of lymphatic
drainage in 17 patients with penile cancer and proven unilateral inguinal lymph node
metastasis. SPECT-CT showed tracer uptake in 4 of the 17 palpable metastastic
nodes, no uptake in three, and rerouting to a neo-sentinel node in 10. These findings
support the observation that metastatic involvement of a sentinel node can lead to
blocked inflow and rerouting to a new sentinel node causing a false-negative
procedure.
In 45 out of 46 patients (98%) with prostate cancer, SPECT-CT led to anatomical
sentinel node localization after transrectal ultrasound guided tracer administration.
Sentinel nodes outside the area of extended pelvic lymphadenectomy were found at
laparoscopy in 16 patients (35%). According to the extra anatomical information for

laparoscopy and additional visualized sentinel nodes SPECT-CT had a mayor contribu
tion in 28 patients (61%). Sentinel node metastases were found in 15 patients (33%).
Real time imaging for intraoperative sentinel node detection The use of a
portable CsI(Na) gamma camera using an innovative device for simultaneous
99m Tc/ 125 I detection for intraoperative laparoscopic sentinel node localization was
evaluated in 16 patients with prostate cancer, 2 with testicular cancer and 2 with renal
cell carcinoma. Six hours after tracer administration laparoscopy was performed and
the portable camera equipped with a 4 mm opening pinhole collimator set to display
both 99m Tc for SN visualisation and 125 I as a pointer for SN seeking. The matching of
both signals, seen on screen during laparoscopy, indicated the site of the sentinel
node, which was subsequently removed and measured (figure 2). The portable
camera contributed to directly identify sentinel nodes in 18 patients (90%).
This procedure was also useful to retrieve para-aortic sentinel nodes. In 12 out of
14 patients with different malignancies and para-aortic drainage preoperative sentinel
node identification by SPECT-CT and real time intraoperative imaging using the
portable gamma camera led to surgical localization and removing of the sentinel
nodes.
More recently this approach was introduced for malignancies of the oral cavity
contributing to identify 20 sentinel nodes in 4 patients. In all patients drainage was
bilateral and all visualized sentinel nodes were found in levels I, II and III of the
neck. Both SPECT-CT and the portable gamma camera were able to detect more
sentinel nodes in the proximity of the injection area.
Figure 2: Axial fused SPECT-CT (A) showing a left para-aortic sentinel node (arrow) which is
subsequently localized at laparoscopy using intraoperative imaging with a portable gamma camera (B).
This sentinel node is real time displayed (C) on screen (arrow) and after resection is controlled for
radioactivity (D) by ex-vivo imaging by means of the gamma camera (arrows).
PET-CT in lung cancer The role of 18 F-FDG PET-CT in the diagnosis of lung cancer
in an approach of out-patient fast track assessment was evaluated in 114 patients with
the suspicion of a malignancy on chest X-ray referred for further analysis to the NKI-
AVL. A malignancy was diagnosed in 94 patients (82%) and in 91 of them PECT-CT
was positive. In 10 out 20 patients with benign pulmonary lesions PET-CT was also
positive. PET-CT led to the diagnosis of disseminated disease in 14 patients. It was
concluded that PET-CT provides acceptable sensitivity and accuracy for rapid
diagnosing lung cancer in new patients with in a 90% of the hypermetabolic lesions
malignancy as outcome.
In a multicenter trial 1059 FDG PET-CT scans of patients with suspected or proven
lung cancer were retrospectively evaluated in order to evaluate the feasibility of FDG
PET-CT limited to the thoracic range. Not scanning above the shoulders and below
the caudal tip of the liver leads to missed proved second primaries in 0.7% and
103
DIAGNOSTIC ONCOLOGY
Publications (continued)
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
Boss DS, Valdés Olmos RA,
Sinaasappel M, Beijnen JH, Schellens JH.
Application of PET/CT in the development
of novel anticancer drugs. Oncologist.
2008;13:25-38
Broeks A, Braaf LM, Huseinovic A,
Schmidt MK, Russell NS, van Leeuwen FE,
Hogervorst FB, Van ‘t Veer LJ. The
spectrum of ATM missense variants and
their contribution to contralateral breast
cancer. Breast Cancer Res Treat.
2008;107:243-8
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. 2008 Sep 26 (epub ahead of
print)
Claes A, Gambarota G, Hamans B,
van Tellingen O, Wesseling P, Maass C,
Heerschap A, Leenders W. Magnetic
resonance imaging-based detection of glial
brain tumors in mice after antiangiogenic
treatment. Int J Cancer 2008;122:1981-6
de Boer JP, Hiddink RF, Raderer M,
Antonini N, Aleman BM, Boot H,
de Jong D. Dissemination patterns in nongastric
MALT lymphoma. Haematologica.
2008;93:201-6
de Jong D, Enblad G. Inflammatory cells
and immune microenvironment in
malignant lymphoma. J Intern Med.
2008;264:528-36
de Jong D, Vasmel WL, de Boer JP,
Verhave G, Barbé E, Casparie MK,
van Leeuwen FE. Anaplastic large-cell
lymphoma in women with breast implants.
JAMA. 2008;300:2030-5

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DIAGNOSTIC ONCOLOGY
Publications (continued)
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 R, 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 (LLBC). J Clin Pathol. 2008
Sep 15 (epub ahead of print)
de Vreeze RS, 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
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. 2008 Sep 26
(epub ahead of print)
de Vreeze RS, Koops W, Haas RL,
van Coevorden F. An unusual case of
hemosiderotic fibrohistiocytic lipomatous
lesion: correlation of MRI and pathologic
findings. Sarcoma. 2008;2008:893918
de Vries NA, Zhao J, Kroon E, Buckle T,
Beijnen JH, van Tellingen O.
P-glycoprotein and breast cancer resistance
protein: two dominant transporters working
together in limiting the brain penetration of
topotecan. Clin Cancer Res 200713:6440-9
Gambarota G, Leenders W, Maass C,
Wesseling P, van der Kogel B,
van Tellingen O, Heerschap A.
Characterisation of tumour vasculature in
mouse brain by USPIO contrast-enhanced
MRI. Br J Cancer 2008;98:1784-9
Garcia-Herrera A, Colomo L, Camós M,
Carreras J, Balague O, Martinez A,
Lopéz-Guillermo A, Estrach T, Campo E.
Primary cutaneous small/medium CD4+
T-cell lymphomas: a heterogeneous group of
tumors with different clinicopathologic
features and outcome. J Clin Oncol.
2008;26:3364-71
incorrect staging with therapeutic consequences in 1.2% of the cases, while reducing
the number of irrelevant findings in 20%, and reducing additional investigations,
stress, and cost in 10%. Limited range of PET-CT scanning may be a feasible
alternative for initial staging of lung cancer, allowing faster patient throughput on
scarce PET resources.
Sequential 18 F-FDG PET-CT was used to evaluate metabolic tumor response in
operable stage I/II non-small cell lung cancer patients receiving preoperative
neoadjuvant erlotinib daily for a period of 3 weeks. In 3 out of 12 patients PET-CT
revealed >50% decrease in tumour uptake, while pathology showed a significant
response in 5 patients. In another study 18 F-FDG PET-CT was used to assess tumor
response in locally advanced non-small cell lung carcinoma patients receiving
Cetuximab, in combination with concurrent chemo- / radiotherapy. In 3 out of 12
patients near or complete metabolic tumor response while in another patient more
than 50% tumor uptake reduction was observed 8 weeks after treatment.
PET-CT in breast cancer In 19 consecutive T2N1 breast cancer patients PET-CT
studies in prone position were evaluated in the context of the adjustment of the
design of a new dedicated mini-PET ring camera aimed for screening and biopsy of
FDG avid lesions (European project MAMMI EU-037555). The hanging breast
diameters varied from 7 to 15 cm and the length from 5 to 14 cm. In 17 out of 19
patients (89%) lesions were measurable for FDG uptake with an average SUVmax of
5.7 (range 2.6-14.4) and tumor/background ratio of 14.2 (4-58). For normal breast
SUVmax was 0.4 (0.2-1.4). Prone position allowed better tumor delineation than
supine position. The dedicated mini-PET detector-ring system, which will be
clinically validated the next year, will be allocated inside a device for hanging the
breast and is expected to reach a spatial resolution near 1.5 mm.
SPECT-CT for tumor imaging SPECT-CT acquired 1 hr after injecting 99m Tc-MIBI
was evaluated in the prediction of early chemotherapy response in 11 patients with
stage IIIB or IV NSCLC. MIBI studies were performed within 24 hours before
starting platinum-containing chemotherapy. The relationship between prechemotherapy
lesion uptake and tumor size change at 4 weeks after chemotherapy
completion was analyzed. The intensity of 99m Tc-MIBI lesion uptake correlated
significantly with reduction in tumor size suggesting that the tracer can be used to
predict early treatment efficacy and disease progression in advanced lung cancer.
The visual and quantitative evaluation of tumor uptake of 99m Tc Hynic-rh-Annexin V
was evaluated in 38 patients with various malignancies. SPECT was acquired before
and within two days after the start of therapy and maximal counts per pixel were
calculated in the tumor volume for every target lesion. Visual evaluation of tumor
uptake was based on a 4-grade score. Good intra-observer and inter-observer
reproducibility were found with significant correlation between changes in tumor
uptake and therapy outcome.
Radionuclide therapy Radionuclide therapy using 131 I-MIBG was evaluated in 12
patients with malignant pheochromocytoma and 7 with paraganglioma. Median
cumulative dose was 22.2 GBq (6.8-81.4). Objective tumor response, assessed by
monitoring of bidirectional tumor size reduction on CT or MRI, was achieved in 47%
of the patients. Biochemical response rate was 67% and symptomatic response 89%.
Overall median follow-up was 29 months and hematological side effects were
observed seen in 26% of the patients.
Treatment of differentiated thyroid carcinoma was modernized by the introduction of
recombinant human TSH (rhTSH) for selected patient groups. In low-risk patients
stimulation with rhTSH allows 131 I ablation of functional thyroid remnants after
thyroidectomy without withdrawal of thyroid hormone suppletion, thus improving
quality of life. In high-risk patients with a relative contraindication for a deep
hypothyroidic state, rhTSH allows high-dose 131 I treatment with a low risk on
complications.

Molecular Imaging Nuclear Medicine
Fijs van Leeuwen, Tessa Buckle, Anne van Leeuwen, Patrick Chin, Henk van de Poel, Emiel Rutgers,
Michiel Sinaasappel, Jos Jonkers, Renato Valdés Olmos
New molecular imaging approaches for tumor staging and response
monitoring One of the main challenges in surgical oncology is intraoperative
detection of preoperatively defined lesions. At present (molecular) imaging is a
rapidly growing research field aimed at non-invasive, but accurately, diagnosing
disease. Consequently, it becomes of increasing importance that the surgical
procedures stay ‘up to date’ and enable the incorporation of such novel imaging
approaches. To achieve such incorporation, we are developing intraoperative imaging
approaches that are complementary to radioactivity based preoperative surgical
planning procedures.
Intraoperative visualization of the sentinel lymph node In mouse models
representative for metastatic breast cancer and prostate cancer, we have investigated
the value of intraoperative fluorescence imaging during sentinel lymph node
dissection procedures. For these studies, we have used the current clinical standard
99m Tc-NanoColl and visible blue dye as reference for near infra red fluorescence
procedures with indocyanine green. Intraoperatively the latter shows great potential
in both breast and prostate cancer. In both breast and prostate models the
fluorescence guidance is significantly better than the one obtained with visible blue
dyes only. Data from our preclinical studies have already prompted application for
clinical pilot studies.
Development of targeted imaging agents for surgical guidance Targeted
intraoperative imaging procedures require imaging agents with a high tumor
specificity. For this purpose we target a common marker for metastatic cancer, the
chemokine receptor CXCR4. In mammary mouse tumor models we investigated the
diagnostic potential of a radiolabeled peptide antagonists agents CXCR4 (figure 3).
Furthermore, nanotechnology is exploited for its potential in high intensity
fluorescence imaging. Nanoparticles such as quantum dots have a bright and narrow
photoemission making them suitable for intraoperative imaging studies. Usually, the
use of heavy metals like Cd and Pb in quantum dots limits their use in vivo. As a
consequence, we are currently investigating alterative ‘non toxic’ quantum dots.
Alternatively we are making use of multivalent dendritic structures in order to
improve the fluorescence intensity and tumor specificity.
Figure 3: CXCR4 targeted imaging agents for surgical guidance: a) Overlay of X-ray and bioluminescent
images accurately depict the location of the primary tumor and lymphatic metastases, B) Distribution (iv
injection) of a 125I-labeled cyclic peptide that serves as an antagonist to CXCR4 revealing large amounts of
unspecific staining, and C) The same imaging agent after locoregional injection, demonstrating a higher
tumor specificity.
105
DIAGNOSTIC ONCOLOGY
Publications (continued)
Gedik GK, Hoefnagel CA, Bais E,
Valdés Olmos RA. 131I-MIBG therapy in
metastatic phaeochromocytoma and
paraganglioma. Eur J Nucl Med Mol
Imaging. 2008 35:725-33
Geurts TW, Balm AJ, van Velthuysen ML,
van Tinteren H, Burgers JA, vanZandwijk N,
Klomp HM. Survival after surgical
resection of pulmonary metastases and
second primary squamous cell lung
carcinomas in head and neck cancer. Head
Neck. 2008 Oct 28 (epub ahead of print)
Geurts TW, van Velthuysen MLF,
Broekman F, Hooft van Huysduynen T,
van den Brekel MWM, van Zandwijk N,
van Tinteren H, Nederlof PM, Balm AJM,
Brakenhoff RH. Clin Cancer Res 2008 (in
press)
Gunn SR, Mohammed MS, Gorre ME,
Cotter PD, Kim J, Bahler DW,
Preobrazhensky SN, Higgins RA,
Bolla AR, Ismail SH, de Jong D, Eldering
E, van Oers MH, Mellink CH, Keating MJ,
Schlette EJ, Abruzzo LV, Robetorye RS.
Whole-genome scanning by array
comparative genomic hybridization as a
clinical tool for risk assessment in chronic
lymphocytic leukemia. J Mol Diagn.
2008;10:442-51
Hartog H, van der Graaf WT, Wesseling J,
van der Veer E, Boezen HM. Measurement
of insulin-like growth factor-1 and insulinlike
growth factor binding protein-3 after
delayed separation of whole blood samples.
Clin Biochem. 2008;41:636-9
Hoebers FJ, Kartachova M, De Bois J,
Van den Brekel MW, Van Tinteren, H,
Van Herk M, Rasch CR, Valdés Olmos RA,
Verheij M. 99mTc Hynic-rh-Annexin V
scintigraphy for in vivo imaging of apoptosis
in patients with head and neck cancer
treated with chemoradiotherapy. Eur J Nucl
Med Mol Imaging. 2008;35:509-18
Hoebers FJ, Pameijer FA, de Bois J,
Heemsbergen W, Balm AJ, Schornagel JH,
Rasch CR. Prognostic value of primary
tumor volume after concurrent
chemoradiation with daily low-dose
cisplatin for advanced-stage head and neck
carcinoma. Head Neck. 2008;30:1216-23

106
DIAGNOSTIC ONCOLOGY
Publications (continued)
Hoefnagel CA, Sivro F. Hoofdstuk
Radionucliden therapie. In:
Van den Broek W, Barneveld P, Lemstra
C, Van Urk P: Leerboek Nucleaire
Geneeskunde, Elsevier 2008:559-577
Hofstra RM, Spurdle AB, Eccles D,
Foulkes WD, de Wind N, Hoogerbrugge N,
Hogervorst FB; for the IARC Unclassified
Genetic Variants Working GroupThe
members of the IARC Working Group are
listed in the Appendix. The International
Agency for Research on Cancer (IARC),
based in Lyon, France, is part of the World
Health Organization. Tumor characteristics
as an analytic tool for classifying genetic
variants of uncertain clinical significance.
Hum Mutat. 2008;29:1292-1303
Holm C, Kok M, Michalides R, Fles R,
Koornstra R, Wesseling J, Hauptmann M,
Neefjes J, Peterse J, Stål O, Landberg G,
Linn S. Phosphorylation of the oestrogen
receptor alpha at serine 305 and prediction
of tamoxifen resistance in breast cancer.
J Pathol. 2008 Sep 23 (epub ahead of print)
Holstege H, Joosse SA, van Oostrom CTM,
Nederlof PM, de Vries A3, and Jonkers J.
Increased frequency of p53 truncating
mutations in BRCA1-associated breast
cancer. Submitted.
Hoogendoorn WE, Hollema H,
van Boven HH, Bergman E,
de Leeuw-Mantel G, Platteel I, Fles R,
Nederlof PM, Mourits MJ, van Leeuwen FE.
Comprehensive Cancer Centers TAMARISKgroup.
Prognosis of uterine corpus cancer after
tamoxifen treatment for breast cancer. Breast
Cancer Res Treat. 2008;112:99-108
Horlings HM, van Laar RK, Kerst JM,
Helgason HH, Wesseling J,
van der Hoeven JJ, Warmoes MO, Floore A,
Witteveen A, Lahti-Domenici J, Glas AM,
Van ‘t Veer LJ, de Jong D. Gene expression
profiling to identify the histogenetic origin of
metastatic adenocarcinomas of unknown
primary. J Clin Oncol. 2008;26:4435-41
Jansen MC, van Wanrooy S,
van Hillegersberg R, Rijken AM,
van Coevorden F, Prevoo W, van Gulik TM.
Assessment of systemic inflammatory
response (SIR) in patients undergoing
radiofrequency ablation or partial liver
resection for liver tumors. Eur J Surg Oncol.
2008;34:662-7
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. Currently we are
investigating tumour characteristics such as pathological parameters, EGFR and
KRAS mutation status and EGFR gene amplification (CISH) of a large series of
NSCLC from our institute and the VU medical centre (Dr Erik Thunissen) and
correlation with response to Iressa therapy.
c-KIT mutation in Gastrointestinal Stromal Tumors (GIST) and response to
imatinib therapy
Both activating c-KIT mutations and mutations resulting in resistance to imatinib
therapy have been described in literature. Mutation analysis of c-KIT and PDGFRA is
performed on small tumor biopsies and preliminary results indeed reveal single or
multiple mutations in primary tumors and resistant recurrences.
HNSCC 2nd tumor versus metastasis by LOH and p53 mutation analysis
T.W. Geurts1 , Loes van Velthuysen, F. Broekman2 , T. Hooft van Huysduynen2 , Michiel van den Brekel,
Nico van Zandwijk, Harm van Tinteren, Petra Nederlof, Fons Balm, Ruud Brakenhoff2 Patients treated for head and neck cancer often develop a lung tumor that can be
diagnosed as distant metastasis (DM) or second primary tumor (SPT). Clinical and
histopathological criteria are unreliable for differentiation, while patterns of allelic
loss (LOH) might be more suited. In this study we use TP53 mutation analysis for
validation of an allelic loss marker panel and a decision algorithm that we have been
using to distinguish DM and SPT in curatively treated head and neck squamous cell
carcinoma (HNSCC) patients with newly diagnosed pulmonary squamous cell
carcinoma.
The TP53 mutation data validate the suitability of the LOH marker panel and decision
algorithm for differential diagnosis of DM and SPT in the lung. LOH analysis can
theoretically be exploited in all cases and is less laborious than mutation analysis.
Gene Expression Profiling to identify the histogenetic origin of metastatic
adenocarcinomas of unknown primary
Hugo Horlings, Ryan van Laar1 , Martijn Kerst, Helgi Helgason, Jelle Wesseling, Annuska Glas3 ,
Laura van ’t Veer, Daphne de Jong
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%.
Our goal was to evaluate the contribution of gene expression profiling for routine
clinical practice in patients with ACUP. Formalin-fixed, paraffin-embedded (FFPE)
samples for a validation series of 84 patients with a known primary adenocarcinoma
and a testing series of 38 patients with ACUP was collected. An extensive
immunohistochemical panel classified 16 of the patients with ACUP, whereas 22
1Academic Medical Center
2VU University Medical Center, Amsterdam
3Agendia BV, Amsterdam

patients remained unclassified for their histogenetic origin. Information about
staging procedures and clinical follow-up were available in all patient cases. The gene
expression–based assay classified the primary site correctly in 70 (83%) of 84 patient
cases of primary and metastatic tumors of known origin, with good sensitivity for the
majority of the tumor classes and relatively poor sensitivity for primary lung
adenocarcinoma. Gene expression profiling identified 15 (94%) of 16 patients with
initial ACUP who were classified by immunohistochemistry, and it made a valuable
contribution to a potential site of origin in 14 of the 22 patients with ACUP.
Therefore it can be concluded that the gene expression platform 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.
These findings are now prospectively explored in a prospective, diagnostic
multicenter trial (EORTC CU-003-CR).
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, de-differentiated,
myxoid, round cell (MRLS), and pleomorphic. Well-differentiated (WLS) and dedifferentiated
liposarcoma is 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 is currently further explored
in a large series of lipomatous tumors encompassing the complete spectrum from
undifferentiated mesenchymal proliferations to terminally differentiated tumors.
Tumor microenvironment in malignant lymphoma
Jan Paul de Boer, Martijn Kerst, Joke Baars, Colette van de Bogaard, Dennis Veldhuizen,
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
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.
This may be partly due to heterogeneous composition of both patient series and
107
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. 2008
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.
2008 Aug 14. (epub ahead of print)
Kappers I, Klomp HM, Burgers JA,
Van Zandwijk N, Haas RL, van Pel R.
Neoadjuvant (induction) erlotinib response
in stage IIIA non-small-cell lung cancer.
J Clin Oncol. 2008;26:4205-7
Kartachova MS, Valdés Olmos RA, Haas,
Hoebers FJ, Van Herk M, Verheij M.
99mTc-HYNIC-rh-annexin-V scintigraphy;
visual and quantitative evaluation of early
treatment-induced apoptosis to predict
treatment outcome. Nucl Med Commun.
2008;29:39-44
Kartachova MS, Verheij M, van Eck BL,
Hoefnagel CA, Valdés Olmos RA.
Radionuclide Imaging of apoptosis in
maliganancies: promise and pitfalls of
99mTc-Hynic-rh-annexin V Imaging. Clin
Med Oncol 2008;1:319-25
Knoops L, de Jong D. The role of the p53
pathway in the treatment of follicular
lymphoma. Cell Cycle. 2008;7:436-9
Korse CM, Bonfrer JM, Aaronson NK,
Hart AA, Taal BG. Chromogranin A as an
Alternative to 5-Hydroxyindoleacetic Acid in
the Evaluation of Symptoms during
Treatment of Patients with Neuroendocrine
Tumors. Neuroendocrinology 2008 October 7
Kroon BB, Hoefnagel CA,
Valdés Olmos RA, Nieweg OE. Regional
lymph nodes at a distance Ned Tijdschr
Geneeskd. 2008;152:1997-2000
Kroon BK, Leijte JA, van Boven H,
Wessels LF, Velds A, Horenblas S,
Van ‘t Veer LJ. Microarray gene-expression
profiling to predict lymph node metastasis
in penile carcinoma. BJU Int. 2008;
102:510-5

108
DIAGNOSTIC ONCOLOGY
Publications (continued)
Leijte JA, Valdés Olmos RA, Nieweg OE,
Horenblas S. Anatomical Mapping of
lymphatic Drainage in Penile Carcinoma
with SPECT-CT: Implications for the
Extent of Inguinal Lymph Node Dissection.
Eur Urol. 2008;54:885-92
Loo CE, Teertstra HJ, Rodenhuis S,
van de Vijver MJ, Hannemann J, Muller SH,
Peeters MJ, Gilhuijs KG. Dynamic contrastenhanced
MRI for prediction of breast cancer
response to neoadjuvant chemotherapy: initial
results.AJR Am J Roentgenol. 2008;191:1331-8
Lukas A, van der Weide M, Boogerd W,
Prevoo W, Zuurmond WW, Sanders M.
Adhesive rachnoiditis following
percutaneous cervical cordotomy--may we
still use lipiodol? J Pain Symptom Manage.
2008;36:e1-4
Marchetti S, de Vries NA, Buckle T, Bolijn MJ,
van Eijndhoven MA, Beijnen JH, Mazzanti R,
van Tellingen O, Schellens JH. Effect of the
ATP-binding cassette drug transporters ABCB1,
ABCG2, and ABCC2 on erlotinib hydrochloride
(Tarceva) disposition in in vitro and in vivo
pharmacokinetic studies employing Bcrp1-/-/
Mdr1a/1b-/- (triple-knockout) and wild-type
mice. Mol Cancer Ther 2008;7:2280-7
Meijnen P, Gilhuijs KGA, Rutgers EJ.Th.
The effect of margins of the clinical
management of ductal carcinoma in situ of
the breast, J. surgical oncology, 2008 (in
press). van Leeuwen FWB, Buckle T.,
Kersbergen A., Rottenberg S., Gilhuijs KGA.
Non-invasive functional imaging of
P-glycoprotein-mediated doxorubicin
resistance in a mouse model for hereditary
breast cancer to predict response, and assign
P-gp inhibitor sensitivity, Eur. J. Nucl.
Med. Mol. Im., 2008 (in press).
Meinhardt W, Valdés Olmos RA,
Van der Poel HG, Bex A, Horenblas S.
Laparoscopic sentinel node dissection for
prostate carcinoma: technical and
anatomical observations. BJU Int.
2008;102:714-7
Monzo M, Navarro A, Bandres E, Artells R,
Moreno I, Gel B, Ibeas R, Moreno J,
Martinez F, Diaz T, Martinez A, Balagué O,
Garcia-Foncillas J. Overlapping expression of
microRNAs in human embryonic colon and
colorectal cancer. Cell Res. 2008;18:823-33
treatments. Therefore, we explored this feature in an immunohistochemicl 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. Biological prognostic indices (BIPI) has been
developed for these patients that stratify specific groups of DLBCL with high
accuracy. Similar studies in FL have been started, including a validation study.
Development of a human in mouse model of breast carcinoma to optimize
alkylating or targeted treatment
Petra ter Brugge, Petra Kristel, Jos Jonkers, Jelle Wesseling
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
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.
However, only a limited number of breast tumor xenografts have been established so
far (Marangoni et al., Clin. Cancer Res. (2007) 13: 3989).
We therefore decided to set up a panel of xenografts in which human breast
carcinomas can be studied in a dynamic and reproducible manner. These xenograft
models can than be used to analyse e.g. tumor progression, invasion, response to
therapy, and acquisition of drug resistance. Furthermore, these models could also be
used to test (combinations of) new targeted therapeutics. We will use our panel of
breast cancer xenografts to analyse the response of homologous recombination
deficient tumors on alkylating agents and PARP inhibitors. Second, we will study
factors that determine sensitivity and resistance to SERMs in estrogen receptor (ER)
positive breast cancer xenografts.
We have recently started to set up the xenograft models. For this, fresh breast cancer
fragments are obtained from the Department of Pathology. Fragments are cut into
small pieces and implanted into mammary fat pads of immunocompromised mice.
So far, pieces from 27 different breast tumors have been implanted. Of these, 13 were
found to be ER-positive. Five of the tumors were positive for HER2 amplification,
including 3 of the ER positive tumors. Of the remaining tumors, 7 showed no
expression of ER, PR of HER2, whereas 5 tumors still have to be classified.
Tumor outgrowth is expected to occur 5 to 8 months after implantation. After
outgrowth, tumors will be serially transplanted followed by analysis of cytogenetic
features, gene expression profile and histology from both the original tumor and the
xenografts. Tumors that can be stably passaged will be used for therapeutic
intervention studies.

In addition to developing our own xenograft panel, we will also use already
established xenograft models that are made available to us by the group of Marie-
France Poupon in the Preclinical Investigation Unit at the Institute Curie in Paris,
France.
Genomic profiling, molecular markers and therapy response in breast cancer
Juliane Hannemann, Jorma de Ronde, Lennart Mulder, Marc van de Vijver, Laura van ’t Veer,
Marieke Straver, Marie-Jeanne Vrancken Peters, Sjoerd Rodenhuis, Jelle Wesseling, Petra Nederlof
The objective of the clinical and pathology part of the project supported by the Center
for Translational Molecular Medicine (CTMM) is to develop clinically useful and
feasible tests (using molecular markers) to predict the response or resistance of
primary breast cancers to specific chemotherapeutic agents or combinations of these.
This is important, since preoperative (neoadjuvant) chemotherapy may lead to
excellent responses in some tumors and to no response at all in others. The tests to
be developed will be based on DNA, mRNA and protein analysis. DNA and mRNAbased
tests should ideally be developed into MLPA tests, in collaboration with MRC
Holland Currently, a collection of pre-chemotherapy biopsies and post-chemotherapy
tissue bank samples have been procured of over 300 patients with primary breast
cancer who received preoperative treatment. The preoperative samples have been
analyzed by the pathology department (Marc van de Vijver, Jelle Wesseling) and
mRNA and DNA have been isolated. Furthermore, 200 mRNA expression arrays
(microarray facility NKI and Agendia BV) have been run for over 100 samples,
homologous recombination deficiency tests have been done. These include singlegene
expression estimates (BRCA1) using quantitative rtPCR, aCGH signature
derived BRCA1-ness classification (Nederlof et al), BRCA1 methylation status and
EMSY amplification detection using MLPA and other techniques and for selected
samples partial sequencing of the BRCA1 gene to detect common mutations. The
analysis of these data is ongoing.
In addition, the gene expression of pre-chemotherapy biopsies has revealed that NKI
70 gene prognosis profile low risk tumors have an extremely low rate of pathological
complete remission, where similarly classified high-risk tumors include all chemoresponsive
tumors (p

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DIAGNOSTIC ONCOLOGY
Publications (continued)
Plon SE, Eccles DM, Easton D, Foulkes WD,
Genuardi M, Greenblatt MS, Hogervorst FB,
Hoogerbrugge N, Spurdle AB, Tavtigian SV;
for the IARC Unclassified Genetic Variants
Working GroupThe members of the
Working Group are listed in the Appendix..
Sequence variant classification and
reporting: recommendations for improving
the interpretation of cancer susceptibility
genetic test results. Hum Mutat.
2008;29:1282-1291
Prevoo W, van den Bosch MA, Horenblas S.
Guideline ‘Renal cell carcinoma’ Ned Tijdschr
Geneeskd. 2008;152:1350; author reply 1350.
(Dutch)
Prevoo W, van den Bosch MA, Horenblas S.
Radiofrequency ablation for treatment of
sporadic angiomyolipoma. Urology.
2008;72:188-91
Schiltz PM, Dillman RO, Korse CM,
Cubellis JM, Lee GJ, De Gast GC. Lack of
elevation of serum S100B in patients with
metastatic melanoma as a predictor of
outcome after induction with an autologous
vaccine of proliferating tumor cells and
dendritic cells. Cancer Biother Radiopharm
2008;23:214-21
Seigers R, Schagen SB, Beerling W,
Boogerd W, van Tellingen O, van Dam FS,
Koolhaas JM, Buwalda B. Long-lasting
suppression of hippocampal cell proliferation
and impaired cognitive performance by
methotrexate in the rat. Behav Brain Res
2008;186:168-75
Smeenk RM, Bruin SC, van Velthuysen ML,
Verwaal VJ. Pseudomyxoma peritonei.
Curr Probl Surg. 2008;45:527-75 (review)
Smeenk RM, van Velthuysen ML, Verwaal VJ,
Zoetmulder FA. Appendiceal neoplasms and
pseudomyxoma peritonei: a population based
study. Eur J Surg Oncol. 2008;34:196-201
Smit LH, Nieweg OE, Mooi WJ, Bonfrer JM,
Haanen JB, Kroon BB, De Gast GC. Value of
serum S-100B for prediction of distant relapse
and survival in stage III B/C melanoma.
Anticancer Res 2008;28:2297-302
Sonke GS, Ludwig I, van Oosten H,
Baars JW, Meijer E, Kater AP, de Jong D.
Poor outcomes of chronic active Epstein-
Barr virus infection and hemophagocytic
lymphohistiocytosis in non-Japanese adult
patients. Clin Infect Dis. 2008;47:105-8
Integration of DNA copy number alterations and prognostic gene expression
signatures in breast cancer patients
Hugo Horlings, Carmen Lai, Dimitry Nuyten, Hans Halfwerk, Petra Kristel, Erik van Beers,
Simon Joosse, Marcel Reinders, Petra Nederlof, Lodewijk Wessels, Laura van ’t Veer,
Marc van de Vijver
Several prognostic gene expression profiles have been identified in breast cancer. In
spite of this progress in prognostic classification, the underlying mechanisms that
drive these gene expression patterns remain unknown. Specific genetic alterations
that can partly be studied by analyzing DNA copy number changes are an important
mechanism of tumor development and progression and are also associated with
changes in gene expression. We carried out array Comparative Genomic
Hybridization (aCGH) in 68 human breast carcinomas were part of the previous
published NKI-295 series, for which gene expression and clinical data were available.
We used a two-class supervised algorithm for the identification of regions of
chromosomal alteration (SIRAC). We identified genes in the altered regions for
which the expression level is significantly correlated with the copy number. The
previously identified poor prognosis 70-gene signature (NKI) is associated with gain
of 3q26-27, 8q22-24 and 17q24-25 and a loss of 14q31; the 70-gene good prognosis
profile is associated with loss at 16q11-24; basal-like tumors with gain of 6p12-24,
10p12-14, 12p13 and losses at 4p15, 5q11-34, 8q24, 10q23-24, 12q13-21 14q12-23,
15q14-25; HER2+ breast tumors have an amplification at 17q12 (where the HER2
gene is located) ((figure 4). These findings help to establish a link between genetic
changes and gene expression signatures that determine tumor behavior in breast
cancer, enabling a better understanding of the tumor biology that causes poor
prognosis.
Figure 4: A summary of the results of the integration of SIRAC algorithm results (DNA copy number
changes) with corresponding gene expression data. We show the aberrations per chromosomal arm with
respect to prognostic gene signatures. Regions of aberration detected have been color-coded based on the
direction of the aberration. Green was used for gain, red for losses and for example HER2, the HER2
positive samples are the class of interest and these tumors are characterized by a gain on 17q, hence the
green block in cell 17q.

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
Previous studies of 70-gene 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 241 patients with 1-3
involved lymph nodes. The 70-gene signature was the strongest predictor of survival
eight years after an initial diagnosis, even after taking into account other important
considerations, such as whether or not these women received adjuvant
chemotherapy. As a result of this work, the MINDACT trial has been amended so
that women with 1-3 involved lymph nodes can be included in the trial.
The 70-gene prognosis-signature has been validated in various series, but so far not
exclusively in postmenopausal patients. However, 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.
Therefore, we validated the signature in 148 patients aged between 55 and 71 years.
This validation study provides evidence that the 70-gene prognosis-signature
(MammaPrint), is also a predictor of disease outcome in postmenopausal breast
cancer patients, especially for early breast cancer-related events. Since the beneficial
effect of chemotherapy in older women predominantly occurs in the first 5 years, the
signature can be of clinical use in selecting postmenopausal women for adjuvant
chemotherapy.
In total for about 1700 NKI diagnosed patients a retrospective 70-gene prognosis
signature was assessed and published in various publications. We have now compiled
all in one database and are evaluating risk assessment by the prognosis signature in
much larger patient groups, thus increasing the power. Ongoing separate analyses
include, her2postive, endocrine responsiveness, tumor diameter, age selected
patients groups. Moreover, the predictiveness of signature subgroups for therapy is
evaluated, e.g., the higher likelihood of 70 gene poor prognostic patients to respond
to chemotherapy.
Validation of Adjuvant! Online
Stella Mook, Marjanka Schmidt, Peter Ravdin, Tony van der Velde, Jelle Wesseling, Sterre Rutgers,
Emiel Rutgers, Laura van ’t Veer
Nowadays, adjuvant treatment recommendations for early stage breast cancer
patients are based on the estimated risk of breast cancer relapse and death and the
expected benefit of adjuvant treatment. Several tool have been developed to make
these estimates for the individual patient. One of these tools is Adjuvant! Online
(www.adjuvantonline.com), which calculates a 10-year survival probability based on
the patient’s age, tumor size, grade and estrogen receptor. In addition, the computer
program also calculates the likely benefit to be expected for adjuvant systemic
treatment. In 2005 Olivotto et al. validated Adjuvant! Online in 4083 patients using
the British Columbia Breast Cancer Outcomes Unit database. They compared
observed outcomes with expected outcomes calculated by Adjuvant!. The 10-year
predicted and observed outcomes were within 1% for the whole group and for most
subgroups predicted and observed outcomes were within 2%.
Adjuvant! Online is already being used by physicians. In addition, in the MINDACT
trial the clinical risk is calculated by Adjuvant! Online. To assess the validity of
Adjuvant! Online for Dutch breast cancer patients we will compare predicted and
observed disease outcome in 2 Dutch cohorts of approximately 10.000 patients in
total.
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DIAGNOSTIC ONCOLOGY
Publications (continued)
Spurdle AB, Couch FJ, Hogervorst FB,
Radice P, Sinilnikova OM; for the IARC
Unclassified Genetic Variants Working
Group The members of the IARC
Unclassified Genetic Variants Working
Group are listed in the Appendix..
Prediction and assessment of splicing
alterations: implications for clinical testing.
Hum Mutat. 2008;29:1304-1313
Straver ME, van Adrichen JC, Rutgers EJTh,
Rodenhuis S, Linn SC, Loo C,
Gilhuijs KGA, Oldenburg HSA,
Wesseling J, Russel NS, Ontonini N,
Vrancken Peeters MTFD, Neoadjuvante
systemische therapie bij het primair
operabel mammcarcinoom: meer voordelen
dan alleen borstsparende behandeling, Ned
Tijdschrift Geneesk 2008;152:2519-2525
Thieblemont C, Rolland D, Baseggio L,
Felman P, Gazzo S, Callet-Bauchu E,
Traverse-Glehen A, Houlgatte R, Fu K,
Weisenburger D, De Jong D, Jaffe ES,
Rosenwald A, Ott G, Coiffier B, Berger F.
Comprehensive analysis of GST-pi
expression in B-cell lymphomas: Correlation
with histological subtypes and survival.
Leuk Lymphoma. 2008;49:1403-6
Tischkowitz M, Hamel N, Carvalho MA,
Birrane G, Soni A, van Beers EH,
Joosse SA, Wong N, Novak D,
Quenneville LA, Grist SA; kConFab,
Nederlof PM, Goldgar DE, Tavtigian SV,
Monteiro AN, Ladias JA, Foulkes WD.
Pathogenicity of the BRCA1 missense
variant M1775K is determined by the
disruption of the BRCT phosphopeptidebinding
pocket: a multi-modal approach.
Eur J Hum Genet. 2008;16:820-32
Valdés Olmos R.A, Vidal-Sicart,
Nieweg OE. SPECT -CT and real-time
intraoperatieve imaging: new tools for
sentinel node localization and radioguided
surgery? Eur J Nucl Med Mol Imaging.
2008 (epub ahead of print)
Valdés Olmos RA, Kroon BB,
Hoefnagel CA, Horenblas S. Penile
carcinoma – Radionuclide Imaging and
PET. In: de la Rosette JJMCH et al:
Imaging in oncological Urology, Springer-
Verlag 2008:345-350

112
DIAGNOSTIC ONCOLOGY
Publications (continued)
Valdés Olmos RA, Van der Ploeg I,
Rutgers E, Nieweg OE. Sentinel nodes
outside the axilla: more accurate staging or
unnecessary morbidity? Rev Senologia
Patol Mam 2008;21:22-26
Valdés Olmos RA, Vogel WV, Hoefnagel CA.
Hoofdstuk Tumoren. In: Van den Broek W,
Barneveld P, Lemstra C, Van Urk P:
Leerboek Nucleaire Geneeskunde, Elsevier
2008:505-557
van den Berg RM, Teertstra HJ,
van Zandwijk N, van Tinteren H, Visser C,
Pasic A, Sutedja TG, Baas P, Golding RP,
Postmus PE, Smit EF. CT detected
indeterminate pulmonary nodules in a
chemoprevention trial of fluticasone. Lung
Cancer. 2008;60:57-61
Van der Ploeg IM, Kroon BB, Antonini N,
Valdés Olmos RA, Rutgers EJ, Nieweg OE.
Axillary and extra-axillary lymph node
recurrences after a tumor-negative sentinel
node biopsy for breast cancer using
intralesional tracer administration. Ann
Surg Oncol. 2008;15:1025-31
Van der Ploeg IM, Nieweg OE, van Rijk MC,
Valdés Olmos RA, Kroon BB. Axillary
recurrence after a tumour-negative sentinel
node biopsy in breast cancer patients; A
systemic review and meta-analysis of the
literature. Eur J Surg Oncol. 2008 (epub
ahead of print)
Van der Ploeg IM, Tanis PJ, Valdés Olmos RA,
Kroon BB, Rutgers EJ, Nieweg OE. Breast
Cancer Patient with Extra-Axillary
Sentinel Nodes Only may be Spared
Axillary Lymph Node Dissection. Ann Surg
oncol. 2008 (epub ahead of print)
Van der Ploeg IM, Valdés Olmos RA,
Kroon BB, Nieweg OE. The Hybrid
SPECT/CT as an additional lymphatic
mapping tool in patients with breast cancer.
World J Surg. 2008;32:1930-4
Van der Ploeg IM, Valdés Olmos RA,
Kroon BB, Nieweg OE. Tumor-positive
sentinel node biopsy of the groin in
clinically node-negative melanoma patients;
superficial or superficial and deep lymph
node dissection? Ann Surg Oncol.
2008;5:1485-91
THE NETHERLANDS CANCER INSTITUTE FAMILY CANCER
CLINIC
Mohamed Achachah, Abderrahim Ajouaou, Priscilla Axwijk, Majella Boutmy - de Lange, Daniela Hahn,
Frans Hogervorst, Irma Kluijt, Mobien Kasiem, Petra Nederlof, Rob Plug, Roelof Pruntel, Anja van Rens,
Efraim Rosenberg, Esther Scheerman, Laura van ’t Veer, Sophie van de Velden, Senno Verhoef,
Gea Wigbout
In 2008 the number of families who have sought genetic advice through our hospital
since the start of the Family Cancer Clinic in 1995 has increased again by more than
10%. Unfortunately the number of clients that can be accepted by the counseling and
laboratory remains maximized as a consequence of limits imposed by the insurance
companies. In 2008 583 new patients were seen for complex genetic counseling.
Most families seen at our family cancer clinic present with a possible genetic
predisposition for breast and/or ovarian cancer. The DNA-diagnostic laboratory has
screened more than 2600 families for germ line mutations in the BRCA1/2 genes
since the start. These families are obtained through our Family Cancer Clinic and the
oncogenetic section of the Department of Clinical Genetics of the Academic Medical
Center. In 270 families a BRCA1 mutation and in 146 families a BRCA2 mutation
was identified. In more than 1300 family members predictive, presymptomatic
testing for these genes was performed and 508 persons were shown to be carrier of
the predisposing mutation. Sequence variants which may or may be not the familial
disposition are a major challenge for the laboratory but with national and
international effort and collaboration we are able to resolve each year a substantial
part of them. Unfortunately, we still have 127 families with unresolved sequence
variants in one of the two genes.
The laboratory has been testing new techniques in order to shorten the through put
time of samples. We started using Conformation Sensitive Capillary Electrophoresis
combined with automated pipetting and analysis. After two years of testing we
shifted to high resolution melting curve analysis which proves to be less complex in
handling and implementation.
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 Simon Joosse and Petra Nederlof, Division of
Experimental Therapy and Diagnostic Oncology). More than 100 a-CGH analyses
have been completed, for high risk families and unclassified variant analysis mostly.
A BRCA2 classifier has been validated at this very moment and the implementation
of this classifier into the diagnostics will hopefully being carried out in 2009.
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 (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.
DEPARTMENT OF RADIOLOGY
Laboratory for Diagnostic Image Research
Kenneth Gilhuijs, Tanja Alderliesten, Christian Siedschlag, Lukas Batteau, Anita Paape,
Kenneth Pengel, Angelique Schlief, Jincey Sriram, Alexander Schmitz, Annemarie Schmitz,
Claudette Loo, Saar Muller, Jelle Teertstra, Wouter Vogel, Fijs van Leeuwen, Patrick Chin,
Tessa Buckle, Anne van Leeuwen
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, and image-guided
therapy.
Automated fusion of MRI and PET to monitor response to neoadjuvant
chemotherapy Contrast-enhanced (CE) MRI and PET/CT of the breast provide
complementary information on neovascularization and tumor cell metabolism.
This information may advance the individualized therapy of patients treated with
neoadjuvant chemotherapy. In a pilot study we assessed the feasibility of fusing
CE MRI and FDG-PET/CT.
Nine patients with breast cancer (ø 23 mm - 58 mm) underwent CE MRI in prone
position using a bilateral MRI breast coil. Wash-in and wash-out kinetics were
established at 90 s and at 450 s, respectively. FDG-PET/CT in prone orientation was
obtained using a replica of the same MRI breast coil. MRI and PET/CT were
automatically aligned by deformable registration using: (a) fiducial control point
alignment constrained by local conservation of volume (FCP) or (b) particle-swarm
optimization using optimization of mutual information (PSO). Accuracy was
assessed by residual misalignment of fiducial landmarks on fat-parenchyma
transitions. SUV and perfusion were correlated.
Average residual misalignment was 5 mm (FCP) and 8 mm (PSO), respectively.
In all cases, asymmetric FDG uptake between left and right breast corresponded with
increased wash-in at MRI. In 4/9 tumors (>40 mm) inhomogeneous FDG uptake
occurred in homogeneous regions of contrast wash-in at MRI. These regions
correlated with increased contrast wash-out.
Figure 5: Left: MRI maximum intensity projection of an invasive ductal cancer, visualizing the
neovascular network. The circles indicate protrusions wihtout aberrant behavior. Right: PET fused with
MRI showing predominant metabolic activity in the protrusions (red), thus complementing MRI.
113
DIAGNOSTIC ONCOLOGY
Publications (continued)
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. 2008 (epub ahead of print)
Van der Ploeg IMC, Nieweg OE,
Valdés Olmos RA. De sentinel node
procedure bij het mammacarcinoom.
Gamma Professional 2008;58:20-23
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. 2008
Oct 8 (epub ahead of print)
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. 2008
Nov 13 (epub ahead of print)
Veltkamp SA, Pluim D, van Tellingen O,
Beijnen JH, Schellens JH. Extensive
metabolism and hepatic accumulation of
gemcitabine after multiple oral and
intravenous administration in mice. Drug
Metab Dispos 2008;36:1606-15
Vogel WV. Hoofdstuk Beeldfusie. In:
Van de Broek W, Barneveld P, Lemstra C,
Van Urk P: Leerboek Nucleaire
Geneeskunde, Elsevier 2008: 207-222
Vollebergh MA, Nederlof PM, Wessels LF,
Schmidt MK, Joosse SA, van Beers E,
Froklage F, Holtkamp M, Schrama JG,
Wesseling J, Hauptmann M, de Bruin M,
Rodenhuis S and Linn SC Predicting
response to alkylating chemotherapy in
breast cancer patients using array
comparative genomic hybridization
(submitted)
Voskuil DW, Vrieling A, Korse CM,
Beijnen JH, Bonfrer JM, van Doorn J,
Kaas R, Oldenburg HS, Russell NS,
Rutgers EJ, Verhoef S, van Leeuwen FE,
Van ‘t Veer LJ, Rookus MA. Effects of
lycopene on the insulin-like growth factor
(IGF) system in premenopausal breast
cancer survivors and women at high
familial breast cancer risk. Nutr Cancer
2008;60:342-53

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DIAGNOSTIC ONCOLOGY
Publications (continued)
Vrieling A, Rookus MA, Kampman E,
Bonfrer JM, Bosma A, Cats A, van Doorn J,
Korse CM,Witteman BJ, van Leeuwen FE,
Van ‘t Veer LJ, Voskuil DW. No effect of red
clover-derived isoflavone intervention on the
insulin-like growth factor system in women
at increased risk of colorectal cancer.
Cancer Epidemiol Biomarkers Prev
2008;17:2585-93
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. 2008
Sep 16 (epub ahead of print)
Vrieling C, de Jong D, Boot H, de Boer JP,
Wegman F, Aleman BM. Long-term results
of stomach-conserving therapy in gastric
MALT lymphoma. Radiother Oncol.
2008;87:405-11
Weigelt B, Horlings HM, Kreike B,
Hayes MM, Hauptmann M, Wessels LF,
de Jong D, Van de Vijver MJ, Van ‘t Veer LJ,
Peterse JL. Refinement of breast cancer
classification by molecular characterization
of histological special types. J Pathol.
2008;216:141-50
Zeleniuch-Jacquotte A, Gu Y, Bruning PF,
Bonfrer JM, Koenig KL, Arslan AA,
Toniolo P, Shore RE. Re: C-reactive protein
and risk of breast cancer. J Natl Cancer Inst
2008;100:443-4
Zuur JK, Muller SH, Vincent A,
Sinaasappel M, de Jongh FH, Hilgers FJ.
Assessment of tracheal temperature and
humidity in laryngectomized individuals
and the influence of a heat and moisture
exchanger on tracheal climate. Head Neck.
2008;30:1072-82
From this pilot study we conclude that automatic deformable co-registration of FDG-
PET/CT and CE MRI is clinically feasible. CE MRI enhances the conspicuity of
asymmetric FDG uptake in left and right breast. Increased wash-out at MRI is
correlated with high SUV in the tumor. Automated PET/MR deformable registration
and multi-modality computer-aided assessment of therapy response will be further
investigated in the CTMM CaRe project, initiated in 2008.
The impact of preoperative MRI on breast-conserving surgery of invasive
cancer: a comparative cohort study This research aimed to assess whether
preoperative contrast-enhanced magnetic resonance imaging (MRI) of the breast
influences the rate of incomplete tumor excision. Methods In a cohort of 349 women
with invasive breast cancer, patients eligible for breast-conserving therapy (BCT) on
the basis of conventional imaging and palpation only (N = 176) were compared to
those who had an additional preoperative MRI (N = 173). Multivariate analysis was
applied to explore associations with incomplete tumor excision. Results MRI detected
larger extent of breast cancer in 19 women (11.0%), leading to treatment change:
mastectomy (8.7%) or wider excision (2.3%). Tumor excision was incomplete in
22/159 (13.8%) wide local excisions in the MRI group and in 35/180 (19.4%) in the
non-MRI group (P = 0.17). Stratified to tumor type, incompletely excised infiltrating
ductal carcinoma (IDC) was significantly associated with absence of MRI: 11/136
(8.1%) versus 2/126 (1.6%) (MRI present) (P = 0.02). No significant factors explained
incomplete excision of other tumor types. Conclusion Preoperative MRI did not
significantly affect the overall rate of incomplete tumor excision, but it yielded
significantly lower rate of incompletely excised IDC. The reduction of incomplete
excisions after MRI was smaller than the rate of a prior treatment change incurred
by MRI.
Accurate definition of the treatment target for non-small cell lung cancer
using PET/CT-pathology matching In ongoing efforts to improve the treatment
target for non-small cell lung cancer (NSCLC) by correlating pretreatment diagnostic
imaging with histopathology, we developed a model to predict the extent of the gross
tumor volume (GTV, primary tumor) and the clinical target volume (CTV, surrounding
microscopic disease) at pathology from PET/CT images.
High-resolution multi-slice CT scans and 18F-FDG-PET scans were obtained from 34
patients with NSCLC prior to lobectomy. The GTVCT was manually delineated and
GTVPET was automatically extracted from PET images by taking the 42% isocontours
of the maximum SUV. These volumes were compared with the reference
pathology volumes (GTVpath). At pathology, microscopic thin sections covering the
tumor and surrounding area of about 2 cm were obtained and their images overlaid
onto the macroscopic images. GTVpath and surrounding microscopic disease were
delineated.
To develop a multivariate model that predicts the histopathology-proven target based
on CT and PET data, we extracted image features such as GTVCT and GTVPET, Max
SUV, and tumor circularity at CT. These features, together with other characteristics
such as tumor type and patient age, were correlated with the histopathology findings
in order to predict the volume of the GTVpath and the presence and extension of
microscopic spread.
The radius of the delineated GTVCT was on average 6 mm larger than the GTVpath
radius. On the other hand, the GTVPET corresponded to GTVpath within 1 mm. We
found microscopic spread in about 50% of our patients. In order to capture all
microscopic spread in 90% of patients, a CTV margin of 15 mm would be required.
This is considerably larger than most other studies have reported.
We conclude that the true GTV for NSCLC patients can be obtained from 18F-FDG-
PET images but is overestimated by CT. Taking lung deformations into account, a 15
mm CTV margin would be required to cover the microscopic spread in 90% of
patients. However, in half the number of patients no surrounding microscopic spread
was found.

Molecular imaging for tumor staging and response monitoring
Fijs van Leeuwen, Tessa Buckle, Ariena Kersbergen, Sven Rottenberg, Jos Jonkers, Saar Muller,
Kenneth Gilhuijs
Functional information provided by molecular imaging can be used to reveal the
molecular basis of a tumor in a non-invasive manner. This can, in turn, be used to
stage tumor progression and monitor tailored therapy response. Studies are currently
performed at the departments of Radiology/Nuclear Medicine and the NKI ‘mouse
cancer clinic’ and use the (advanced) mouse models present at the NKI.
Staging an detection DCIS In a mouse model representative for ductal carcinoma
in situ (DCIS), diagnostic approaches are being studied that can be used to identify
tumor progression from an initial preinvasive to invasive lesions. For these studies
we use a combination of PET and MRI. PET can be used to visualize changes in the
molecular biology of the tumor e.g. increase in proliferation (18F-FLT) or metabolism
(18F-FDG). Contrast enhanced MRI provides complementary information regarding
changes in tumor size and vascular physiology. Finally we wish to exploit such a
complementary imaging approach in concurrent PET/MRI imaging.
Analysis of drug resistance and response Using ‘spontaneous’ mouse models for
hereditary breast cancer with a known response to therapy we investigate
endogenous and non-invasive markers for response. Here computer aided diagnosis
of resistance induced by Mdr1 drug transporter activity has been used to noninvasively
predict therapeutic resistance to doxorubicin. Since this tumor model also
has a well documented sensitivity/resistance pattern (sensitive and resistant) against
docetaxel, it has also been used in docetaxel response investigations based on
apoptosis imaging with 99mTc Annexin V. Unfortunately, the latter approach proved
not to be response-specific and does not allow non-invasive prediction of
chemotherapeutic response. Present we are investigating other endogenous markers
that may allow early prediction of the therapeutic efficacy.
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Division head, Sjoerd Rodenhuis
Sjoerd Rodenhuis 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
John Haanen MD 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
Hilde Rosing PhD Academic staff
Jan Schellens MD PhD Academic staff
Jan Schornagel MD PhD Academic staff
Marianne Smits MD PhD Academic staff
Gabe Sonke MD Academic staff
Babs Taal MD PhD Academic staff
Wim Ten Bokkel Huinink 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 stafff
Nico Van Zandwijk MD PhD Academic staff
Karin Beelen Temporary staff
Emile Kerver MD PhD Temporary staff
Peter Kunst MD PhD Temporary staff
Alet Mager Temporary staff
Lidwine Tick MD PhD Temporary staff
Catrien Tromp-van Driel Temporary staff
David Boss PhD student
Carola Damen PhD student
Maarten Deenen PhD student
Thomas Dorlo PhD student
Anne Charlotte Dubbelman PhD student
Claudia Heijens PhD student
Marie-Christine Gast PhD student
Robert Jansen PhD student
Ron Keizer PhD student
Stijn Koolen PhD student
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
Background and objectives
The division of Clinical Pharmacology remains involved in research in different
phases of anticancer drug development. This concerns: I) Pharmaceutical
formulation, II) Bioanalytical method development and implementation in clinical
pharmacological studies, III) Early clinical phase I/II studies and IV) supportive care
studies in patients with breast cancer.
Research activities of the division of Clinical Pharmacology of the department of
Medical Oncology, the department of Pharmacy & Pharmacology and the department
of Experimental Therapy (group Schellens) are closely integrated.
In 2008, 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 2008
I Pharmaceutical formulation
Formulation research and clinical manufacture of small molecule experimental
anticancer agents has continued with the anti-metastatic compound NAMI-A for
which a suitable pharmaceutical formulation was developed and the first batches for
clinical trial use were manufactured.
Clinical manufacture (Thalidomide tablets) of oral formulations of anticancer agents
has continued. A pharmaceutical development program of oral taxanes was initiated
and the first formulation composed of a solid dispersion of docetaxel has entered the
clinic. Optimization of this formulation will continue in 2009. Currently a similar
formulation of paclitaxel is in development which is expected to be ready for clinical
testing in the course of 2009.
A first formulation project aimed at the GMP-manufacture of ready-to-label
molecular imaging agents has been started with bevacizumab as its first product.
The Amsterdam BioTherapeutics Unit (AmBTU) is now fully operational for the
manufacture of GMP-grade DNA-plasmids. A lyophilized pharmaceutical
formulation of pDERMATT (plasmid DNA Encoding Recombinant Mart-1 and
Tetanus Toxin fragment-c) for the intradermal administration by tattooing was
developed for a phase I clinical trial which started by the end of 2008 in our institute.
The next project, the pharmaceutical development and manufacture of HPV E6 and
E7 pDNA vaccines for clinical trial use 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. The ultimate aim of this program
is, of course, to take promising vectors to the clinic.
II Bioanalytical method development and implementation in PK studies
A new linear ion trap Mass Spectrometer (MS) has been purchased for our laboratory.
The new instrument (LTQ XL) offers multiple dissociation techniques generating
more fragments and extending the low mass range. The high resolution enables
isolation of isobaric mass peaks for cleaner MSn spectra. Furthermore, the ultra fast

polarity switching allows high sensitivity analysis of unknowns. The instrument will
be used for quantitative analysis of multiple analytes, metabolic profiling and
proteomic research. We have also decided to extend our triple quadruple MS
equipments with the purchase of a highly sensitive API 4000 (Sciex) MS detector.
E7080 is a new experimental tyrosine kinase inhibitor and assays have been validated
in whole blood, plasma, urine, and feces for the quantification of the parent drug and
its four metabolites. These assays will be used to support a mass balance study with
the drug early next year. For bendamustine, a bifunctional mechlorethamine
alkylating drug, we are in the process of setting up assays for the quantitative analysis
of the drug and its known metabolites in several biomatrices for the analysis of
samples to be collected in a mass balance study. For both mass balance studies, urine
and fecal samples will be used to generate structural information of potential new
metabolites using the novel LTQ XL MS instrument.
New formulations for apaziquone (EO9) have been developed and we were involved
in stability studies and the bioanalysis of EO9 and its metabolite EO5a. Porous
graphitic carbon material as stationary phase has been used to quantify capecitabine
and four metabolites (5]-deoxy-5-fluorocytidine, 5]-deoxy-5-fluorouracil, 5-fluorouracil
and dihydro-5-fluorouracil) in plasma to support clinical studies of the drug. The
same inert material was used to separate mono-, di-, and tri-phosphates of
gemcitabine and dFdU in human PBMCs and the method has been successfully
validated. For these assays, stable isotopic-labeled internal standards were
synthesized from the labeled nucleosides.
Other new bioanalytical methods for anti-cancer drugs that were developed and
validated are: vincristine and actinomycine D, 2-amino-1-methyl-6phenylimidazo[4,5-b]pyridine
(PhIP), tamoxifen, dasatinib, sunitinib and sorafenib.
Sample analysis were performed for clinical trials within and outside the institute
and included topotecan, irinotecan and its active metabolite SN-38, trabectedin,
indibulin, paclitaxel, docetaxel, gemcitabine and dFdU, gemcitabine triphosphate,
and cyclophosphamide in combination with fludarabine.
For trastuzumab, a humanized monoclonal antibody (MAb), an assay has been
developed to quantify the intact MAb using liquid chromatography coupled to
fluorescence detection. For the isolation of trastuzumab from the plasma matrix, an
anti-idiotype MAb generated after rabbit immunization was used.
During the bioanalysis of Miltefosine, a drug used in the treatment of the parasitic
disease Leishmaniasis, it became clear that the capsules given to the patients
contained no active ingredients. Based on our findings the Bangladeshi government
has started an investigation.
III Early clinical phase I/II studies
III-1 Novel approaches to improve oral bioavailability
We have demonstrated that the oral applicability of commonly used anticancer drugs
is limited by the affinity for one or more of the ABC transporters expressed in the
epithelial layer of the gastro-intestinal tract, in particular P-glycoprotein (Pgp;
ABCB1), Breast Cancer Resistance Protein (BCRP; ABCG2), MRP2 (ABCC2) and/or
affinity for cytochrome P450 3A (CYP3A). Based on our proof of concept trial
demonstrating high systemic exposure to orally applied docetaxel as a drinking
solution 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% of that achieved after
intake of the drinking solution of docetaxel that we applied previously. Importantly
however, the interpatient variation of systemic exposure to docetaxel was substantially
lower after intake of the capsule (%CV of 93% after intake of the drinking solution
and 36% after intake of the capsules). In addition, the intrapatient variability in
systemic exposure to docetaxel after intake of ModraDoc001 plus ritonavir was low
(%CV 20%). This represents an excellent basis for the swift further clinical
development of this concept. Currently, other boosting agents are being tested in
combination with ModraDoc001, such as ketoconazole and grapefruit juice. We have
also continued studies with oral paclitaxel (ModraPac).
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Nienke Lankheet PhD student
Jelte Meulenaar PhD student
Johannes Moes PhD student
Roos Oostendorp PhD student
Susanne Quaak PhD student
Rob Ter Heine PhD student
Bas Teunissen PhD student
Ly Tran PhD student
Liia Vainchtein PhD student
Joost Van Den Berg PhD student
Jolanda Van Den Hoven 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
Annelies Boekhout Nurse practitioner
Ria Dubbelman Nurse practitioner
Joke Foekema Nurse practitioner
Emmy Harms Nurse practitioner
Marjo Holtkamp Nurse practitioner
Marianne Keessen Nurse practitioner
Maria Kuiper Nurse practitioner
Henk Mallo Nurse practitioner
Annemarie Nol Nurse practitioner
Albert Olijve Nurse practitioner
Margaret Schot Nurse practitioner
Wilma Uyterlinde Nurse practitioner
Jana Van Der Sar Nurse practitioner

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Figure 1: Plasma concentration-time profiles of a representative patient who was treated with oral
docetaxel formulated as capsules ModraDoc001 (denoted as MODEA) on two occasions and with an oral
drinking solution on a third occasion, on all accasions taken together with one single dose of 100 mg of the
boosting agent ritonavir.
III-2 Pharmacology (PK/PD, ADME, mass balance) of novel anticancer drugs
Currently, we are performing twenty-eight phase I/II, pharmacological and proof of
concept studies. We recruited over 220 new patients in these studies in 2008. Sixteen
of these studies are two- or multicenter studies. Representative examples are
described below.
a. Studies with the poly-ADP-ribose polymerase (PARP1) inhibitor AZD2281
In a two-center phase I trial we test the safety, optimal dose and schedule of
AZD2281. The drug is given daily BID in combination with three-weekly carboplatin
and paclitaxel, or in combination with weekly paclitaxel. This study is based on our
recent experience with AZD2281 given as single agent. AZD2281 is taken orally daily
without interruption at doses of 100-200 mg BID. We have demonstrated promising
anticancer activity of the combination of carboplatin and AZD2281. However,
prolonged mild bone marrow suppression made 3-weekly dosing unfeasible. For this
reason the ‘bone marrow sparing’ agent paclitaxel was added to the three weekly
administration of carboplatin. Preliminary results reveal that this strategy is feasible
and active. Weekly paclitaxel at 80 mg/m 2 is feasible in combination with AZD2281
and was found to be active. Current studies aim to assess the activity in a subgroup of
patients with the genotype and phenotype of homologous recombination deficiency.
b. Phase I study with an oral gemcitabine prodrug
Gemcitabine is an attractive candidate for oral application. Based on the experience
obtained in a phase I study with oral gemcitabine, applied every other day for 21 days
followed by one week of rest, we started a new phase I trial with a gemcitabine
prodrug. Since oral gemcitabine is swiftly deanimated presystemically, a prodrug 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 have revealed excellent safety and relevant systemic
exposure to gemcitabine after oral administration. Significant activity was observed in
two patients with advanced mesothelioma who are now on study for more than 10
months and who clearly showed disease progression prior to start of intake of oral
gemcitabine prodrug. Complete and persistent PSA normalization has been observed
in advanced and hormonal and docetaxel refractory prostate cancer. In this patient,
the oral gemcitabine prodrug is given in combination with erlotinib.
c. Studies with angiogenesis inhibitors
Two phase I studies with the orally available angiogenesis inhibitors E7080 and Bay
57-9352 are ongoing. The main mechanism of action of these three molecules is
inhibition of VEGFR 1 and 2. In dose-escalating studies, E7080 is applied as a single
agent and Bay 57-9352 is given in combination with irinotecan and capecitabine.
Hypertension and proteinuria are the main toxicities of these agents. We started a
third trial with an angiogenesis agent this year. It is pazopanib given in combination
with topotecan. Of interest, significant antitumor activity has been observed in all

three studies. In several 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 were
noted.
d. Other phase I studies
We finalized this year the three-center phase I study with the orally available and
irreversible HER1, -2 and -4 inhibitor PFS-00299804. This is a first-in-man study
initiated in our institute. The optimal dose-level for further testing is 40 mg/day for
21 days q day 28. Moderate to significant but overall manageable skin and gastrointestinal
toxicity was observed. Other phase I studies are being performed with the
aurora kinase B inhibitor AZD1152, showing neutropenia as dose-limiting activity.
Significant antitumor activity was observed in carcinoma of unknown primary site.
III -3 Pharmacokinetic and pharmacodynamic modelling and simulation
Pharmacokinetic and pharmacodynamic (PK/PD) modeling and simulation were
used to study the influence of polymorphisms in drug metabolizing enzymes on the
pharmacokinetics and toxicity of high-dose chemotherapy with cyclophosphamide,
thiotepa and carboplatin. Polymorphisms in glutathione-S-transferases (GST) play a
role in the variability in thiotepa pharmacokinetics. However, the allele frequencies
were low in the population. Polymorphisms in genes encoding for aldehyde
dehydrogenase (ALDH) were related to the occurrence of severe liver toxicity and
hemorrhagic cystitis, suggesting a role for genotyping in preventing toxicity after
intensive alkylating chemotherapy.
Data from a phase I dose escalating trial of the experimental oral anticancer agent
E7820 were analyzed using PK/PD modeling. The influence of food on the PK of this
agent was assessed. The relationship between exposure to E7820 and alfa-2-integrin
expression on platelets (a possible biomarker for E7820) was described by an indirect
effect model, enabling the assessment of different dosing schedules on the levels of
this biomarker.
The program of oral docetaxel was supported by the development of a population
pharmacokinetic (PK) model to evaluate and quantify the influence of ritonavir on
the PK of docetaxel. Thirty-six patients were included in two studies, and PK data
were collected. The absolute bioavailability of docetaxel increased 2-fold from 14 to
29% by ritonavir co-administration. The inhibition and re-activation of CYP3A4 by
ritonavir occurred instantaneously and was best described by an equilibrium constant
(K eq) of 0.068 mL/µg. A maximum inhibition of docetaxel clearance of more than
90% was reached. A PK model describing both the PK of orally and iv. administered
docetaxel in combination with ritonavir was successfully developed. Coadministration
of ritonavir leads to increased oral absorption and to a reduced
elimination rate of docetaxel.
III-4 Pharmacogenomics to identify patients at risk for toxicity or undertreatment
Pharmacogenetic screening of patients treated with 5-FU/Capecitabine
The genotype of the DPYD gene in patients treated with 5-FU or capecitabine was
determined, in order to relate the genotype and phenotype to severe 5-FU-induced
toxicity. The previously developed diagnostic real-time PCR assay was applied in
routine clinical practice demonstrating the feasibility of daily assessment of the
DYPD*2 genotype confirming the results of high-throughput screening of this
mutation in order to test the prevention of severe 5-FU-related toxicity. To date, a
population of more than 600 patients has been genotyped prior to start of
capecitabine therapy, amongst which seven heterozygotes have been identified.
III-5 Proteomics
We finalized recruitment in proteomics studies aimed to identify and prospectively
validate unique protein profiles in serum of patients with breast, gastric, colorectal
and renal cell cancer. The analysis employing the ProteinChip SELDI-TOF MS
system (Ciphergen) demonstrated unique protein profiles discriminating patients
with advanced disease from normal controls in all these diseases. In line with all
other proteomics studies published to date we did not identify unique oncoproteins.
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IV Supportive care studies in patients with breast cancer
We continued the originally single center study to identify the best therapeutic
intervention in female patients with menopausal complaints induced by treatment
for breast cancer. This year, we opened the study also in the Albert Schweitzer
Hospital in Dordrecht to increase the recruitment rate in this trial. Currently, 75 of
the planned 100 patients have been recruited. It concerns a prospective double-blind
randomized placebo-controlled intervention study with placebo, venlafaxine (Efexor ® )
and clonidine hydrochloride.
We continued the multicenter cardiac protection trial with the angiotensine II (ATI)
receptor antagonist candesartan in patients with Her 2 positive breast cancer who
received adjuvant treatment with trastuzumab. This is a double blind randomized
trial versus placebo. The study is open in 18 Dutch hospitals and 40 of the 200
planned patients have been registered.
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 is leading in the development of investigator-initiated phase II/III
trials to improve the treatment with small molecule receptor tyrosine kinase
inhibitors (RTKI), mTOR kinase inhibitors and combinations of cytokines and antiangiogenesis
drugs.
Melanoma
Participation in phase III clinical studies:
We participated in two phase III clinical trials in which a fully human monoclonal
antibodies against ‘cytotoxic lymphocyte antigen-4’ (CTLA4) is the study drug.
Blockade of CTLA4, a molecule expressed on activated T lymphocytes with
immunomodulating effects on the immune response (safe guard against
overstimulation) has been shown to increase cancer-specific immune responses in a
number of malignancies. Only HLA-A*0201-positive patients can be enrolled since
the treatment comprises two gp100 peptide vaccines that have binding affinity only
for HLA-A2 molecules. Patients are randomized between peptide vaccine only, MDX-
010 or ipilimumab only or the combination. The NKI-AVL has enrolled 11 patients in
this study so far.
Translational research with DNA vaccination in melanoma patients:
This year we received approval from both the CCMO and the Ministry of
Environmental affairs for the start of a phase I gene therapy study with a DNA
vaccine that was produced within our own GMP facility (Amsterdam Biotherapeutics
Unit (Am-BTU). The vaccine will be delivered into the epidermis of patients with
stage IV melanoma using a tattoo or permanent make-up device (see Division of
Immunology). We plan to enroll the first patient in 2008.
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. Ours will be the first trial in Europe comparing TIL treatment to standard
Dacarbazine chemotherapy. As of September 2008 the Division of Immunology and
Medical Oncology is working closely together to prepare for this high impact trial.
Renal cell carcinoma
In the renal cell carcinoma program we closely collaborate with Dr Axel Bex from
the urology-oncology group and with Dr Florry Vyth-Dreese who supervises the

monitoring of clinical immunotherapy studies (in collaboration with the Clinical
Laboratory, headed by Dr. Wim Nooijen).
We participated 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.
The overall results in a group of more than 82 patients has been analyzed in
collaboration with VUMC (Van den Veldt et al. Br J Cancer). The main finding of this
study was that also in a unselected patient population suntinib treatment results in
progression-free survival of about 9 months. Importantly, half of the patients
required dose reductions. Based on these results a Dutch study is being designed that
will base sunitinib dosing on pharmacokinetics in order to optimize both in terms of
efficacy and toxicity sunitinib treatment.
An investigator-initiated study to define the optimal time point for tumor
nephrectomy in primary metastatic renal cell carcinoma patients is ongoing. This
study is has now also been approved by the EORTC executive board as a randomized
phase III study that will start in 2009. 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 two phase I studies: sunitinib + IL-21 as first line
treatment and sorafenib + IL-2 as second line treatment for metastatic clear cell renal
cell cancer.
BREAST CANCER THERAPY AND RESPONSE PREDICTION
Sjoerd Rodenhuis, Sabine Linn, Gabe Sonke, Jos Beijnen, Corine Ekhart, Juliane Hannemann,
Marjo Holtkamp, Alwin Huitema, Ingrid Mandjes, Paula Nederlof, Margaret Schot,
Marc Van de Vijver, Laura Van ‘t Veer, Jelle Wesseling.
The objective of this program is to develop and improve potentially curative therapy
for patients with locoregional or oligometastatic breast cancer. For all these studies,
close collaborations are maintained with many other clinical departments and
research divisions. The Molecular Pathology Department is central in the response
prediction studies. In 2008, about 175 patients were entered in 8 ongoing adjuvant
medical studies in breast cancer, and in six studies in advanced disease.
Preoperative chemotherapy
The preoperative chemotherapy program continues to accrue approximately 75
patients per year and now includes data and tissues of over 300 patients. This
program is the core of an expanding multidisciplinary research effort to optimize
response prediction (e.g. with mRNA expression arrays) and to improve response
monitoring (e.g., with contrast-enhanced MRI) to allow a change in chemotherapy
regimen when the development of a pathologic complete remission appears to be
unlikely. A CTMM project based on this clinical work, but with many contributions
of basic research laboratories, began in November 2007. It involves a collaboration of
the institute with 2 academic partners (the Academic Medical Center in Amsterdam
and the Erasmus Medical Center in Rotterdam) and three industrial partners
(Organon, Philips and MRC Holland).
A distinct group of breast cancers lack the ability to repair double-strand DNA breaks
through homologous recombination (HR). For these tumors, bifunctional alkylating
agents function as targeted toxins (as do PARP inhibitors), since tumor cells are
highly sensitive while other cells are not. This situation is present in breast cancers of
patients who carry a BRCA1 or BRCA2 mutation, but has also been reported in about
30% of sporadic breast cancers. Tumors of patients in the neoadjuvant program are
tested for HR defects, and if present, are eligible for treatment in a randomized phase
II study of intermediate dose alkylator therapy. Preliminary findings indicate that part
of the HR deficient tumors can be identified through aGCH analysis.
HER2 positive breast cancers strongly benefit from Trastuzumab-based systemic
treatment, but this is not yet standard in the neo-adjuvant setting. Up-front use of
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Trastuzumab may also allow the avoidance of anthracyclines at least for a subgroup
of tumors. A multi-centre phase II study investigating the response to nonanthracycline
based chemotherapy incorporating Trastuzumab is currently recruiting
patients and collecting tissues. Preliminary results from our center show promising
pathologic complete response rates (figure 2).
Figure 2: Pathological complete response rate of breast and axilla following preoperative chemotherapy
with trastuzumab, carboplatin and paclitaxel in HER2-positive locally advanced breast cancer.
Abbreviations: CR (b+a): pathological complete response of both breast and axilla; CR (breast):
pathological complete response of the primary tumor; ER: estrogen receptor.
Microarrays, prognosis and response prediction Microarray technology is
gaining credibility as an important diagnostic tool in guiding treatment decisions in
breast cancer. Our institute has developed a 70-gene prognostic signature that
outperformed classic clinico-pathologic prognostic factors and remained a strong
independent prognostic factor in multivariate analysis. In collaboration with
AGENDIA, we compared the performance of the signature to predict response to
neoadjuvant chemotherapy. All pathologic complete remissions were observed in
tumors with a high-risk profile, confirming the expectation that the more slowly
proliferating low-risk tumors may be less sensitive to chemotherapy.
In 2004, the MATADOR study was initiated and this study is currently open in 31
centers in the Netherlands; ~240 patients have been included. The primary objective
of this study is to identify predictive gene expression profiles for a disease-free
survival benefit of either a docetaxel-containing regimen (docetaxel-doxorubicincyclophosphamide)
or an accelerated doxorubicin-cyclophosphamide regimen.
In collaboration with Laura Van ‘t Veer and the Erasmus University (Rotterdam) we
have validated and refined a predictive profile for tamoxifen resistance in the
metastatic setting (see also under Division of Experimental Therapy). We are
currently investigating the utility of this profile for the adjuvant setting.
We have recently started a preoperative single centre trial to study responsiveness of
hormone-receptor positive breast cancers to Tamoxifen, Anastrozole or Anastrozole
in combination with Fulvestrant in postmenopausal patients. Prior to primary
surgery, patients are exposed at least two weeks to hormonal agents. Blood and tumor
tissue is collected before and after treatment for molecular studies, 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 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.

THORACIC ONCOLOGY
Paul Baas, Sjaak Burgers, Michel Van den Heuvel, Jose Belderbos, Wieneke Buikhuisen,
Houke Klomp, Peter Kunst, Petra Nederlof, Jan Schellens, Michel Wouters
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, which is given for 3 weeks. This study is led by the department of thoracic
surgery (Houke Klomp). Preliminary findings indicate that such a short course of
targeted therapy causes a significant degree of apoptosis at pathological examination.
In patients with advanced NSCLC, a pharmacogenomics study has now included 100
patients who have received cisplatin and gemcitabine as first-line chemotherapy. The
correlation between plasma levels and metabolism drugs and the presence of Single
Nucleotide Polymorphisms (SNP’s) is currently being analyzed. In patients with
unexpected metastasis in N2 lymph nodes during surgery, the effect of mediastinal
RT is under evaluation in the European phase III LUNGART study. A total of 700
patients will be required.
Two vaccination studies have been initiated in 2008 for patients with advanced stages
of NSCLC: A phase IB study of maintenance therapy with Selectikine after a short
course of RT; and a randomized phase II study of Stimuvax with or without
radiotherapy in HLA-A2 positive patients (figure 3). Both studies build on the
postulated synergetic effects of simultaneous radiotherapy and immunotherapy.
In addition, patients will be enrolled in a phase III vaccination study with Lucanix.
Figure 3: Working mechanism of Stimuvax.
In second line NSCLC, we have participated in a randomized study of erlotinib and
placebo vs. erlotinib and sunitinib, a tyrosine kinase inhibitor which blocks both the
VEGF receptor and the EGF receptor.
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 is planned and will start beginning of 2009. In the first
six weeks after registration, single agent Sunitinib is given and patients are closely
monitored for response/progression with PET-CT scans.
Pleural disease In 2007, the pleural fluid 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 with biomarker research employing both frozen serum
and pleural fluid samples. Currently we have patient data and matched samples in
over 300 patients.
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MEDICAL ONCOLOGY
Malignant Pleural Mesothelioma (MPM) The national randomized phase III
maintenance study of thalidomide after standard chemotherapy in MPM has now
accrued 80% of the target population. 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.
The EORTC phase II study 08931 tested the toxicity of induction chemotherapy
followed by extra-pleural-pneumonectomy and hemi-thoracic irradiation. The toxicity
data will be analyzed by the EORTC in the first quarter of 2009.
For second and third line therapy we offer patients a randomized phase III study of
Vorinostat vs. placebo. This international study has passed its first endpoint at 220
out of 660 planned patients and since no toxicity was observed the second stage has
been initiated.
For 2009, a randomized phase I-II study will examine 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 also be measured, in addition
to the changes in vascular flow analysis by MRI.
GASTROENTEROLOGY
Henk Boot, Annemieke Cats, Marianne Smits, Babs Taal, Hans Bonfrer, Maarten Deenen,
Edwin Jansen, Tiny Korse, Jan Schellens, Marcel Verheij
Neuroendocrine tumours (NET) Urinary excretion of 5-HIAA, the degradation
product of serotonin, is widely available as a marker in NET, but has a low sensitivity
and the 24-hour collection is inconvenient for patients with diarrhea. The serum
marker Chromogranin A (CgA) has the advantage of being positive in NETs of
various origin, but a thorough evaluation during follow-up is lacking. In our study
gastrointestinal NET patients (n=39) were monitored 3-monthly for one year during
treatment. Significant correlations were found between CgA and symptoms such as
physical functioning (p=0.01) and overall quality of life (p=0.03), in contrast to
5-HIAA. Similarly, Cox regression showed an association between CgA levels and
survival time (p=0.02), and not for 5-HIAA levels.
In NET fibrosis of the tricuspid heart valve (carcinoid heart disease or CHD) is a well
known complication probably as a result of exposure to serotonin produced by liver
metastases. This complication develops in 20-70% and is a major cause of death in
patients with carcinoid syndrome. To detect this in an early phase and to select
patients for heart valve surgery we evaluated the role of NT-proBNP, a natriuretic
peptide available as a marker of left ventricular dysfunction, and CgA.
Figure 4: ROC-curve demonstrating the accuracy of
CgA and NT-proBNP distinguishing patients with
severe CHD from patients without or with mild CHD.
Among 102 NET-patients, CHD was present at cardiac ultrasound in 14 patients
(14%). In 12 of these patients (86%) NT-proBNP was elevated, with a specificity of
80% (figure 4). In the univariate logistic regression analysis NT-proBNP (p=

survival (p

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RADIOTHERAPY
Division head, group leader, Marcel Verheij
Berthe Aleman MD PhD Academic staff
Harry Bartelink MD PhD Academic staff
Jose Belderbos MD PhD Academic staff
Eugène Damen PhD Academic staff
Roel De Boer PhD Academic staff
Luc Dewit MD PhD Academic staff
Paula Elkuizen PhD Academic staff
Rick Haas MD PhD Academic staff
Olga Hamming-Vrieze MD Academic staff
Guus Hart MSc Academic staff
Wilma Heemsbergen MSc 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
Corrie Marijnen MD PhD Academic staff
Harry Masselink MD Academic staff
Ben Mijnheer PhD Academic staff
Luc Moonen MD PhD 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
Joep Stroom PhD Academic staff
Bart Van Bunningen MD 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
Marcel Verheij MD PhD Academic staff
Frits Wittkämper PhD Academic staff
Monique Bloemers MD Temporary staff
Gerben Borst MD Temporary staff
Desiree Van Den Bongard MD PhD
Temporary staff
Birgit Hollmann MD Temporary staff
Bas Kreike MD Temporary staff
Oene Van Meer MD Temporary staff
Philip Meijnen MD PhD Temporary staff
Dimitry Nuyten MD Temporary staff
Gabriele Speijer Temporary staff
Roel Steenbakkers MD PhD Temporary staff
Ben Vanneste MD Temporary staff
Francine Voncken MD Temporary staff
Martijn Eenink PhD Post-doc
Marnix Maas PhD Post-doc
DIVISION OF RADIOTHERAPY
In 2008 our department continued to invest in its major research themes: improving
treatment accuracy, efficacy and safety. Intensity-modulated radiotherapy leads to
superior dose distributions and is applied in an increasing number of indications.
Optimal geometrical precision before and during treatment is achieved by cone-beam
CT-based image guidance, now routinely in use on 5 of our 9 linear accelerators.
Both technologies have been pivotal for allowing dose escalation and the clinical
implementation of extreme hypofractionated radiation schedules, e.g. in early stage
lung cancer and solitary brain tumors/metastases. To further enhance radiation
response new combined modality approaches are explored using both classical
chemotherapeutic agents and novel biological response modifiers. These new
generation biologicals have become available through our own research as well as
through collaborations with pharmaceutical companies, and are making their way
from the lab to the clinic as potential radiosensitizers. Given the increasing
complexity and potential toxicity of these treatments, we have given high priority to
dose verification by in vivo dosimetry. All curative treatments are currently verified by
means of EPID dosimetry, minimizing the risk of over- and underdosage.
Functional imaging becomes increasingly important in tumor staging, target
delineation and response prediction. In collaboration with the departments of
Radiology and Nuclear Medicine we therefore evaluate the value of several imaging
modalities in these processes, including MRI spectroscopy, 18F-FAZA-PET, 99mTc-
MIBI-SPECT and 99mTc-Annexin V scintigraphy. The integration of both anatomical
and functional information in our treatment protocols creates the possibility to adapt
therapy at an early stage and on an individual basis.
In September 2008, the European Comprehensive Cancer Centres Alliance (ECCCA)
was founded during an international symposium at the NKI-AVL. This collaboration
between three European cancer centers (Institut Gustave Roussy, Karolinska
Institutet and NKI-AVL), brings together powerful technological platforms of
genomics/proteomics based selection assays and preclinical evaluation tools to
identify promising agents for combined application in early clinical trials. There are
currently three clinical trial recruiting patients on a jointly basis.
The Radiotherapy department is also an active partner in a new initiative in proton
therapy. The Holland Particle Therapy Center (HollandPTC) project is a joint
initiative of Erasmus MC, NKI-AVL, Leiden University MC and TU Delft, and aims at
establishing a particle therapy center in Delft for both patient care, research and
technological development.
IMAGE ACQUISITION AND PROCESSING
A Al-Mamgani1 , Anja Betgen, Jose Belderbos, Suzanne Van Beek, Roman Bohoslavsky, Jan De Boer,
Josien De Bois, X Chai2 , Eugene Damen, H Dees-Ribbers, Joop Duppen, Rick Haas,
Wilma Heemsbergen, David Jaffray3 , Edwin Jansen, Rianne De Jong, Simon Van Kranen,
Corrie Marijnen, Vanessa Mexner, Danny Minkema, Jasper Nijkamp, Erik-Jan Rijkhorst, Floris Pos,
Simon Rit, Christoph Schneider, Monique Smitsmans, Corine Van Vliet-Vroegindeweij, Marnix Witte,
Jochem Wolthaus, Lambert Zijp, Coen Rasch, Joos Lebesque, Peter Remeijer, Jan-Jakob Sonke,
Marcel Van Herk
Cone beam CT reconstruction Respiratory motion induces artifacts in conebeam
(CB) CT images of the thorax which can prevent an accurate localization of
lung tumors at treatment time for patient positioning. Respiration-correlated
reconstruction has been used to correct for this motion but requires a long
acquisition time. We therefore developed an online motion-compensated CBCT
reconstruction technique based on a prior model of the respiratory motion obtained
from the 4D planning CT with deformable registration. The motion was compensated
1 EMC Rotterdam / 2 Academic Medical Center
3 University of Toronto/Princess Margaret Hospital, Ontario, Canada

during filtered-backprojection of scans made on the treatment machine. The method
was evaluated on 26 CBCT scans of 3 patients acquired on an Elekta Synergy system.
Visual observation of reconstructed MC-CBCT images showed that most of the
respiratory motion artifacts were removed obtaining image quality comparable to
static CBCT, even with a 1 min acquisition time protocol where 4D CBCT is hardly
usable (see figure 1). Using the off-line computed motion model, the computational
time 67 s for the 1 min acquisition protocol (concurrent with CB acquisition),
providing a high quality image within a few seconds after the end of the acquisition.
Figure 1: Effect of motion compensation reconstruction for a typical lung cone beam CT scan used for
image guided radiotherapy. a) 3D reconstruction without motion compensation for a 4 minute acquisition
time (intended for 4D acquisition). b) Motion compensated reconstruction (4 min. acquisition time).
Note the improved sharpness of tumor and diaphragm. c) Motion compensated reconstruction (1 min.
acquisition time). The image quality is more than acceptable for image guidance of the tumor and
normal structures.
Image guidance Recently, promising results have been reported for stereotactic
body radiotherapy (SBRT) of primary and metastatic liver cancer. For conventional
RT of liver cancer, the bony anatomy is often used as a surrogate for tumor location,
which is too imprecise for SBRT. We implanted markers close to the lesion as a
surrogate for tumor location and applied four dimensional (4D) cone beam CT
guidance for registration and correction of the marker location. Four patients with
metastasic liver cancer were included in this study so far (2 – 4 markers per patient).
Patients were treated with 15 fractions of 3 Gy with an online or offline bony anatomy
setup correction protocol using CBCT guidance. Retrospectively, markers were
localized using locally rigid registration on 4D CBCT. Baseline variations were
quantified by the difference in the (time weighted average) position of the markers
and the bony anatomy. The results were analyzed in terms of group mean (M),
standard deviation. The average peak-to-peak tumor amplitude assessed by 4D CBCT
was 1.2 cm [range 0.7 cm - 1.6 cm]. Interfraction baseline shifts were about 1-2 mm
in each direction. Baseline variations of 1-2 mm (1 SD) have been observed over the
course of RT of liver cancer patients.
Correction strategies Current image guided systems provide efficient on-line
correction of target positioning. Significant motion, however, can occur between
target and organs-at-risk (OARs), for instance for lung tumors (baseline shifts).
Then, proper target positioning may lead to overdosage of OARs. We devises a
practical image guidance system that can deal with such differential motion. During
planning for lung SBRT patients, the cord was expanded by 1 cm, but sometimes less
expansion was required to reach an acceptable plan. The guidance system registration
regions, placed on spinal column and target. Image registration is performed for
both regions providing setup error data of cord and target. The software checks the
proposed correction against predefined limits for both regions. When the correction
would move the high dose region outside limits towards the OAR, a warning is given.
In a separate review procedure, the operator ‘fades’ between correct target and OAR
positioning to reach a compromise with both regions within limits. The system was
applied in 86 patients. In 10 patients, the proximity of the tumor and OAR required
reduced OAR expansions. In 8 patients, a compromise had to be made because the
baseline shift exceeded the OAR margin during one or more fractions. In all cases,
it was possible to find a correction that satisfied both target and OAR constraints.
This study showed that especially in hypofractionated radiotherapy of lung tumors,
differential motion of tumor and OAR may lead to serious overdosages when OARs
are not considered in the guidance procedure.
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RADIOTHERAPY
Anton Mans PhD Post-doc
Leah McDermott PhD Post-doc
Vanessa Mexner PhD Post-doc
Anke Van Mourik PhD Post-doc
Juup Nijholt PhD Post-doc
Erik-Jan Rijkhorst PhD Post-doc
Simon Rit PhD Post-doc
Jan-Jakob Sonke PhD Post-doc
Mine Temurhan PhD Post-doc
Rajko Topolnjak PhD Post-doc
Markus Wendling PhD Post-doc
Marnix Witte PhD Post-doc
Johan De Boer MSc PhD student
Simon Van Kranen MSc PhD student
Monique Smitsmans MSc PhD student
Jochem Wolthaus MSc PhD student
Jonathan Yang MSc PhD student
Robin Kalisvaart Physician Assistant
Margriet Kwint Physician assistant
Gül Yüksel Physician assistant
Anja Betgen Technical staff
Josine De Bois Technical staff
Joop Duppen Technical staff
Angelo Mencarelli Technical staff
Tom Minderhoud Technical staff
Jasper Nijkamp MSc Technical staff
Agnieszka Olszewska MSc Technical staff
Carmen Panneman MSc Technical staff
Carolien Peters Technical staff
Kenneth Pengel Technical staff
Lennert Ploeger Technical staff
Edwin Roosjen Technical staff
Maddalena Rossi Technical staff
René Tielenburg Technical staff
Lambert Zijp MSc Technical staff

128
RADIOTHERAPY
Publications
Image acquisition and processing
Fitton I, Steenbakkers RJ, Gilhuijs K,
Duppen JC, Nowak PJ, van Herk M,
Rasch CR. Impact of anatomical location
on value of CT-PET co-registration for
delineation of lung tumors. Int J Radiat
Oncol Biol Phys 2008;70:1403-1407
Rit S, Wolthaus J, van Herk M, Sonke JJ.
On-the-fly motion-compensated cone-beam
CT using an a priori motion model. Med
Image Comput Comput Assist Interv Int
Conf Med Image Comput Comput Assist
Interv 2008;11:729-736
Smitsmans MH, Pos FJ, de Bois J,
Heemsbergen WD, Sonke JJ,
Lebesque JV, van Herk M. The influence of
a dietary protocol on cone beam CT-guided
radio therapy for prostate cancer patients.
Int J Radiat Oncol Biol Phys 2008;71:
1279-1286
Sonke JJ, Lebesque J, van Herk M.
Variability of four-dimensional computed
tomography patient models. Int J Radiat
Oncol Biol Phys 2008;70:590-598
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.
2008 Nov 27. (epub ahead of print)
Van Herk M. Will IGRT live up to its
promise? Acta Oncol 2008;47:1186-1187
Construction of a midposition CT scan for planning of lung cancer patients using
deformable registration. Lower lobe lung tumors move with amplitudes of up to 2 cm
due to respiration. To reduce respiration imaging artifacts in planning CT scans, 4D
imaging techniques are used. Currently, we use a single (midventilation) frame of the
4D data set for clinical delineation of structures and radiotherapy planning. A single
frame, however, often contains artifacts due to breathing irregularities. The aim of
this work is to develop a framework for the acquisition of a good quality scan
representing all scanned anatomy in the mean position by averaging transformed
(deformed) CT frames, i.e. canceling out motion. 4D and inspiration breath-hold
(BH) CT scans were acquired for 13 patients. An iterative multiscale motion estimation
technique was applied to the 4D CT scan, similar to optical flow but using image
phase (gray-value transitions from bright to dark and vice versa) instead. From the
(4D) deformation vector field (DVF) derived, the local mean position in the
respiratory cycle was computed to deform all structures of the original 4D CT scan to
this mean position. A 3D midposition (MidP) CT scan was then obtained by averaging
of the deformed 4D CT scan. The shape of the tumor, with respect to that of the BH
CT scan, was better represented by the MidP reconstructions than any of the 4D CT
frames (including MidV; reduction of ‘shape differences’ was 66%). The MidP scans
contained about one-third the noise of individual 4D CT scan frames. Tumor shape
and position in the MidP CT scan represents that of the BH CT scan better than
MidV CT scan and, therefore, was found to be appropriate for treatment planning.
Geometrical Uncertainties in Soft Tissue Sarcomas of Extremities Standard of
care for almost all extremity soft tissue sarcomas (ESTS) patients is surgery, followed
by radiotherapy (RT). Several studies however, show benefit of preoperative RT,
especially concerning late morbidity endpoints such as fibrosis and joint stiffness.
The purpose of this study was to quantify volume and positional changes of the Gross
Target Volume (GTV) during preoperative RT and investigate the possibility for more
advanced correction strategies. Ten patients with lower limb ESTS were included in
this retrospective study. Planning CT-scan and 2 CBCT-scans acquired at the start of
the treatment and after five weeks were used for delineation of the GTV. Delineation
was performed after a registration on bony anatomy, which was used as a surrogate
for tumor position during treatment for setup correction. In four patients volumes
did not change more than 3%. However, significant changes were discovered in six
patients. In three patients an increase of the GTV volume was measured (10%, 19%
and 26%) and in three patients a decrease was noted (12%, 17% and 23%). The COG
of these delineations changed for the last CBCT with a mean vector length of 0.39
cm (range 0.03 – 0.86 cm). The changes in the increasing volumes are always away
from the bones, and vice versa in the decreasing volumes in the direction of the
bones. Clinically applied margins were adequate to account for these uncertainties.
To further reduce morbidity, reduction of geometrical uncertainties and margins are
essential. This might require soft tissue guidance because the bony anatomy is not
always a good surrogate for GTV position and adaptive re-planning to account for
volume changes.
Figure 2: Comparison of the so-called mid-ventilation image, and a novel concept: the mid-position image.
a) The mid-ventilation image is used clinically as most representative image of a breathing patient. It is
selected from a respiration correlated CT scan by choosing that (animation) frame that is closest to the
mean position of the tumor. b) the mid-position image is constructed by deforming all pixels of all frames
to their mean position and then averaging the results. This image gives an optimal representation of the
patients’ anatomy and has a much better image quality.

Target shape variability in rectum cancer patients Changes in filling of rectum
and bladder cause large geometrical uncertainties during radiotherapy of rectum
cancer patients. The purpose of this study was to quantify the interfraction CTV
shape changes of rectum cancer patients treated pre-operatively with 5x5 Gy. We
assessed the interfraction CTV shape variability on Cone-Beam CT (CBCT) scans
acquired just prior to irradiation for 27 patients (prone position). We focused on
mesorectum first adnd delineated this structure on the planning CT and on each
CBCT scan. In addition, rectum and bladder were delineated. It turns out that
interfraction shape variability is substantial in certain parts of the mesorectum: The
systematic (]) and random (]) errors varied substantially for different regions of the
mesorectum: ] and ] of up to 7 mm were found on the anterior upper part of the
mesorectum. At the lateral and dorsal borders of the mesorectum the errors were
relatively small except for below the os coccyx (tail bone). The large systematic and
random errors in the upper-middle part were mainly caused by differences in rectum
filling and not so much by differences in bladder filling. Errors below the os coccyx
can be explained by differences in muscle tension.
Margins for deformable objects Image guided radiotherapy in combination with
advanced registration techniques revealed substantial deformation for tumor sites
such as head-and-neck, rectum and lung. Classical margin recipes (e.g. M=2.5S+
0.7s), assume the target to be a single rigid object, which is not true in the presence
of deformations. The purpose of this study was to derive margins for deforming
objects. The framework of the classical margin recipe was extended by analyzing
multiple points in space (differential motion) or on a spherical surface with
deformations perpendicular to the surface. All points move with a Gaussian
distribution with a constant standard deviation ] and a single correlation r between
the distributions, which could be varied. By generating many possible instances the
margin required to encompass all points for 90% of the instances was derived as a
function of the number of points, r and ] (Monte Carlo simulations). In this analysis,
we focused on the effect of the systematic error. The effect of random errors is
unchanged compared to a single rigid object. Complete target coverage for 90% of
the population depended on the number of points and the their correlation. If points
move with r=1, the solution for classical 90% coverage probability was retrieved, i.e.
a margin of 2.5 ] for multiple targets and 1.3] for deformations (single sided 1D
probability distribution). For the fully uncorrelated situation, margins depended on
the number of points. For deformations 20 points results in a margin of 2.5], 200
points in 3.2]. For a situation with multiple rigid targets, the margins required
increased from 2.8] (2 targets) to 3.1] (5 targets). These data show that applying
classical margins for deformations is incorrect for two reasons. First, target coverage
is unaffected for surface-points moving inwards. Second, with more than one point
the probability of having all points within classical margins simultaneously
decreases. We expect that larger tumors need more points to describe observed
deformations and margins will increase accordingly.
High precision objectives for IMRT optimization using Pinnacle Although
triangulated surface meshes are available in the Pinnacle treatment planning system,
the objective functions for IMRT optimization rely on volumes described as contours
on the CT slices. A set of replacement objectives was developed to exploit the
increased definition of mesh-based volumes, enabling high precision IMRT
optimization. A customized set of objectives that perform Monte Carlo volume
integration of triangulated regions of interest (ROIs) resulting in a higher accuracy of
the cost value evaluation. For a head and neck cancer patient, we first optimized
applying the original Pinnacle functions until a minimum of the total cost was
achieved. Next, we applied our new functions and continued optimization. The
higher accuracy of the cost evaluation did require more computation time during
initialization (a few minutes), but the optimization process was not noticeably slowed
down. PTV coverage was improved somewhat without violating organs at risk (OARs)
dose constraints because the OARs are described with a higher resolution. However,
the differences are small unless a very high spatial accuracy is required, such as for
the optic nerve.
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Advanced analysis of clinical trial results Trials with centrally collected digital
planning data allow, in principle, relating dose distribution details with clinical
outcome. However, this type of analysis is rare because of the difficulty of comparing
dose distributions between patients. The purpose of this study is to relate the dose to
the anatomy outside the prostate with observed and predicted failure free survival.
CT, delineations and 3D dose cubes of the Dutch 68 vs. 78 Gy prostate trial were
collected digitally for all patients. Failure at 4y was defined according to the Phoenix
definition (clinical or biochemical). Maps of the dose surrounding prostate and
rectum were created for each patient, giving the dose at different distances (up to
4 cm) from the organ surface, in fixed directions from the center of the organ. In this
way, map points have reasonable anatomical correspondence. Average dose maps
were computed for patient subgroups with and without failure, and differences of
these maps were computed. Both rectum and prostate based-maps show areas with
significant dose differences at some distance from the prostate. A very significant
(p

EPID DOSIMETRY
Priscilla Camargo, Anton Mans, Leah McDermott, Jan-Jakob Sonke, Joep Stroom, Marcel Van Herk,
Markus Wendling, Ben Mijnheer
EPID dosimetry has been clinically used for IMRT verification in our department,
both pre-treatment and in vivo, since 2005. In recent years, the method has gradually
been applied to more treatment sites, and starting this year, the plans of all our IMRT
patients (~2000/year) are verified with EPID dosimetry. Basically EPID dosimetry
serves therefore two purposes: (1) it is our only form of patient-specific IMRT
verification, used for all curative patient treatments; (2) with a few exceptions (too
large fields with respect to the EPID dimensions, about 3%, and single high dose
fractions, about 1%) it is applied in vivo thus guaranteeing the correct dose delivery to
the patient. Three years of clinical experience have proven the system to be
sufficiently accurate and highly efficient (25 min/plan, 50 plans/week, 1 radiographer).
EPID dosimetry provides a safety net as the final check that all patients are correctly
treated, given the fact that several types of errors have been detected that would have
led to erroneous patient treatments if undetected.
In our standard procedure, the dose in a plane parallel to the EPID intersecting the
isocentre is reconstructed per beam from EPID images by means of a back-projection
algorithm and compared to the planned dose distribution with the g-evaluation
method (3%/3mm). No inhomogeneity correction is used and the EPID should be
aligned with the central axis of the linac. Verification of the treatment of breast and
lung cancer needed special attention, as our standard dose reconstruction based on
EPID transmission measurements was not accurate enough for these two cancer sites.
In our department, breast treatments (~700 patients/year) consist of two opposing,
non-wedged, tangential half fields, each with an open and IMRT part. When lymph
nodes have to be treated as well, quarter fields are used. To apply our method to
breast cancer treatments, two modifications are made: (1) the dose specification point
is used, since the isocentre is located on a field edge, and (2) an off-axis correction is
applied, taking into account the modified pixel response due to spectral changes
when the EPID is shifted. For quarter fields, a large EPID shift is necessary to image
the field without irradiating the edge of the detector. When applying these corrections,
in vivo EPID dosimetry for breast treatments shows now good results.
Our current back-projection dose-reconstruction algorithm uses water-based scattercorrection
kernels and does therefore not account for tissue inhomogeneities, which
gives large deviations for lung cancer treatments. With in aqua vivo dosimetry, the
dose is reconstructed in the patient as if the patient would have consisted entirely of
water. By using in aqua vivo EPID dosimetry, dose verification for lung cancer
patients during the actual treatment is possible, except for the tissue inhomogeneity
correction of the TPS. The method was proven to be accurate within g criteria of 3%
and 3 mm. This method has been implemented clinically and is applied for all our
lung cancer treatments with curative intent.
If the patient is in a different position, the radiation beams directions alter and in
some cases the dose distribution will change from the planned dose. Whether or not
EPID dosimetry picks up this deviation depends on the change in transmission for
each field. We therefore studied the direction and magnitude of the shifts for which
an alert would be raised for a single field and typical lung IMRT plans based on in
aqua vivo EPID dosimetry. For this purpose an anthropomorphic thorax phantom was
irradiated and shifted in ±3 orthogonal directions. From the absolute dose reconstructed
inside the patient it could be concluded that EPID in aqua vivo dosimetry raises an
alert for positioning differences of ~1-2 cm for typical lung cancer treatment plans.
A start has been made with adapting our model for the use of EPID dosimetry in
wedged beams. Because of the change in photon energy spectrum when placing a
wedge in the beam, some of the parameters in our model, e.g. the beam hardening
correction, have to be modified to get a better prediction of the reconstructed dose
distribution in patients irradiated with wedged beams.
Publication (continued)
Epid dosimetry
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RADIOTHERAPY
McDermott LN, Wendling M, Nijkamp J,
Mans A, Sonke JJ, Mijnheer BJ, van Herk M.
3D in vivo dose verification of entire hypofractionated
IMRT treatments using an
EPID and cone-beam CT. Radiother Oncol
2008;86:35-42
Van Elmpt W, McDermott L, Nijsten S,
Wendling M, Lambin P, Mijnheer B.
A literature review of electronic portal
imaging for radiotherapy dosimetry.
Radiother Oncol 2008;88:289-309

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RADIOTHERAPY
Publication (continued)
Treatment planning
Wolthaus JW, Sonke JJ, van Herk M,
Belderbos JS, Rossi MM, Lebesque JV,
Damen EM. Comparison of different
strategies to use four-dimensional computed
tomography in treatment planning for lung
cancer patients. Int J Radiat Oncol Biol
Phys 2008;70:1229-1238
Wolthaus JW, Sonke JJ, van Herk M,
Damen EM. Reconstruction of a timeaveraged
midposition CT scan for
radiotherapy planning of lung cancer
patients using deformable registration. Med
Phys 2008;35:3998-4011
TREATMENT PLANNING
Harry Bartelink, Jose Belderbos, Gerben Borst, Joop Duppen, Paula Elkhuizen, Martijn Eenink,
Barry Doodeman, Wilma Heemsbergen, Mariëlle Van Heumen, Suzanne Den Hollander,
Rianne De Jong, Annemarie Lakeman, Emmy Lamers, Corrie Marijnen, Vanessa Mexner,
Danny Minkema, Anke Van Mourik, Floris Pos, Coen Rasch, Jan-Jakob Sonke, Mine Temürhan,
Corine Van Vliet-Vroegindeweij, Eugene Damen
Lung
Shift invariance of the dose distribution for online corrections of stereotactic
body radiotherapy of lung cancer patients Substantial baseline variation (motion
of the mean tumor position relative to the bony anatomy) has been observed in lung
cancer patients treated with stereotactic body radiotherapy (SBRT). We therefore use
online corrections based on the actual tumor position. This procedure assumes shift
invariance of the dose distribution for small corrections. In a treatment planning
study on five patients this assumption was tested. Clinical treatment plans were
recalculated in Pinnacle after shifting the isocenter and the planning target volume
(PTV), without re-optimizing. Shifts (0.5 cm, 1.0 cm and 1.5 cm) were applied in
12 equiangular spaced directions of the sagittal plane. The main objective for the
original plans was that at least 95% of the PTV volume received 54 Gy (3 x 18 Gy).
As expected, increasing shifts led to increasing deterioration of the PTV coverage,
but varied over the patient group. For half of the patients, D95 dropped up to 1 Gy for
0.5 cm shifts, 1-2 Gy for 1.0 cm shifts and 1-3 Gy for 1.5 cm shifts. For the other half,
D95 dropped up to 1-4 Gy, 2-5 Gy and 3-6 Gy, respectively. Tumor locations near the
thoracic wall show the largest deterioration, due to the effect of a varying penumbra
width when the PTV is moved towards or from the thoracic wall. From the results we
concluded that in case of lung tumors located free from the thoracic wall, baseline
shifts up to 1.0 cm are acceptable without re-optimization. In other cases, the
robustness of the treatment plan to correct for baseline shifts must be validated.
Gastroenterology
An improved drinking protocol to decrease irradiated volume of small bowel
during radiotherapy of rectal cancer The amount of irradiated small bowel can be
reduced by treating patients with a full bladder. The effect of two drinking protocols
on bladder volume was evaluated. Old protocol (OP, 28 patients): patients were
instructed to empty their bladder one hour before start of treatment and subsequently
drink 250 ml of water. New protocol (NP, 9 patients): patients were instructed to void
one hour before start of treatment and drink 350 ml of water, in addition with the
instruction to drink 2 liters of fluids per day during the course of treatment. The
bladder was delineated on the planning CT and on cone-beam CT (CBCT) images,
which were acquired for each fraction (fractionation schedule: 5 x 5 Gy). Average
bladder volumes increased from 240 ml for the OP to 336 ml for the NP. In a
multivariate analysis, this difference was significant (p = 0.02), while patient sex, age
and position (prone or supine) were not. However, day-to-day variations in bladder
volume increased with the average volume, and a significant negative time trend in
bladder volume was seen during the course of the treatment. Moreover, bladder
filling on the planning CT was on average larger than during the course of treatment
(293 ml vs. 233 ml, p = 0.001). From these results it was concluded that NP increases
bladder volume during treatment. NP results in an increase of inter-fraction
variability in bladder volume, which should be taken into account when determining
required PTV margins.

Breast
Unraveling influences of patient error sources on breast IMRT plans by
means of deformable registration Setup error and tissue deformation during
treatment of breast cancer patients lead to variations in position and shape of the
target volume and alter the originally planned dose distribution. We determined the
sensitivity of 3 breast RT planning strategies for these error sources by evaluation of
accumulated dose. For 5 patients, 3 plans were made with identical tangential,
glancing beam setup: a non-IMRT technique with wedges and two IMRT techniques.
For the SimpleIMRT technique 80% of the dose was delivered by the tangentials,
while additional computer-optimized segments delivered the remaining 20%. In the
FullIMRT technique, 100% of the dose was delivered by computer-optimized
segments. The dose was accumulated over 5 fractions, taking the separate and
combined errors into account. To that end, the dose was recalculated on the original
CT after setup translations (T), a deformed CT (see below) (D), and a deformed CT
after setup translations (T&D), and compared to the original plan (O). The original
CT was deformed to the CBCT of the day to account for shape changes. Resulting
deformation fields were used to generate a deformed CT for recalculating the dose
distributions, and to deform these distributions back into the original planning CT
for accumulation.
Table 1 Coverage (V95 mean (sd)) of original dose (O) and dose including different types error sources
(T, D, T&D) for different planning strategies (1-3). Results are preliminary in view of ongoing analyses.
V95 O T D T&D
Correction protocol Original plan Offline Online Offline
Wedge 95(3) 91(4) 93(4) 90(3)
SimpleIMRT 98(2) 95(2) 97(1) 94(1)
FullIMRT 97(1) 95(2) 94(1) 93(1)
The impact of error sources is reflected in reduction of the V95 compared to the
original plan (Table 1). Translations represent the larger error source for the wedged
and SimpleIMRT plans. For FullIMRT deformations seem to have a larger impact.
Underdosage was mainly located near the skin. From these results we conclude that
the impact of error sources on breast RT depends on planning technique. Full IMRT
plans do not have glancing beams and are therefore more susceptible to deformation
errors of the breast contour. The difference between D and T&D reveals the gain that
can be achieved by clinically using an online instead of an offline correction
Interobserver variability in target volume delineation of boost volume in
breast irradiation across institutions Thirteen radiation oncologists from eleven
different institutions delineated the volumes of surgical area, CTV boost and PTV
boost of two left-sided and three right sided breast cancer patients. All observers used
a written instruction and had access to all clinical available information. Three
patients had no seroma (patient 1-3), one patient had some seroma (patient 4) and
one patient had a clearly visible seroma (patient 5). The conformity index (CI) of two
delineated structures (defined as the ratio of the overlapping volume and the
encompassing total delineated volume) was used to quantify the difference in
delineation between observers. For all patients a median delineation was defined by
the volume consisting of all pixels that were delineated by at least 50% of the
observers. To pinpoint specific areas of variation the median was subdivided in
several anatomic regions: caudal, cranial, lateral, medial, dorsal, ventral and skin.
A large variety in the delineated volumes was seen. In general, some observers
consistently delineated larger volumes than the median volume for all patients, while
others were consistently delineating smaller than the median volumes. The CI values
showed a correlation with the presence of seroma. When seroma was hardly present,
average CI-values below 0.5 were found, whereas in the case of a clearly visible
seroma an average CI-value of 0.8 was found. Hardly any variations over the different
anatomic regions were found. We concluded that significant interobserver variations
could be found in the delineated volumes. The presence of seroma resulted in low
interobserver variations.
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134
RADIOTHERAPY
Publication (continued)
Clinical application of image guided
radiotherapy
Belderbos JS, Kepka L, Spring Kong FM,
Martel MK, Videtic GM, Jeremic B. Report
from the International Atomic Energy
Agency (IAEA) consultants’ meeting on
elective nodal irradiation in lung cancer:
non-small-Cell lung cancer (NSCLC). Int J
Radiat Oncol Biol Phys 2008;72:335-342
Kappers I, Belderbos JS, Burgers JA,
van Zandwijk N, Groen HJ, Klomp HM.
Non-small cell lung carcinoma of the
superior sulcus: favourable outcomes of
combined modality treatment in carefully
selected patients. Lung Cancer 2008;59:
385-390
Videtic GM, Belderbos JS, Spring Kong FM,
Kepka L, Martel MK, Jeremic B. Report
from the International Atomic Energy
Agency (IAEA) consultants’ meeting on
elective nodal irradiation in lung cancer:
small-cell lung cancer (SCLC). Int J Radiat
Oncol Biol Phys 2008;72:327-334
Effect of interobserver variation on dose distributions of simultaneous
integrated boost (SIB) breast irradiation Delineating a boost target volume for
breast cancer is difficult and interobserver variation is a fact. We investigated how
much tumor coverage of a dose distribution planned on an initial boost planning
target volume (PTV b) changes when evaluating on a PTV b of other physicians. CTscans
of 5 patients after breast conserving surgery were used. Three physicians
delineated the tumor bed and subsequently expanded to CTV and PTV. Two
simultaneous integrated boost (SIB) IMRT planning techniques were used (PTV b
31 x 2.38Gy): SIB 1: Simultaneous inverse optimization of 2 tangential glancing open
fields combined with 2 tangential IMRT fields for the breast and 2 tangential open
fields for PTV b; SIB 2: like SIB1 but with 1 orthogonal and 2 tangential IMRT fields
for PTV b. Coverage of the PTV b used for planning was >98% in all cases. SIB 2
resulted in a much tighter dose distribution. Coverage of PTVb in the presence of
interobserver variation remains more than 90% in the majority of the cases for SIB 1.
For SIB 2 a minimum of 80% coverage is observed for most plans. From these results
we concluded that SIB 1 used at our institute is relatively robust against interobserver
variation.
CLINICAL APPLICATION OF IMAGE GUIDED RADIOTHERAPY
Anja Betgen, Eugene Damen, Marcel Van Herk, Juup Nijholt, Peter Remeijer, Maddalena Rossi,
Jan-Jakob Sonke, Jochem Wolthaus, Marieke Van Zwienen, Floris Pos, Coen Rasch, Jose Belderbos
Stereotactic body radiation therapy (SBRT) for non-small cell lung cancer
(NSCLC) patients or pulmonary metastases SBRT is characterized by the delivery
of one or a few fractions (hypofraction) with a high dose per fraction and a high
accuracy. With this treatment regime high local control rates can be reached in early
stage lung cancer patients. At the NKI-AVL, SBRT has been introduced since mid-
2005 to treat medically inoperable primary NSCLC or pulmonary metastases. We
analyzed the response rate and toxicity of SBRT from July 2005 till November 2007
in 70 patients treated for 74 tumors. All patients (median age 73 years) had
peripheral tumors outside the proximal bronchial tree (diameter ≤ 5 cm) and were
irradiated, frameless, with 3 fractions of 18 Gy (within 8-10 days). Dose prescription
was according to the RTOG 0236 protocol. Stage was T1 in 85% and T2 in 15% of the
patients. In 45% of the patients pathological proof of NSCLC was available. In 55%
of patients a growing tumor on chest X-ray or CT scan was diagnosed with high
FDG-PET uptake and a contra-indication for trans-thoracic puncture. Inoperability
was due to poor pulmonary function tests in 44% of patients, heart disease in 14%,
combination of both in 15%, previous thoracotomy in 7%, other malignancy in 8%,
poor condition in 7% and thoracotomy refusal in 5%. Patients received a 4D midventilation
CT approach in treatment planning. Prior to- and after the irradiation,
4D- cone-beam CT (4D-CBCT) was used to verify and correct patient set-up errors
and tumor baseline variation.
Acute toxicity (with a median follow-up time of 9 months) was very mild. Late toxicity
revealed radiation pneumonitis grade 2 in 8% and grade 3 in 1% of the patients.
The local and loco-regional control in our SBRT group was excellent (90%).
A B
Figure 4: A: 5/2008 central tumor right hilar side
B: 8/2008 4 weeks after completion of chemo-radiation:complete metabolic response, however uptake in the
esophagus due to radiation esophagitis

We investigated the accuracy of frameless SBRT using 4D-CBCT guidance by
determining the intra-fraction stability in 58 patients. The time from the patient
entering until leaving the treatment room was on average 50 min. The residual interfraction
group mean, systematic and random errors for all directions were < 1mm.
Even with considerable tumor motion the PTV-margins can safely be kept relatively
small allowing patients with larger tumor volumes to benefit from the advantages of
SBRT. For all patients, post treatment validation scans did not show unacceptable
deviations confirming the stability of our frameless approach.
We compared the overall survival in the SBRT treated patients with a very similar
patient group treated with conventional fractionation from our dose-escalation (DE)
trial [Belderbos et al. 2006]. The 28 patients from the DE trial were selected based on
a small Gross Tumor Volume (GTV < 65cc). These patients were irradiated to a dose
of 81-95 Gy (2.25 Gy per fraction; within 6 weeks). The 12 and 18 months overall
survival in the patients treated with SBRT (n=67) compared to the DE patients was
69% (SBRT) versus 68% (DE) and 60% (SBRT) versus 53% (DE). SBRT is a safe and
effective alternative (compared to conventional fractionated radiotherapy) for
peripheral early stage NSCLC or pulmonary metastases.
STUDIES OF RADIATION RESPONSE IN TUMORS AND
ORGANS AT RISK
Abrahum Al-Mamgani1 , Jose Belderbos, Gerard Bengua2 , Gerben Borst, M Dielwart3 ,
Michael Hauptmann4 , W Heemsbergen, Michel Van den Heuvel5 , Luca Incrocci1, M Ishikawa2 ,
Peter Levendag1 , Jasper Nijkamp, Rikaya Onimaru2 , Stephanie Peeters, Wim Van Putten1 , H Shirato2 ,
A Slot6 , Jan-Jakob Sonke, Joos Lebesque
Prostate cancer
The Dutch dose escalation randomized trial We recently updated the Dutch dose
escalation randomized trial of 68 Gy versus 78 Gy with 669 prostate cancer patients.
The primary endpoint was freedom from failure (FFF), defined as clinical or
biochemical failure (BF). Two definitions of BF were used; the ASTRO-definition
(3 consecutive rises of PSA level) and the Phoenix definition (the nadir plus 2). After
a median follow-up of 70 months, FFF (ASTRO) was significantly better in the 78-Gy
arm compared with the 68-Gy arm (7-year FFF rate is 54% vs. 47%; p= 0.04). FFF
(Phoenix) was also significantly better in the 78-Gy arm with 7-year FFF rate of 56%
vs. 45% (p = 0.03) (figure IX.5) However, no differences in freedom from clinical
failure (FFCF) and overall survival were observed. The incidence of late genitourinary
(GU) toxicity grade ≥ 2 was similar in both arms (40% vs. 41% at 7 years; p= 0.6),
while the cumulative incidence of late gastrointestinal (GI) toxicity grade ≥ 2 was
increased in the 78-Gy arm (35% vs 25% at 7 years; p= 0.04).
We further performed a subgroup analysis, in which we divided the patients into two
dose groups according to the actually given dose:

136
RADIOTHERAPY
dose radiotherapy. For patients, who received hormonal therapy, FFF was significantly
improved for the high-dose group compared to the low-dose group (67% vs. 45%,
p=0.05), indicating that high dose radiotherapy should also be given to this group of
patients.
Figure 5: Kaplan-Meier curve of 7-year rates of freedom from failure (FFF) by dose randomization defined
according to the Phoenix definition (the nadir plus 2 µg/L after RT)
The role of intensity modulated radiation therapy in reducing toxicity The
results of a subgroup of 78 patients were studied. They were treated in the NKI-AVL
to a total dose of 78 Gy with either a 3D-conformal radiotherapy technique with a
sequential boost (SEQ) or a simultaneous integrated boost using IMRT (SIB-IMRT).
The 5-year freedom from biochemical failure (Phoenix) was not worse in the SIB-
IMRT patients (70% vs. 61% for the SEQ, p = 0.3). However, there was a significant
lower incidence of acute grade ≥ 2 GI toxicity in patients treated with SIB-IMRT
compared to SEQ (20% vs. 61%, p = 0.001). For acute GU toxicity and late GI and
GU toxicity, the incidences were lower with SIB-IMRT, but these differences were not
statistically significant.
Lung
We cooperated with the Department of Radiology at the Hokkaido University School
of Medicine (Sapporo, Japan) where they have an extensive experience of treating
pulmonary lesions using Stereotactic Body Radiotherapy (SBRT). The aim of the
study was to find the relation between the incidence radiation pneumonitis (RP) and
dose volume parameters.
Linear quadratic (LQ) model and hypofractionated lung irradiation We first
evaluated the validity of the LQ model for the high doses per fraction, given in SBRT.
The lung radiation dose for 128 SBRT patients was calculated as the equivalent dose
given in fractions of 2 Gy (i.e. Normalized Total Dose), according to the LQ model
using a range of a/b values. The mean lung dose was calculated for a/b ratios of 1 Gy
(MLD 1), 2 Gy (MLD 2), 3 Gy (MLD 3), 4 Gy (MLD 4), 5 Gy (MLD 5), 7.5 Gy (MLD 7.5) 10 Gy
(MLD 10) and infinity (i.e. physical dose, MLD phys). In addition, other dose volume
parameters like the volume lung receiving more than a threshold dose of x Gy (V x)
and the MLD calculated with a modification of the LQ model (MLD LQL) were evaluated.
Strong correlations were observed between all these parameters. Fitting the Normal
Tissue Complication Probability (NTCP) model of Lyman to the observed incidence
of RP, the MLD 3 had the highest log likelihood (best NTCP fit) compared to the MLD
corrected with the other a/b ratios and the MLD LQL. However, these differences were
not statistically significant. The MLD3 was significantly better predicting RP than the
V 5, V 13 (p 0.025) and V 20 but was not significantly better the V 40.

Comparing the dose-effect relationship between the incidence of radiation
pneumonitis (RP) after SBRT and conventional fractionated radiation therapy
(CFRT) For both SBRT (n=128) and CFRT (n=142) patients, RP grade 2 or higher was
scored. The incidence of RP was 10.9 % and 17.6 % for the SBRT and CFRT patients,
respectively. The dose-response relationship for RP for SBRT was not significantly
different from the dose-response relationship for CFRT (p=0.37). No difference in
incidence in any dose range was observed between SBRT and CFRT (p>0.1).
Breast cancer
Ninja Antonini, Liesbeth Boersma1 , Eugene Damen, Paula Elkhuizen, Guus Hart, Heather Jones,
Bas Kreike, Ben Mijnheer, Nicola Russell, Laura Van ’t Veer, Marc Van de Vijver, Conny Vrieling,
Harry Bartelink
The impact of an extra dose of 16 Gy was investigated in 251 patients with involved
surgical margins in patients who underwent breast-conserving therapy and were part
of the EORTC ‘boost versus no boost’ trial. Thirty-seven patients initially relapsed
locally. At 10 years, the cumulative incidence of local recurrence was 17.5% (95% CI:
10.4-24.6%) versus 10.8% (95% CI: 5.2-16.4%) for the low and high boost dose
groups, respectively (HR=0.83, 95% CI: 0.43-1.57, p>0.1). Overall, 64 patients have
died (25.5%), 47 of them of breast cancer, without a difference in duration of survival
between the two groups (HR=0.97, 95% CI=0.59-1.5, p>0.1). In summary: we could
not demonstrate a statistically significant difference in local control or survival
between the high boost dose of 26 Gy and the low boost dose of 10 Gy in patients
with microscopically incomplete excision of early breast cancer.
In the same trial 5178 conservatively treated early breast cancer patients had a
complete excision. Previously we showed that a 16 Gy boost dose significantly
improved local control, but increased the risk of breast fibrosis. To investigate
predictors for the long-term risk of fibrosis, Cox regression models of the time to
moderate or severe fibrosis were developed on a random set of 1797 patients with
and 1827 patients without a boost, and validated in the remaining set. The median
follow-up was 10.7 years. The risk of fibrosis significantly increased (p

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RADIOTHERAPY
Publication (continued)
Meijnen P, Oldenburg HS, Peterse JL,
Bartelink H, Rutgers EJ. Clinical outcome
after selective treatment of patients
diagnosed with ductal carcinoma in situ of
the breast. Ann Surg Oncol 2008;15:235-243
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. 2008
Sep 21 (epub ahead of print)
Poortmans PM, Collette L, Horiot JC,
Van den Bogaert WF, Fourquet A, Kuten
A, Noordijk EM, Hoogenraad W,
Mirimanoff RO, Pierart M,
Van Limbergen E, Bartelink H; On behalf
of the EORTC Radiation Oncology and
Breast Cancer Groups. Impact of the boost
dose of 10Gy versus 26Gy in patients with
early stage breast cancer after a
microscopically incomplete lumpectomy:
10-year results of the randomised EORTC
boost trial. Radiother Oncol. 2008 Aug 15
(epub ahead of print)
Poortmans PM, Collette L, Bartelink H,
Struikmans H, Van den Bogaert WF,
Fourquet A, Jager JJ, Hoogenraad W,
Müller RP, Dubois JB, Bolla M,
Van Der Hulst M, Wárlám-Rodenhuis CC,
Pierart M, Horiot JC; On behalf of the
EORTC Radiation Oncology and Breast
Cancer Groups.The addition of a boost dose
on the primary tumour bed after lumpectomy
in breast conserving treatment for breast
cancer. A summary of the results of EORTC
22881-10882 ‘boost versus no boost’ trial.
Cancer Radiother 2008;12:565-570
Mechanisms, modulation and prediction of
radiation-induced cell death
Hoebers FJ, Kartachova M, de Bois J,
van den Brekel MW, van Tinteren H,
van Herk M, Rasch CR, Valdés Olmos
RA, Verheij M. 99mTc Hynic-rh-Annexin
V scintigraphy for in vivo imaging of
apoptosis in patients with head and neck
cancer treated with chemoradiotherapy. Eur
J Nucl Med Mol Imaging 2008;35:509-518
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 Cell Biology I) 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 these in vitro experiments, a proof-of-concept study was performed in a
mouse xenograft KB carcinoma model showing complete and sustained tumor
regression after Perifosine and radiation. These in vitro and in vivo observations
prompted us to conduct a clinical phase I study defining the MTD, toxicity profile
and optimal scheduling of the combined treatment of oral administration of
Perifosine and radiotherapy in patients with locally advanced inoperable solid
tumors. Next, we initiated an international multicenter placebo-controlled phase II
study in locally advanced NSCLC, randomizing 168 patients between radiation +
placebo and radiation + daily Perifosine (completed ultimo 2007). In the first 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 collaboration with different
groups we evaluate other radio-enhancing agents. In a separate project with J Borst
(Division Immunology IV), we study the interaction between death receptor ligands
(TRAIL) 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. TRAIL is of particular interest for combination therapies,
since TRAIL and radiation or chemotherapy activate partially distinct apoptosis
signaling pathways, while a molecular basis for synergy lies in the sensitization of
cells by radiation to TRAIL-induced apoptosis. 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 in
collaboration with Dr. Henning Walczak, DKFZ, Germany. 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.
In collaboration with the University of Florida and the division of Radiobiology at the
Free University of Amsterdam, we have initiated preclinical efficacy testing of (-)
Gossypol, a potent small-molecule inhibitor of Bcl-XL, Bcl-2 and Mcl-1. Gossypol
binds to the BH3 domains of these, and possibly other Bcl-2 family members, and
induces apoptosis. Because overexpression of these anti-apoptotic molecules confers
radioresistance and is often associated with poor prognosis, inhibition of Bcl-2/
Bcl-XL/Mcl-1 represents a promising strategy to increase tumor response to radiation.
In a panel of human leukemic and head and neck cancer cell lines Gossypol was
found to increase radiation-induced cell death. Currently, this combination approach
is evaluated in vivo and in a recently initiated clinical phase I/II study in locally
advanced head and neck cancer.

Another strategy that aims at enhancing the tumor response to radiation focuses on
inhibition of poly(ADP-ribose)polymerase (PARP), an important DNA repair enzyme.
APO866 is a highly specific non-competitive inhibitor of nicotinamide phosphoribosyltransferase
(NAPRT), a key enzyme in the regulation of NAD+ biosynthesis from the
natural precursor nicotiname. Inhibition of NAPRT progressively depletes NAD+.
Cells that have a high turnover of NAD+, e.g. through its cleavage by PARP, rely on
the readily available nicotinamide pathway for NAD+ synthesis and undergo apoptosis
after inhibition of NAPRT. Indeed, inhibition of NAD+ by APO866 has been reported
to induce apoptosis in human leukemic, hepatocarcinoma cells in vitro and various
types of tumor xenografts in vivo. We have studied the effect of APO866 in the human
leukemic cell lines U937 and Jurkat T and found a dose- and time-dependent induction
of cell death as measured by the MTT assay. Reduction of cellular NAD+ levels was
essential for these effects. In addition, we demonstrated the radiosensitizing effect of
nanomolar concentrations of APO866 in a clonogenic survival assay. These results
indicate that this NAD+-depleting and PARP-inhibiting compound is effective in
compromising DNA repair when used in combination with radiation.
Prediction of tumor response To evaluate whether early treatment-induced cell
death can be visualized in vivo and whether it predicts clinical outcome, we evaluated
a novel in vivo apoptosis imaging technique in collaboration with the department of
nuclear medicine (Valdés Olmos, Division XIII). This 99mTc-Annexin V scintigraphy
(TAVS) is based on the exposure of phosphatidylserine (PS) at the outer leaflet of the
plasma membrane lipid bilayer during the early phase of apoptosis. The human
endogenous protein annexin V has a high affinity for PS, providing a detection
method for apoptotic cells in vitro and in vivo. We have gained experience with this
imaging technique 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. Recently, we
examined a group of 11 patients (20 lesions) with advanced NSCLC who received
99m Tc-MIBI single photon emission computed tomography (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 99m Tc-MIBI uptake and change in tumor size (r 2 =0.53, p

140
RADIOTHERAPY
Publication (continued)
Combination of radiotherapy and
chemotherapy
Ackerstaff AH, Balm AJ, Rasch CR,
de Boer JP, Wiggenraad R, Rietveld DH,
Gregor RT, Kröger R, Hilgers FJ. First-year
quality of life assessment of an intra-arterial
(RADPLAT) versus intravenous chemoradiation
phase III trial. Head Neck. 2008
Oct 28 (epub ahead of print)
Hoebers FJ, Pameijer FA, de Bois J,
Heemsbergen W, Balm AJ, Schornagel JH,
Rasch CR. Prognostic value of primary
tumor volume after concurrent
chemoradiation with daily low-dose
cisplatin for advanced-stage head and neck
carcinoma. Head Neck 2008;30:1216-23
Jansen EPM, Verheij M. Radiotherapy of
gastric cancer. In: Gastrointestinal
Oncology: A Critical Multidisciplinary
Approach. Editors: J Jankowski,
R Sampliner, D Kerr, Y Fong, Blackwell
Publishing 2008;164-166
Topolnjak R, van Vliet-Vroegindeweij C,
Sonke JJ, Minkema D, Remeijer P,
Nijkamp J, Elkhuizen P, Rasch C. Breast-
Conserving Therapy: Radiotherapy Margins
for Breast Tumor Bed Boost. Int J Radiat
Oncol Biol Phys. 2008;72:941-948
Verheij M. The role of radiotherapy in the
treatment of gastric cancer. Asian Hospital
& Healthcare Management 2008;17:44-46
Verheij M, Bartelink H.
Chemoradiotherapy. In: Encyclopedia of
Cancer 2008. Editor M Schwab
Zuur CL, Simis YJ, Verkaik RS,
Schornagel JH, Balm AJ, Dreschler WA,
Rasch CR. Hearing loss due to concurrent
daily low-dose cisplatin chemoradiation for
locally advanced head and neck cancer.
Radiother Oncol 2008;89:38-43
COMBINATION OF RADIOTHERAPY AND CHEMOTHERAPY
Berthe Aleman, Harry Bartelink, Jose Belderbos, Henk Boot, Annemieke Cats, Otilia Dalesio,
Luc Dewit, Michel Van Den Heuvel, Edwin Jansen, Corrie Marijnen, Maurits Swellengrebel,
Renato Valdes Olmos, Coen Rasch, Marcel Verheij
Gastroenterology – gastric cancer Combined radiotherapy and chemotherapy
(CRT) has improved treatment outcome and organ preservation in a growing number
of solid tumors (uterine cervix, lung, esophagus, head and neck, anorectal). More
recently, 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 collaboration with our department of Gastroenterology
and the Christie Hospital in Manchester, UK. 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/m2 for cisplatin and 650
mg/m2 bid for capecitabine. Very recently we analyzed cisplatin in a weekly schedule
in this combination. The maximum tolerated dose (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/m 2 the
MTD was not reached. Until now, we have treated over 170 gastric cancer patients
with CRT. In the first 91 patients that had an adequate follow up in only 16% in-field
recurrences were found, which compares favorably with historical surgical series.
Furthermore, with long-term follow up using serial renography to monitor kidney
function, a 27% and 52% decrease in left renal function is 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; cone beam CT scan on the linear accelerator) in the postoperative treatment
of gastric cancer to minimize normal tissue toxicity. We have started accrual in a large
international phase III study (CRITICS) which has been granted by the Dutch Cancer
Society (CKTO), 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 100 patients have been randomized.
In addition Sweden has joined the trial. 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 We retrospectively evaluated acute and perioperative
toxicity and efficacy of preoperative chemoradiotherapy using the oral 5-FU
prodrug capecitabine in locally advanced rectal cancer. Between June 2004 and
February 2008, 147 patients with T3-4/N2 rectal cancer were treated with radiation
(25x2 Gy) in combination with capecitabine (825 mg/m 2 , day 1-33). Surgery was
performed approximately 6 weeks later in one of 11 regional hospitals. The population
consisted of 86 males and 61 females, with a median age of 63 years. Dosis reduction
of chemotherapy occurred in 12% and 4% did not complete all radiotherapy fractions.
Grade 3-5 toxicity was seen in 22.4%; 1 patient died due to pancytopenia and mucositis.
Fifteen patients (10%) developed grade 3 diarrhoea and 17 patients (12%) grade 3-4
radiation dermatitis. Of the 147 patients, 138 underwent a laparotomy, while resection
was performed in 131 patients. 62 patients underwent an abdominal perineal
resection (APR), 47 a low anterior resection (LAR) and 22 patients a Hartmann’s
procedure. Extra visceral resection was necessary in 15 of these patients (11%).
Anastomotic leakage was seen in 12 of 47 patients (26%) who underwent a LAR,
while perineal wound complications developed in 20 of 62 perineal resection
patients (33%). In 21% of all operated patients re-operation was necessary while
1patient died due to postoperative complications (1%). A pathological complete
response was achieved in 15 patients (11%). In 6 patients (5%, 2 LAR and 4 APR) a
positive circumferential resection margin was found. All exenterations were microscopically
radical. These results demonstrate that preoperative chemo radio therapy
with oral capecitabine is an effective treatment in locally advanced rectal cancer,
associated with an acceptable acute but significant peri-operative morbidity.

NSCLC Combining concurrent chemotherapy with radiotherapy has been a step
forward in the treatment of locally advanced NSCLC. Recently a meta-analysis based
on individual patient data was performed to compare sequential and concurrent
chemo-radiation (CRT) in inoperable NSCLC patients. The patients included in
EORTC study 08972 and M03IVC were included in this analysis. In 1301 randomized
patients the 3 year survival was 24.8% for patients treated with concurrent chemoradiotherapy
versus 18.2% in sequentially treated patients (p = 0.0026).The locoregional
control was significantly better in the concurrent arm (HR 0,76, 95% CI
[0,62-0,94], p = 0,011) while the chance to develop distant metastases was not
different in both arms.
Despite this improvement after concurrent chemo-radiotherapy, prognosis remains
poor emphasizing the need for more effective strategies. The epidermal growth factor
receptor monoclonal antibody (Cetuximab) has been shown to be an active agent in
metastasized NSCLC. We therefore tested the feasibility of combining Cetuximab
with concurrent CRT. In a phase I trial In patients presenting with inoperable locally
advanced NSCLC Cetuximab (loading dose 400 mg/m 2 on day 1, followed by a weekly
dose of 250 mg/m 2 ) was added to our chemo-radiotherapy regimen (66 Gy in
24 fractions and daily dose Cisplatin 6 mg/m 2 ) . Between April and July 2008,
12 patients were treated within this trial. The addition of Cetuximab to our concurrent
chemo-radiotherapy with daily dose Cisplatin was well tolerated with dermatitis and
radiation esophagitis being the most common side effects (maximum score 3 according
to CTC Version 3.0). No unexpected toxicity occurred. Early response monitoring,
using a repeat PET-CT scan, 4 weeks after the last dose of radiotherapy was done in
10 patients and showed a metabolic response in 5 patents (3 complete and 2 partial
metabolic responses) and one patient with progressive disease. We will continue in
the near future with a multi-centre phase II trial randomizing patients to concurrent
chemo-radiation with or without Cetuximab.
Publication (continued)
Other
141
RADIOTHERAPY
Bartelink H, Begg AC. Prognostic and
predictive markers in oncology.
Introduction. Semin Radiat Oncol
2008;18:73-74
Bossema ER, Marijnen CA,
Baas-Thijssen MC, van de Velde CJ,
Stiggelbout AM. Evaluation of the
Treatment Tradeoff Method in Rectal
Cancer Patients: Is Surgery Preference
Related to Outcome Utilities? Med Decis
Making 2008;28:888-898
Broeks A, Braaf LM, Huseinovic A,
Schmidt MK, Russell NS, van Leeuwen FE,
Hogervorst FB, Van ‘t Veer LJ. The
spectrum of ATM missense variants and
their contribution to contralateral breast
cancer. Breast Cancer Res Treat
2008;107:243-248
de Boer JP, Hiddink RF, Raderer M,
Antonini N, Aleman BM, Boot H,
de Jong D. Dissemination patterns in nongastric
MALT lymphoma. Haematologica
2008;93:201-206
de Bruin EC, van de Velde CJ,
van Krieken JH, Marijnen CA, Medema JP.
Epithelial human leukocyte antigen-DR
expression predicts reduced recurrence rates
and prolonged survival in rectal cancer
patients. Clin Cancer Res 2008;14:1073-1079
De Bruin ML, Huisbrink J, Hauptmann M,
Kuenen MA, Ouwens GM, van ‘t Veer MB,
Aleman BM, van Leeuwen FE. Treatment-
related risk factors for premature menopause
following Hodgkin lymphoma. Blood
2008;111:101-108
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. 2008 Sep 26.
(epub ahead of print)
de Vreeze RS, Koops W, Haas RL,
van Coevorden F. An unusual case of
hemosiderotic fibrohistiocytic lipomatous
lesion: correlation of MRI and pathologic
findings. Sarcoma 2008;2008:893918

142
RADIOTHERAPY
Publication (continued)
de Vreeze RS, 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;72:1480-1487
den Dulk M, Krijnen P, Marijnen CA,
Rutten HJ, van de Poll-Franse LV, Putter H,
Meershoek-Klein Kranenbarg E,
Jansen-Landheer ML, Coebergh JW,
van de Velde CJ. Improved overall survival
for patients with rectal cancer since 1990:
the effects of TME surgery and pre-operative
radiotherapy. Eur J Cancer 2008;44:
1710-1716
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. 2008 Oct 16 (epub
ahead of print)
Hooning MJ, Aleman BM, Hauptmann M,
Baaijens MH, Klijn JG, Noyon R,
Stovall M, van Leeuwen FE. Roles of
radiotherapy and chemotherapy in the
development of contralateral breast cancer.
J Clin Oncol 2008;26:5561-5568
Hoving S, Heeneman S, Gijbels MJ,
te Poele JA, Russell NS, Daemen MJ,
Stewart FA. Single-dose and fractionated
irradiation promote initiation and
progression of atherosclerosis and induce
an inflammatory plaque phenotype in
ApoE(-/-) mice. Int J Radiat Oncol Biol
Phys 2008;71:848-857
Kappers I, Klomp HM, Burgers JA,
van Zandwijk N, Haas RL, van Pel R.
Neoadjuvant (induction) erlotinib response
in stage IIIA non-small-cell lung cancer.
J Clin Oncol 2008;26:4205-4207
Kunkler IH, Canney P, van Tienhoven G,
Russell NS; MRC/EORTC (BIG 2-04)
SUPREMO Trial Management Group.
Elucidating the role of chest wall irradiation
in ‘intermediate-risk’ breast cancer: the
MRC/EORTC SUPREMO trial. Clin
Oncol (R Coll Radiol) 2008;20:31-34
Lange MM, Maas CP, Marijnen CA, Wiggers T,
Rutten HJ, Kranenbarg EK, van de Velde CJ;
Cooperative Clinical Investigators of the Dutch
Total Mesorectal Excision Trial. Urinary
dysfunction after rectal cancer treatment is
mainly caused by surgery. Br J Surg
2008;95:1020-1028
Messick TE, Russell NS, Iwata AJ, Sarachan KL,
Shiekhattar R, Shanks JR, Reyes-Turcu FE,
Wilkinson KD, Marmorstein R. Structural basis
for ubiquitin recognition by the Otu1 ovarian
tumor domain protein. J Biol Chem
2008;283:11038-11049
Nijkamp J, Pos FJ, Nuver TT, de Jong R,
Remeijer P, Sonke JJ, Lebesque JV. Adaptive
radiotherapy for prostate cancer using kilovoltage
cone-beam computed tomography: first clinical
results. Int J Radiat Oncol Biol Phys
2008;70:75-82
Pieterse AH, Baas-Thijssen MC, Marijnen CA,
Stiggelbout AM. Clinician and cancer patient
views on patient participation in treatment
decision-making: a quantitative and qualitative
exploration. Br J Cancer 2008;99:875-882
Rutten HJ, den Dulk M, Lemmens VE,
van de Velde CJ, Marijnen CA. Controversies of
total mesorectal excision for rectal cancer in
elderly patients. Lancet Oncol 2008;9:494-501
Steggerda MJ, van der Poel HG, Moonen LM.
An analysis of the relation between physical
characteristics of prostate I-125 seed implants
and lower urinary tract symptoms: bladder
hotspot dose and prostate size are significant
predictors. Radiother Oncol 2008;88:108-114
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. Primary
systemic therapy in patients with operable
primary breast cancer: more benefits than breastconserving
therapy. Ned Tijdschr Geneeskd.
2008;152:2519-25
Thomas G, Ali S, Hoebers FJ, Darcy KM,
Rodgers WH, Patel M, Abulafia O, Lucci JA
3rd, Begg AC. Phase III trial to evaluate the
efficacy of maintaining hemoglobin levels above
12.0 g/dL with erythropoietin vs above 10.0 g/dL
without erythropoietin in anemic patients
receiving concurrent radiation and cisplatin for
cervical cancer. Gynecol Oncol 2008;108:317-325
van Sandick JW, Kappers I, Baas P, Haas RL,
Klomp HM. Surgical treatment in the
management of malignant pleural
mesothelioma: a single institution’s experience.
Ann Surg Oncol 2008;15:1757-1764
Voskuil DW, Vrieling A, Korse CM, Beijnen JH,
Bonfrer JM, van Doorn J, Kaas R,
Oldenburg HS, Russell NS, Rutgers EJ,
Verhoef S, van Leeuwen FE, van ‘t Veer LJ,
Rookus MA. Effects of lycopene on the insulinlike
growth factor (IGF) system in
premenopausal breast cancer survivors and
women at high familial breast cancer risk. Nutr
Cancer 2008;60:342-353
Vrieling C, de Jong D, Boot H, de Boer JP,
Wegman F, Aleman BM. Long-term results of
stomach-conserving therapy in gastric MALT
lymphoma. Radiother Oncol 2008;87:405-411
Weichselbaum RR, Ishwaran H, Yoon T,
Nuyten DS, Baker SW, Khodarev N et al.
An interferon-related gene signature for DNA
damage resistance is a predictive marker for
chemotherapy and radiation for breast cancer.
Proc Natl Acad Sci USA 2008;105:18490-18495
Weigelt B, Kreike B, Reis-Filho JS. Metaplastic
breast carcinomas are basal-like breast cancers:
a genomic profiling analysis. Breast Cancer Res
Treat 2008,September 25 (epub ahead of print)
Wildiers H, Kunkler I, Biganzoli L, Fracheboud J,
Vlastos G, Bernard-Marty C, Hurria A,
Extermann M, Girre V, Brain E, Audisio RA,
Bartelink H, Barton M, Giordano SH, Muss H,
Aapro M; International Society of Geriatric
Oncology. Management of breast cancer in
elderly individuals: recommendations of the
International Society of Geriatric Oncology.
Lancet Oncol. 2007;8:1101-115 (review)
Thesis
Heemsbergen WD. Radiation treatment in
prostate cancer: Balancing between tumor
control and toxicity. University of Amsterdam,
Amsterdam 2008

DIVISION OF SURGICAL ONCOLOGY
GYNAECOLOGY
Marc van Beurden, Willemien van Driel, Lotti Lubsen-Brandsma, Jan Lange, Manon van Seters
The department focuses on treatment and screening for ovarian cancer, on
immunology and treatment for (premalignant) vulvar neoplasms and on the
development of new treatment for premature menopausal symptoms.
Ovary A randomized phase III clinical trial for stage III ovarian carcinoma has started
investigating the effect of secondary debulking surgery with or without hyperthermic
intraperitoneal cisplatinum. This multicenter study started inclusion in march 2007
and currently 5 centers in the Netherlands are participating. Within short time it is
expected that this will expand with 4 additional centers in Belgium and France.
During interval debulking patients are randomized between interval debulking alone
or adding infusion of cisplatinum under hyperthermic condition at the end of surgery.
Primary endpoint of this study is progression free survival (KWF CKTO2006-16).
Participation in a randomised, multicenter, phase III study of erlotinib versus
observation in patients with no evidence of disease progression after first line,
platinum-based chemotherapy for high-risk stage I and stage II-IV ovarian epithelial,
primary peritoneal, or fallopian tube cancer is finished in 2008 and results are
expected in 2010. Primary endpoint will be progression free survival; secondary
endpoint will be overall survival, safety and quality of life (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.
285 of the 325 patients needed are randomized (KWF NKI 2006-3470).
Vulva Participation in the GROINSS-V II study, an international multicenter
observational study on patients with vulvar cancer during which patients with
positive lymph nodes are treated with radiation instead of inguinofemoral
lymphadenectomy is ongoing. 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 nonblinded,
randomized controlled trial (M06PRO Kousen study). This study is finished
in September 2008 and results will be available in 2009.
The efficacy of a immune respons modifier, imiquimod 5% cream, has been
inevstigated in a randomized placebo controlled trial in patients with VIN. Thirty-five
percent obtained a clinical and histological complete remission. There was a strong
association between viral clearance and histologisc regression (P

144
SURGICAL ONCOLOGY
Adriaan Timmers DDS Academic staff
Corina Van As-Brooks PhD Academic staff
Michiel Van den Brekel MD PhD
Academic staff
Joost van Tongeren MD Resident
C Verhagen MD Research physician
Karel Zuur MD Reseach physician
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
Joris Leijte Research physician
Niel Joshi MD Fellow
Niels Graafland Research physician
Gynaecology
Marc Van Beurden MD PhD Academic staff
Willemien Van Driel MD PhD Academic staff
Jan Lange MD Academic staff
Lotti Lubsen–Brandsma MD Academic staff
Manon van Seters MD Research fellow
Plastic and Reconstructive Surgery
Joris Hage MD PhD Academic staff (head)
Brigit Drost MD Academic staff
Leonie Woerdeman MD PhD Academic staff
Marieke Van der Berg MD Academic staff
Martine Van Huizum MD Academic staff
Kalam Ahmed MD Research fellow
Esther Moerman MD Fellow
Jan Kortmann MD Research fellow
Mona Mazgani MD Fellow
Tomas Bulut Research fellow
Wouter Van der Pot MD Fellow
Anesthesiology
Peter Schutte MD Academic staff (head)
Dirk Buitelaar MD Academic staff
Katina Efthymiou MD Academic Staff
Christiaan Keijzer MD PhD Academic staff
May Ronday MD Academic staff
Michael Srámek MD PhD Academic staff
Tino Stoppa MD Academic staff
Julia Ten Cate MD Academic staff
Ingeborg Vergouwe MD Academic staff
Dermatology
Wietze Van der Veen MD PhD Academic
staff (head)
Inka Nieuweboer-Krobotova MD
Academic staff
Sanne Vos MD Academic staff
Biljana Zupan-Kajcovski MD Academic staff
GASTRO-INETSTINAL CANCER
Theo Ruers, Frits van Coevorden, Vic Verwaal, Johanna van Sandick, Marie-Jeanne Vrancken Peeters,
Ewout Courrech Staal, Sjoerd Bruin
Oesophageal cancer In January 2008, Ewout Courrech Staal started as a full-time
research physician on a thesis-project ‘Multimodality treatment of oesophageal
cancer’. A retrospective database was constructed including 1499 patients who
presented at the NKI-AVL between 1995-2007 with primary, recurrent or metastatic
oesophageal cancer. Of the 87 patients who were operated, 56 patients received neoadjuvant
treatment. A microscopic radical resection was achieved in 96%. Pathologic
response was complete in 25%. Postoperative mortality was 1%, and 5-year overall
survival rate 42%. Thus, results of low-volume oesophageal cancer surgery in our
institute meet high-quality standards. In an other study, different regimens of
concurrent chemoradiotherapy were studied. Overall, tolerance was acceptable
(77 of 94 patients [82%] completed the planned regimen). In September 2008,
a prospective study was initiated to investigate whether gene expression and/or
serum protein profiling can improve individual treatment planning in patients with
non-metastatic oesophageal cancer.
Gastric cancer Participation in the so-called CRITICS study (A multicenter
randomized phase III trial of neo-adjuvant chemotherapy followed by surgery and
chemotherapy or by surgery and chemoradiotherapy in resectable gastric cancer) has
led to an increasing number of patients referred for gastric surgery.
Colorectal cancer The CGH analysis of colorectal tumors with metastasis to liver
peritoneum and colorectal tumor without metastasis showed a loss in chromosome
20q in those patients who have liver metastasis while there was no loss in chromosome
20q in patients without liver metastasis. The genes associated with preferred metastasis
to the liver will further be investigated.
Furthermore new grading of psuedomyxoma peritonei was established by connecting
the results of pathologic characteristics like mitotic activity, atypia and amount of
mucous to survival. The results showed that histology with > 90 % mucous, limited
mitotic activity and no mitotic activity were related to superior outcome, all other
patients should be regarded as metastasized colorectal cancer. These results sharpen
the indication of cytoreduction and HIPEC.
The randomized trial comparing cytoreduction followed by HIPEC to systemic
chemotherapy in patients with peritoneal metastasis of colorectal origin was updated,
showing a 45% 5-year survival in those selected patients who underwent a complete
cytoreduction.
Interim results of a randomized multicentre European study investigating the effect
of radiofrequency ablation (RFA) for unresectable colorectal liver metastases
(EORTC 40004) were presented at ASCO this year. This study, under guidance of
dr T. Ruers, showed a superior disease free survival after combined treatment of RFA
plus chemotherapy versus chemotherapy alone (median DFS 17 mo versus 10 mo).
A number of new studies are started. These studies are: in vivo scanning with radioactive
labeled bevacuzimab, accelerated growth of synchronous colorectal liver metastasis,
tumor destruction and dendritic cell activation in situ leading towards in vivo loaded
DC vaccines.
BREAST CANCER
Emiel Rutgers, Hester Oldenburg, Marie-Jeanne Vrancken Peeters, Omgo Nieweg, Marieke Straver,
Jelle Wesseling, Hans Peterse, Claudette Loo, Leonie Woerdeman, Reinie Kaas, Philip Meijnen
Neomamma data base Over the past 7 years we have treated 329 patients with
breast cancer according to different regimes of primary systemic therapy (PST).
We have focused on the axillary response. An axillary pCR was seen in 20%, thus
residual axillary disease was seen in 80% of the patients that underwent ALND.
Axillary pCR was higher in patients with a positive sentinel node, namely 32%.
Multivariate analysis identified two predictors of axillary pCR: subtype according to
receptor-status and response of the primary breast tumor.

Future plans are to optimize response monitoring by using PET-CT scan in addition
to MRI.
New localization method for breast tumors This year we have introduced the
radioactive jodium seed to localize both areas of DCIS and carcinoma in the breast.
In a pilot study of 10 patients we have shown that this is a safe procedure; average
migration of the seed was 0.62 mm (SD 0.43mm) with a minimum of 0.18 mm and
a maximum migration of 1.3 mm.
Future plans are to use radioactive seed localization in both the breast and axilla for
surgery in the context of PST.
Prophylactic mastectomy in BRCA 1-2 mutation carriers 260 BRCA1/2 carriers
had uni- (n = 115) or bilateral (n = 145) prophylactic mastectomy. In 12 carriers DCIS
was diagnosed and in one a small invasive breast cancer. Afterwards in one carrier a
breast cancer was detected. During follow up (average 5 years), only in one patient
after contralateral prophylactic mastectomy, a cancer was foud in the axilla.
Prophylactic mastectomy is highly effective in preventing breast cancer in women at
very high risk, so continued surveillance is not warranted.
Effectiveness of cognitive behavioural therapy and physical exercise on
climacteric symptoms in women with breast cancer experiencing treamentinduced
menopause (EVA project) Patient accrual and data collection for this
randomized multicenter study started, as planned, in January 2008. To date,
268 women have been randomly allocated to the CBT group, (n=69), the physical
exercise group (n=65), the combined intervention group (n=67), or to the control
group (n=67). Patient recruitment, intervention, and data collection (including
follow-up) will continue until approximately medio 2010.
LYMPHATIC MAPPING
Omgo Nieweg, Iris Van der Ploeg, Maartje Van Rijk, Niels Graafland, Joost Leijte, Pieter Tanis,
Renato Valdés Olmos, Emiel Rutgers, Simon Horenblas, Michiel Van den Brekel, Cornelis Hoefnagel,
Bin Kroon
The aims of our research program are to outline indications and contra-indications,
to refine the technique of lymphoscintigraphy, of surgery and pathology, and to
determine implications in terms of more individualized treatment, survival and
regional tumor control. Reduction of the extent of the operations was a theme of
research in 2008.
Sentinel node biopsy became standard of care in breast cancer patients before
consensus on the technique was reached and without randomized studies having
shown a similar or decreased axillary recurrence rate. A systematic review and metaanalysis
of the literature was performed of 48 studies concerning 14,959 patients
with a tumour-negative sentinel node biopsy and no subsequent axillary node
dissection. With a median follow-up of 34 months, the axillary recurrence rate was
found to be 0.3%, which is well within the desired range. This percentage was 0.25 in
our own patient population.
The extent of a groin dissection in melanoma patients in whom sentinel node biopsy
reveals metastasis is controversial. The high quality lymphoscintigrams at the
Netherlands Cancer Institute enable the extent of a completion groin lymph node
dissection to be determined by the location of the higher-tier nodes. Eighteen patients
had femoro-inguinal second-echelon nodes on their lymphoscintigram and
underwent a superficial lymph node dissection only. The median follow-up duration
was 61 months. One of the eighteen patients developed a deep (obturator) lymph
node recurrence after twelve months. Revision of the lymphoscintigram showed that
the images had been interpreted incorrectly and that the second-echelon node was
located in the obturator area after all. This result suggests that the strategy to
determine the extent of the groin dissection based on the location of the second-tier
nodes is valid.
Publications
145
SURGICAL ONCOLOGY
Balm AJM, Smeele LE, Lohuis PJ.
Optimizing exposure of the posterolateral
maxillary and pterygoid region: the lower
cheek flap. Eur J Surg Oncol. 2008,34:
699-703
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. 2008 Jul 8.
Bex A, Haanen JB, Vyth-Dreese FA,
Horenblas S, De Gast GC. Cytokine
therapy response as a selection criterion for
cytoreductive nephrectomy in metastatic
renal clear-cell carcinoma of intermediate
prognosis. Results and conclusions from a
combined analysis. Urol Int. 2008;80:367-71
Bex A. Review: treatment of small cell
carcinoma of the urinary bladder: can we
learn from small cell lung cancer? Clin Adv
Hematol Oncol. 2008 May;6:385-6. Review.
No abstract available
Bleeker MC, Heideman DA, Snijders PJ,
Horenblas S, Dillner J, Meijer CJ. Penile
cancer: epidemiology, pathogenesis and
prevention. World J Urol. 2008 Jul 8
Borgemeester MC, van den Brekel MWM,
van Tinteren H, Smeele LE, Pameijer FA,
van Velthuysen ML, Balm AJM.
Ultrasound-guided aspiration cytology for
the assessment of the clinically N0 neck:
factors influencing its accuracy. Head Neck
2008;30:1505-1513
Bueno-de-Mesquita JM, Linn SC, Keijzer R,
Wesseling J, Nuyten DS, van Krimpen C,
et al. Validation of 70-gene prognosis
signature in node-negative breast cancer.
Breast Cancer Res Treat 2008 Sep 26
Buitelaar D, Huitink J, Oldenburg H,
Rutgers E, Schutte P, van Tinteren H. Field
Block: an additional technique of potential
value for breast surgery under general
anaesthesia. Eur J Anaesthesiology
2008;25:253-255
Buitelaar D, Huitink J, Oldenburg H,
Rutgers E, Schutte P, van Tinteren H.
Field block: an additional technique of
potential value for breast surgery under
general anaesthesia. Eur J Anaesthesiol
2008;25:253-5

146
SURGICAL ONCOLOGY
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 supraregional
tumor positive lymph nodes
only. Eur J Surg Oncol. 2008 Aug 20.
Dirven R, Hilgers FJM, Plooij JM, Maal TJJ,
Bergé SJ, Verkerke GJ, Marres HAM. 3D
Stereophotogrammetry for the assessment of
tracheostoma anatomy. Acta Otolaryngol.
2008;128:1248-1254
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. 2008 Oct 28. [Epub
ahead of print]
Hage JJ, Woerdeman LAE. The design of
the lateral thoracodorsal flap. Ann Plast
Surg 2008;61:302-305
Hage JJ. Heraklas on knots - Sixteen
surgical nooses and knots from the first
century A.D. World J Surg 2008;32:
648-655
Hennink S, Wouters MW, Klomp HM,
Baas P. Necrotizing pneumonitis caused by
postoperative pulmonary torsion. Interact
Cardiovasc Thorac Surg. 2008;7:144-5
Hilgers FJM, Soolsma J, van den Brekel MWM,
Ackerstaff AH, Balm AJM, Tan IB.
A thin tracheal silicone washer solving
periprosthetic leakage in laryngectomees;
direct results and long-term clinical effects.
Laryngoscope 2008;118:640-645
Hilgers FJM, van den Brekel MWM,
van Tinteren H. Views on Acton el al.
‘Investigation of tracheoesophageal voice
prosthesis leakage patterns: Patient’s selfreport
versus clinician’s confirmation’, Head
Neck 2008;30:618-621. Letter to the editor.
Head Neck 2008;30:161660-1661
Ho VK, van der Heiden-van der Loo,
Rutgers EJ, van Diest PJ, Hobbelink MG,
Tjan-Heijnen VC, et al. Implementation of
sentinel node biopsy in breast cancer
patients in the Netherlands. Eur J Cancer
2008;44:683-91
THORACIC CANCER SURGERY
Houke Klomp, Ingrid Kappers, Rachel Numan, Lotte Nieuwenhuis, Jeffrey Schaap, Michel Wouters,
Johanna van Sandick
Clinical research was focused on neoadjuvant treatment with EGFR tyrosinekinase
inhibitors in operable non-small cell lung cancer (NSCLC), optimalization of
treatment strategies in locally advanced NSCLC and in thoracic sarcomas,
multimodality treatment for pleural mesothelioma, local treatment for oligometastatic
disease and introduction and evaluation of a tailored care path in thoracic surgery.
Preoperative EGFR tyrosinekinase inhibitor treatment in selected patients with
NSCLC (females, non-smokers, adenocarcinoma) proved to be feasible. In a pilot
study, (near) complete responses were observed within 3 weeks. This study will
continue in unselected patients and further tests will be performed to validate
pathologic tumour response.
In locally advanced stage IIIA-N2 NSCLC, the role of surgery is a hot topic.
Evaluation of induction chemotherapy followed by either surgical resection or
radiotherapy, showed that outcome was favourable after lobectomy, but not after
pneumonectomy (5-yr survival 43% versus 16%, 5-yr survival after radiotherapy 16%).
In malignant pleural mesothelioma, introduction of neoadjuvant chemotherapy
(followed by extra-pleural pneumonectomy and hemithoracic radiotherapy) showed
disappointing results as to (local) disease control.
Evaluation of all resections for chest wall sarcomas in the NKI and DDHK from
1980-2006 showed that full-thickness surgical resection is a safe and effective
procedure, even for recurrent chest wall sarcoma. Favourable overall survival
supports repeated attempts to obtain local control.
In patients with limited pulmonary metastatic disease, increased use of minimally
invasive treatment proved to be safe in terms of procedure-related events and did not
significantly affect disease control rates.
Base line data from the prospective evaluation of clinical recovery after thoracic surgery
showed a strong association of experienced pain during the first postoperative days
and length of hospital stay as well as long-term quality of life. This is one of many
items to be re-evaluated after adjustment of the thoracic surgery care path.
SARCOMA
Frits van Coevorden, Ronald de Vreeze, Houke Klomp, Rick Haas, Luc Dewit, Martijn Kerst,
Daphne de Jong, Hester van Boven, Petra Nederlof, Joris Hage, Gerard Schaap (AMC),
Frank de Nies (OLVG)
Clinical aspects and development of mixed-type liposarcoma: A radiological,
histological, immunohistochemical and molecular biological analysis The WHO
divides liposarcoma in the morphological subtypes well-differentiated, dedifferentiated,
myxoid, round cell, and pleomorphic. Recent cytogenetic studies
found two alterations. Where well-differentiated and de-differentiated liposarcoma is
characterized by amplification, myxoid and round cell liposarcoma is characterized
by a translocation. In this study we analyzed mixed-type liposarcomas which show a
radiological and histomorphological combined pattern of these two subgroups.
Preoperative radiotherapy, shown in the Canadian randomized trial, to have a
comparable local control to postoperative radiotherapy, can be considered in
radiosensitive tumors as myxoid/roundcell liposarcoma, whereas in well
differentiated liposarcoma radiotherapy will only be considered in recurrent tumors..
Differentiation between subtypes is important to indicate prognosis and choose the
appropriate treatment. In this study we found that when MRI images suggest a
mixed-type liposarcoma, biopsies for immunohistochemical and molecular biological
analyses are needed to characterize these tumors in order to choose the appropriate
treatment. The heterogeneity in these mixed liposarcomas is probably explained by
different maturation levels within a single developmental pathway from an
undifferentiated stem cell.

Multifocal myxoid liposarcoma; metastasis or second primary? A molecular biological
analysis. For details of this study see contribution of the DOD.
HEAD AND NECK ONCOLOGY AND SURGERY
Fons Balm, Michiel van den Brekel, Marcel Copper, Frans Hilgers, Martin Klop, Peter Lohuis,
Ludi Smeele, Bing Tan, Annemieke Ackerstaff, Adrian Begg, Guido van den Broek, Jan Paul de Boer,
O Hamming-Vrieze, Frank Hoebers, Saar Muller, Coen Rasch, Tom Geurts, Boudewijn Plaat,
Renske Scheenstra, Margot Tesselaar, Louise van Velthuysen
Pulmonary rehabilitation after total laryngectomy The short-term effect of three
different HME devices on endotracheal temperature and humidity was tested in a
randomized cross-over study in 14 laryngectomized individuals. The endotracheal
temperature and humidity were measured without HME, with the ‘Normal, the
‘HiFlow’ and the ‘Micron HME’ (with integrated bacterial filter).The absolute
humidity during maximal inspiration without HME was 17.4 mg/l (mean inhalation
breath length 1.3 sec). Breathing with Normal HME, HiFlow HME and Micron HME
increased the humidity values with 5.5 mg/l (P

148
SURGICAL ONCOLOGY
Publications (continued)
Kaas R, Muller SH, Hart AA, Rutgers EJ.
Stage of breast cancers found during the
surveillance of women with a familial or
hereditary risk. Eur J Surg Oncol
2008;34:501-7
Kaas R, Rutgers EJ. [Systematic breast selfexamination
is not a useful screening
procedure, except in hereditary or familial
increased risk of breast cancer]. Ned
Tijdschr Geneeskd 200825;152:2317-8
Kappers I, Belderbos JS, Burgers JA,
van Zandwijk N, Groen HJ, Klomp HM.
Non-small cell lung carcinoma of the
superior sulcus: favourable outcomes of
combined modality treatment in carefully
selected patients. Lung Cancer. 2008
Mar;59:385-90.
Kappers I, Klomp HM, Burgers JA,
Van Zandwijk N, Haas RL, van Pel R.
Neoadjuvant (induction) erlotinib response
in stage IIIA non-small-cell lung cancer.
J Clin Oncol. 2008;26:4205-7
Karim RB, de Lint CA, van Galen SR,
van Rozelaar L, Nieuwkerk PT,
Askarizadeh E, Hage JJ. Long-term effect of
polyalkylimide gel injections on severity of
facial lipoatrophy and quality of life of
HIV-positive patients. Aesth Plast Surg
2008;32:873-878
Keijzer C, Perez RS, de Lange JJ.
Inspiratory Carbon Monoxide and
Compound A Concentrations During
Desflurane and Sevoflurane Anesthesia in
Humans: An Observational Study. The
Open Anesthesiology Journal 2008;2:26-29
Kerst JM, Pos FJ, Visser O, Horenblas S.
[New developments in the treatment of
muscle-invasive bladder cancer] Ned
Tijdschr Geneeskd. 2008 Jan 26;152:187-92.
Review. Dutch
Kroon BBR, Hoefnagel CA,
Valdés Olmos RA, Nieweg OE. Regionale
lymfekliergebieden op afstand. Ned Tijdschr
Geneeskd 2008;152:1997-2000
Kroon BBR, Noorda EM, Vrouenraets BC,
Van Slooten GW, Nieweg OE. Isolated
limb perfusion for melanoma. Surg Oncol
Clin N Am. 2008;17:785-794.
6,2 years), who underwent a selective or (modified) radical SalvND (only) for
suspected regional residual or recurrent disease after Cisplatin containing concurrent
chemoradiation (70 Gy). Cisplatin regimen: intra-arterially 4x150mg/m 2 (n=21) or
3x100mg/ m2 i.v. (n=15) and 6mg/m 2 i.v. (n=14). Postoperative complications
occurred in 25 patients and were mostly wound related (38%): infection in 28%,
delayed wound healing in 10%. Swallowing deteriorated in 12% after SalvND. In
patients with complications, surgery was more extensive (in duration and number of
dissected levels) and peroperative blood loss was more pronounced (p

UROLOGIC ONCOLOGY
Simon Horenblas, Wim Meinhardt, Axel Bex, Henk Van der Poel
Research in urologic oncology has been centered around the following themes:
Improved staging of urologic tumors, organ and function sparing, fundamental
research in prostate, kidney and penis cancer, improved quality control of treatment
results.
Improved staging of urologic tumors Research has been focussed on the role of
sentinel node biopsy in prostate, testicular and penile cancer. In all above mentioned
tumors, 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 experience with penile cancer,
where the false negative rate dropped from 20% to an acceptable 5.1%, led to a prize
winning publication. New aspects of research in sentinel nodes has been the use of
SPECT /CT scans and intra-operative imaging with a mobile gamma camera
(Sentinella®). These adjuncts have been able to increase accuracy of detection of
sentinel nodes. In prostate cancer no false negative findings were observed. The
SPECT/CT scan together with the Sentinella was effective in localizing sentinel nodes
in a variety of anatomical locations, otherwise not removed during standard lymph
node dissection. 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 tumors are mostly been treated outside of NKI-AVL. For
renal cancer a sentinel node study is ongoing since 2007 (N06SNR). The removed
sentinel node is also used for advanced studies on immunological changes. This part
of research is being conducted together with the department of immunology.
Research is also focussed on better delineation of the primary tumor, especially
prostate cancer during robotic surgery. This is being investigated by dr Van der Poel
and H Wijkstra (AMC) using transrectal ultrasound and microbubble enhanced
vascular imaging. In animal experiments the use indocyanine green was shown to
improve sentinel node visualization intraoperatively.
Organ and function sparing While nephrectomy has been standard treatment for
renal cancer, this is increasingly being replaced by partial nephrectomy and
percutaneous Radio Frequency Ablation (RFA). These modalities play an important
role in the integrative approach of metastatic renal cancer. Together with the dept. of
urology of the Academic Medical Center in Amsterdam the value of contrast
enhanced ultrasound is being assessed as staging modality and during follow up
instead of the currently used CT-scan.
The role of cytoreductive surgery is being investigated in a prospective phase II study
with neo-adjuvant Sutent (tyrosine kinase inhibitor), in collaboration with the
department of immunology (N06SUN). An EORTC-study, under guidance of Axel
Bex and John Haanen, investigating the sequence of nephrectomy in metastatic renal
cancer will be launched this year. In bladder cancer we continue to expand our
experiences with the so called Sexuality Preserving Cystectomy and Neobladder
(SPCN). In a recent analysis 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 has been
introduced. A randomized study comparing standard and robotic cystectomy is
anticipated if funding can be found.
In prostate cancer the introduction of the Da Vinci robot led to an increase of prostate
cancer patients and operations. First results show good tumor margin free rates,
continence and potency rates. All patients are carefully monitored using EORTC-
QLQ questionnaires.
Translational research
Prostate cancer
Cell signalling in hormone refractory prostate cancer is studied in a variety of in vitro
and in vivo mouse modelling systems. Pim1 expression in advanced prostate cancer
was shown to increase proliferation but suppress cell invasiveness explaining the
149
SURGICAL ONCOLOGY
Publications (continued)
Kroon BK, Leijte JA, Van Boven H,
Wessels LF, Velds A, Horenblas S,
Van ‘t Veer LJ. Microarray gene-expression
profiling to predict lymph node metastasis
in penile carcinoma. BJU Int. 2008
Aug;102:510-5
Langenhoff BS, Krabbe PF, Ruers TJ.
Efficacy of follow-up after surgical treatment
of colorectal liver metastases. Eur J Surg
Oncol 2008 Aug 6
Leijte JA, Gallee M, Antonini N,
Horenblas S. Evaluation of current TNM
classification of penile carcinoma. J Urol.
2008;180:933-8;discussion 938
Leijte JA, Horenblas S. Shortcomings of the
current TNM classification for penile
carcinoma: time for a change? World J
Urol. 2008 Aug 9
Leijte JA, Kirrander P, Antonini N,
Windahl T, Horenblas S. Recurrence
patterns of squamous cell carcinoma of the
penis: recommendations for follow-up based
on a two-centre analysis of 700 patients.
Eur Urol. 2008 Jul;54:161-8
Leijte JA, Valdés Olmos RA, Nieweg OE,
Horenblas S. Anatomical mapping of
lymphatic drainage in penile carcinoma
with SPECT-CT: implications for the extent
of inguinal lymph node dissection. Eur
Urol. 2008;54:885-90
Leijte JAP, Valdés Olmos RA, Nieweg OE,
Horenblas S. Anatomical mapping of
lymphatic drainage in penile carcinoma
with SPECT/CT: implications for the extent
of inguinal lymph node dissection.
Eur Urol 2008;54:885-892
Li WW, Visser O, Ubbink DT, Klomp HM,
Kloek JJ, de Mol BA. The influence of
provider characteristics on resection rates
and survival in patients with localized nonsmall
cell lung cancer. Lung Cancer.
200860:441-51
Linthorst Homan MW, de Korte J,
Grootenhuis MA, Bos JD, Sprangers MA,
van der Veen JP. Impact of childhood
vitiligo on adult life. Br J Dermatol
2008;159:915-20

150
SURGICAL ONCOLOGY
Publications (continued)
Linthorst Homan MW, Sprangers MA, de
Korte J, Bos JD, van der Veen JP.
Characteristics of patients with universal
vitiligo and health-related quality of life.
Arch Dermatol 2008 Aug;144:1062-4
Loonen MPJ, Hage JJ, Kon M. Plastic
surgery classics – Characteristics of 50 topcited
articles in four plastic surgery journals
since 1946. Plast Reconstr Surg
2008;121:320e-327e
Lordick F, Ruers T, Aust DE, Collette L,
Downey RJ, El Hajjam M, et al. European
Organisation of Research and Treatment of
Cancer (EORTC) Gastrointestinal Group:
Workshop on the role of metabolic imaging
in the neoadjuvant treatment of
gastrointestinal cancer. Eur J Cancer 2008
Sep;44:1807-19
Meijnen P, Oldenburg HS, Peterse JL,
Bartelink H, Rutgers EJ. Clinical outcome
after selective treatment of patients
diagnosed with ductal carcinoma in situ of
the breast. Ann Surg Oncol 2008
Jan;15:235-43
Meijnen P, Peterse JL, Antonini N,
Rutgers EJ, van de Vijver MJ.
Immunohistochemical categorisation of
ductal carcinoma in situ of the breast.
Br J Cancer 2008 Jan 15;98:137-42
Meijnen P, Rutgers EJ. Meta-analysis of
sentinel node biopsy in ductal carcinoma in
situ of the breast (Br J Surg 2008;95:547-
554). Br J Surg 2008 Oct;95:1305
Meinhardt W, Valdés Olmos RA,
Van der Poel HG, Bex A, Horenblas S.
Laparoscopic sentinel node dissection for
prostate carcinoma: technical and
anatomical observations. BJU Int. 2008
Sep;102:714-7
Mol AJ, Geldof AA, Meijer GA,
Van der Poel HG, Van Moorselaar RJ. New
experimental markers for early detection of
high-risk prostate cancer: role of cell-cell
adhesion and cell migration. J Cancer Res
Clin Oncol. 2007 Oct;133:687-95
Mulier S, Ni Y, Jamart J, Michel L,
Marchal G, Ruers T. Radiofrequency
ablation versus resection for resectable
colorectal liver metastases: time for a
randomized trial? Ann Surg Oncol 2008
Jan;15:144-57
variety of Pim1 expression levels found in human prostate cancer tissue. In preclinical
analysis a synergistic antitumor effect of the combination of cyclophosphamide and
mTOR inhibition was observed. This is the basis for a phase I clinical study in men
with hormone refractory prostate cancer.
Penis cancer
The role of HPV in penis cancer was further investigated. 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. In a prospective study, serum and cervical
scrapings are being collected and investigated to look into the role of HPV
transmission between patients with penile cancer and partners.
Renal cancer
Renal cancer tissue from the N06SUN trial exposed to the active metabolite of the
tyrosine kinase inhibitor sunitinib is investigated in the Department of Immunology
and in cooperation with the Angiogenesis Laboratory of the Department of Pathology
at the University of Maastricht to establish an angiogenic profile of the post-treatment
tissue in correlation to activated and suppressed immune cells infiltrating tumor
tissue. These investigations are further substantiated by material from phase II trials
of presurgical sunitinib and bevacizumab at the MD Anderson Cancer Center,
Houston, TX and the St. Batholomews Hospital, London.
RECIST criteria of anatomical response may not be adequate to document response
of tumors treated with tyrosine kinase inhibitors. Dynamic contrast enhanced MRI of
the primary tumors pre- and posttreatment are correlated to microvessel density to
investigate if changes on imaging reflect a biological response.
Improved quality control of treatment results Using prospective data collection,
almost all uro-surgical treatments at the NKI-AVL can be analysed now. Together with
the Comprehensive Cancer Center Amsterdam a comparative study has been finished
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. Despite difference, this was not statistically
significant due to relatively small numbers. The same analysis has been performed
nation wide. This time statistically significant differences between mortality rates of
low and high volume hospitals were found.
ANAESTESIOLOGY AND INTENSIVE CARE
Peter Schutte, Dirk Buitelaar, Katina Efthymiou, Christiaan Keijzer, May Ronday, Michael Srámek,
Tino Stoppa, Julia ten Cate, Ingeborg Vergouwe
Inspiratory Carbon Monoxide and Compound A Concentrations During Desflurane
and Sevoflurane Anesthesia in Humans: An Observational Study
All modern vapor anesthetics are capable of carbon monoxide (CO) production as a
result of interaction with desiccated strong base containing carbon dioxide absorbents.
In desiccated absorbents, desflurane produces the highest concentrations of CO.
Sevoflurane is known to produce the nephrotoxic compound A (CA) independently
from water content of the carbon dioxide absorbent. The purpose of this study was to
register the average CO concentrations in forty patients receiving anesthesia with
desflurane or sevoflurane after implementation of a safety protocol adapted from
Woehlck et al.. This protocol was developed to prevent desiccation of the strong base
containing absorbent Drägersorb 800 Plus®. Methods: In 40 patients a low-flow
anesthesia was maintained using an oxygen/air mixture with either sevoflurane or
desflurane in combination with the CO2 absorbent Drägersorb 800 plus®. CO and
CA production was measured in the inspiratory limb of the anesthesia machine
using a portable gas chromatograph. Results: No carbon monoxide was measured in
any of the desflurane or sevoflurane anesthesia’s. The mean concentration of CA for
anesthesia with sevoflurane was 17.1 ± 5.5 parts per million. Conclusion: With the

introduction of a safety protocol no carbon monoxide was measured in anesthesia
performed with desflurane or sevoflurane. Compound A is almost continuously
detected in anesthetic procedures with the use of sevoflurane in very low
concentrations. Implementation of a simple safety protocol possibly prevents
desiccation of the absorbent and could subsequently reduce the risk of carbon
monoxide intoxication.
Comparison of postoperative throat and neck complaints after use of the i-gel® and
the La Premiere® disposable laryngeal mask. A double blinded randomized
controlled trial.
The i-gel® supraglottic airway device has been designed to match the peripharyngeal
anatomy without the use of an inflatable cuff. We therefore hypothesized that the
i-gel® should have less postoperative throat and neck complaints compared to a
standard disposable laryngeal mask (La Premiere®).
Methods: 244 patients were randomized to receive either the i-gel® (i-gel) or La
Premiere ® standard laryngeal mask (LM) for airway management. Ease of
insertion, anatomical positioning and leak pressure of the devices were registered.
Patients were interviewed postoperatively by trained recovery room personnel who
were blinded for the used device, for throat or neck complaints after 1, 24 and 48
hours. Results: Sore throat occurred in 6, 6 and 5% of the patients in the i-gel group
after 1, 24 and 48 hours. For the laryngeal mask this was 32*,47* and 24*%
(*=p

152
SURGICAL ONCOLOGY
Publications (continued)
Peeters CF, de Waal RM, Wobbes T,
Ruers TJ. Metastatic dormancy imposed by
the primary tumor: does it exist in humans?
Ann Surg Oncol 2008 Nov;15:3308-15
Pengel KE, Loo CE, Teertstra HJ, Muller SH,
Wesseling J, Peterse JL, et al. The impact
of preoperative MRI on breast-conserving
surgery of invasive cancer: a comparative
cohort study. Breast Cancer Res Treat 2008
Sep 21
Ploeg AJ, Lardenoye JW, Vrancken Peeters MP,
Hamming JF, Breslau PJ. Wound
complications at the groin after peripheral
arterial surgery sparing the lymphatic
tissue: a double-blind randomized clinical
trial. Am J Surg 2008 Oct 15
Prevoo W, Van den Bosch MA, Horenblas S.
[Guideline ‘Renal cell carcinoma’] Ned
Tijdschr Geneeskd. 2008 Jun
7;152:1350;author reply 1350. Dutch
Rutgers EJ, Pusztai L, Bernards R. Are
short-term or long-term recurrence rates
more important in breast cancer screening?
Ann Intern Med 2008 Sep 2;149:357-8
Rutgers EJ. Sentinel node biopsy:
interpretation and management of patients
with immunohistochemistry-positive
sentinel nodes and those with micrometastases.
J Clin Oncol 2008 Feb
10;26:698-702
Santegoets LA, van Seters M,
Heijmans-Antonissen C, Kleinjan A,
van Beurden M, Ewing PC, et al. Reduced
local immunity in HPV-related VIN:
Expression of chemokines and involvement
of immunocompetent cells. Int J Cancer
2008;123:616-22
Schagen SB, Boogerd W, Muller MJ,
Huinink WT, Moonen L, Meinhardt W,
Van Dam FS. Cognitive complaints and
cognitive impairment following BEP
chemotherapy in patients with testicular
cancer. Acta Oncol. 2008;47:63-70
Scheer MG, Sloots CE, van der Wilt GJ,
Ruers TJ. Management of patients with
asymptomatic colorectal cancer and
synchronous irresectable metastases. Ann
Oncol 2008 Nov;19:1829-35
Both patients and panelists expressed an overall preference for the adhesive side as of
1 week after surgery. Patients’ visual analogue scale scores for scar comfort and scar
appearance and panelists’ visual analogue scale scores for aesthetic outcome were
significantly better for the adhesive side after 6 weeks and 6 months (p < 0.05), as
was the total Hollander Wound Evaluation Scale score of the panelists after 6 weeks
(p < 0.02). The total Patient and Observer Scar Assessment Scale score after
6 months was significantly better for the adhesive side according to the patients
(p < 0.01), but not according to the panelists (p = 0.11).
We concluded that 2-octyl-cyanoacrylate is a sound alternative for wound closure.
Skinning and resurfacing of the clitoris for vulvar intraepithelial neoplasia
Partial or total clitoris amputation for vulvar intraepithelial neoplasia (VIN) affects
quality of life and sexual function and is likely to constitute over-treatment as
superficial excision only of the involved epithelium would suffice. Thus, we applied
skinning clitorectomy and replacement of clitoral skin as an organ-sparing surgical
therapy for clitoral VIN.
Seven consecutive patients were treated from July 2003 through February 2008.
Only the skin of the glans clitoridis was resected and the spongiosum was
subsequently covered with either a thin skin flap from the inner aspect of the
remaining preputium or minor labium, or a split thickness skin graft.
In all patients, the pre-operative diagnosis of VIN was confirmed histopathologically.
Additionally, micro-invasive carcinoma was found in two. Preoperative sexual
function was largely preserved and, after a mean follow-up of 21 months, no recurrence
or invasion of the original lesion was observed in any of the patients.
We concluded that skinning clitorectomy and skin replacement should be advocated
as a sound organ-sparing alternative for clitoral amputation in the treatment of
clitoral VIN.
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 in situ: relevance for
immunotherapy
Clinical studies have shown that immunotherapy of melanoma by vaccination or
adoptive transfer of effector T-cells can mediate tumor regression. However, a
significant 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. This model is currently
tested and offers the opportunity to predict the potential clinical outcome of
immunotherapy for melanoma patients.

Publications (continued)
Scheer MG, Stollman TH, Boerman OC,
Verrijp K, Sweep FC, Leenders WP, et al.
Imaging liver metastases of colorectal cancer
patients with radiolabelled bevacizumab: Lack of
correlation with VEGF-A expression. Eur J
Cancer 2008 Sep;44:1835-40
Scheer MG, Stollman TH, Vogel WV,
Boerman OC, Oyen WJ, Ruers TJ. Increased
metabolic activity of indolent liver metastases
after resection of a primary colorectal tumor.
J Nucl Med 2008 Jun;49:887-91
Schouten van der Velden AP, Punt CJ,
van Krieken JH, Derleyn VA, Ruers TJ. Hepatic
veno-occlusive disease after neoadjuvant
treatment of colorectal liver metastases with
oxaliplatin: a lesson of the month. Eur J Surg
Oncol 2008 Mar;34:353-5
Seters M van, Beckmann I,
Heijmans-Antonissen C, Beurden M van,
Ewing PC, Zijlstra FJ, et al. Disturbed patterns
of immunocompetent cells in usual-type vulvar
intraepithelial neoplasia. Cancer Res
2008;68:6617-22
Seters M van, Beurden M van, ten Kate FWJ,
Beckmann I, Ewing PC, Eijkemans MJC,
Kagie MJ, et al. Treatment of vulvar
intraepithelial neoplasia with topical
imiquimod. New Eng J Med 2008;358:1465-73
Smeenk RM, Bruin SC, van Velthuysen ML,
Verwaal VJ. In brief. Curr Probl Surg 2008
Aug;45:522-5
Smeenk RM, Bruin SC, van Velthuysen ML,
Verwaal VJ. Pseudomyxoma peritonei.
Curr Probl Surg 2008 Aug;45:527-75
Smeenk RM, van Velthuysen ML, Verwaal VJ,
Zoetmulder FA. Appendiceal neoplasms and
pseudomyxoma peritonei: a population based
study. Eur J Surg Oncol 2008 Feb;34:196-201
Smit LHM, Nieweg OE, Mooij WJ,
Bonfrer JMG, Haanen JBAG, Kroon BBR,
De Gast GC. Value of serum S100-B for
prediction of distant relapse and survival in
stage III B/C melanoma. Anticancer Res,
2008;28:2297-2302
Soolsma J, van den Brekel MWM,
Ackerstaff AH, Balm AJM, Tan IB, Hilgers FJM.
Long-term results of Provox ActiValve, solving
the problem of frequent Candida- and
‘underpressure’-related voice prosthesis
replacements. Laryngoscope 2008;118:252-257
Steggerda MJ, Van der Poel HG, Moonen LM.
An analysis of the relation between physical
characteristics of prostate I-125 seed implants
and lower urinary tract symptoms: bladder
hotspot dose and prostate size are significant
predictors. Radiother Oncol. 2008 Jul;88:108-14.
Stollman TH, Scheer MG, Leenders WP,
Verrijp KC, Soede AC, Oyen WJ, et al. Specific
imaging of VEGF-A expression with radiolabeled
anti-VEGF monoclonal antibody. Int J Cancer
2008 May 15;122:2310-4
Studer UE, Collette L, Whelan P, Albrecht W,
Casselman J, De Reijke T, Knönagel H, Loidl W,
Isorna S, Sundaram SK, Debois M; EORTC
Genitourinary Group. Using PSA to guide
timing of androgen deprivation in patients with
T0-4 N0-2 M0 prostate cancer not suitable for
local curative treatment (EORTC 30891).
Eur Urol. 2008 May;53:941-9
Tanis PJ, Nieweg OE, van den Brekel MWM,
Balm AJM. Dilemma of clinically node-negative
head and neck melanoma: outcome of ‘watch
and wait’ policy, elective lymph node dissection,
and sentinel node biopsy – a systemic review.
Head Neck 2008;30:380-389
Tournoy KG, Burgers SA, Annema JT,
Vermassen F, Praet M, Smits M, Klomp HM,
van Meerbeeck JP, Baas P. Transesophageal
endoscopic ultrasound with fine needle
aspiration in the preoperative staging of
malignant pleural mesothelioma. Clin Cancer
Res. 2008 Oct 1;14:6259-63
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, (e-pub ahead of print)
Van den Berg JH, Nuijen B, Beijnen JH,
Vincent A, van Tinteren H, Kluge J,
Woerdeman LA, Hennink WE, Storm G,
Schumacher T, Haanen JB. Optimization of
intradermal vaccination by DNA tattooing in
human skin. Hum Gene Ther 2008;Nov 25
[Epub ahead of print]
Van der Bogt KE, Vrancken Peeters MP,
van Baalen JM, Hamming JF. Resection of
carotid body tumors: results of an evolving
surgical technique. Ann Surg 2008
May;247:877-84
153
SURGICAL ONCOLOGY
Van der Ploeg IM, Kroon BB, Antonini N,
Valdes Olmos RA, Rutgers EJ, Nieweg OE.
Axillary and extra-axillary lymph node
recurrences after a tumor-negative sentinel
node biopsy for breast cancer using
intralesional tracer administration. Ann
Surg Oncol 2008 Apr;15:1025-31
Van der Ploeg IM, Tanis PJ, Valdes Olmos RA,
Kroon BB, Rutgers EJ, Nieweg OE.
Breast cancer patients with extra-axillary
sentinel nodes only may be spared axillary
lymph node dissection. Ann Surg Oncol
2008 Nov;15:3239-43
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 2008 Aug 20
Van der Ploeg IMC, Nieweg OE, Antonini N,
Valdés Olmos RA, Rutgers EJTh, Kroon BBR.
Axillary and extra-axillary lymph node
recurrences after a tumor-negative sentinel
node biopsy for breast cancer using intralesional
tracer administration Ann Surg
Oncol 2008;15:1025-1031
Van der Ploeg IMC, Nieweg OE,
Valdés Olmos RA, Kroon BBR. Tumorpositive
sentinel node biopsy of the groin in
clinically node-negative melanoma patients:
superficial or superficial and deep lymph
node dissection? Ann Surg Oncol
2008,15:1485-1491
Van der Ploeg IMC, Nieweg OE, Van Rijk MC,
Valdés Olmos RA, Kroon BBR. Axillary
recurrence after a tumour-negative sentinel
node biopsy in breast cancer patients:
A systematic review and meta-analysis of
the literature. Eur J Surg Oncol
2008;34:1277-1284
Van der Ploeg IMC, Tanis PJ, Valdés Olmos RA,
Kroon BBR, Rutgers EJTh, Nieweg OE.
Breast cancer patients with extra-axillary
sentinel nodes only may be spared axillary
lymph node dissection. Ann Surg Oncol
2008,15:3239-3243
Van der Ploeg IMC, Valdés Olmos RA,
Kroon BBR, Nieweg OE. The hybrid
SPECT/CT as an additional lymphatic
mapping tool in patients with breast cancer.
World J Surg 2008;32:1930-1934

154
SURGICAL ONCOLOGY
Publications (continued)
Van der Ploeg IMC, Valdés Olmos RA,
Nieweg OE, Rutgers EJTh, Kroon BBR,
Hoefnagel CA. The hidden sentinel node
and SPECT/CT in breast cancer patients.
Eur J Nucl Med Mol Imaging (e-pub ahead
of print)
Van der Ploeg IMC, Van Rijk MC, Nieweg OE,
Kroon BBR. Sentinel lymph node. In:
Encyclopedia of cancer. Second edition.
Schwab M, Ed. Springer, Heidelberg 2008.
ISBN 978-3-540-36847-2
Van der Poel HG, De Blok W, Bex A,
Meinhardt W, Horenblas S. Peroperative
transrectal ultrasonography-guided bladder
neck dissection eases the learning of robotassisted
laparoscopic prostatectomy.
BJU Int. 2008 Sep;102:849-52
Van Der Poel HG, Moonen L, Horenblas S.
Sequential treatment for recurrent localized
prostate cancer. J Surg Oncol. 2008 Apr
1;97:377-82
Van der Poel HG. Editorial comment on:
marked gene transcript level alterations
occur early during radical prostatectomy.
Eur Urol. 2008 Feb;53:344
Van der Poel HG. Editorial comment on:
Oncological and functional results of
antegrade radical retropubic prostatectomy
for the treatment of clinically localised
prostate cancer. Eur Urol. 2008 Mar;53:561-
2. No abstract available
Van der Poel HG. Editorial Comment on:
Predictive Value of the Differential
Expression of the Urokinase Plasminogen
Activation Axis in Radical Prostatectomy
Patients. Eur Urol. 2008 Jun 23
Van der Veldt AA, Meijerink MR, van den
Eertwegh AJ, Bex A, De Gast G, Haanen
JB, Boven E. Sunitinib for treatment of
advanced renal cell cancer: primary
tumor response. Clin Cancer Res. 2008
Apr 15;14:2431-6
Van der Zee J, Kroon BBR. Isolated limb
perfusion for malignant melanoma;possibly
better results with high dose hyperthermia.
Int J Hyperthermia. 2008;24:602-603
Van Driel WJ, Dalesio OB, van der Donk E.
OVHIPEC-studie in combinatie met
het gebruik van het ALEA eCRFsysteem.
NTvO 2008;5:326-30
Van Rossum MA, Jongmans P,
Van As-Brooks CJ, Hilgers FJM. De ontwikkeling
van een evidence-based therapieprogramma voor
het verbeteren van spraakverstaanbaarheid bij
tracheoesofageale sprekers. Stem-, Spraak-, en
Taalpathologie 2008;16:112-123
Van Sandick JW, Kappers I, Baas P, Haas RL,
Klomp HM. Surgical treatment in the
management of malignant pleural
mesothelioma: a single institution’s experience.
Ann Surg Oncol 2008;15:1757-64
Van Sandick JW, Kappers I, Baas P, Haas RL,
Klomp HM. Surgical treatment in the
management of malignant pleural
mesothelioma: a single institution’s experience.
Ann Surg Oncol. 2008 Jun;15:1757-64
Verwaal VJ, Bruin S, Boot H, van Slooten G,
van Tinteren H. 8-year follow-up of randomized
trial: cytoreduction and hyperthermic
intraperitoneal chemotherapy versus systemic
chemotherapy in patients with peritoneal
carcinomatosis of colorectal cancer. Ann Surg
Oncol 2008 Sep;15:2426-32
Verwaal VJ, Kusamura S, Baratti D, Deraco M.
The eligibility for local-regional treatment of
peritoneal surface malignancy. J Surg Oncol
2008 Sep 15;98:220-3
Voskuil DW, Vrieling A, Korse CM, Beijnen JH,
Bonfrer JM, van Doorn J, et al. Effects of
lycopene on the insulin-like growth factor (IGF)
system in premenopausal breast cancer survivors
and women at high familial breast cancer risk.
Nutr Cancer 2008 May;60:342-53
Weum S, de Weerd L, Klein S, Hage JJ
Behandling av bløtdelsdefekter med
perforatorlapper. Tidsskr Nor Lægeforen
2008;128:313-315
Wouters M. Outcome-based referral to improve
quality of care in upper GI surgery. J Surg
Oncol. 2008 Oct 1;98:307-8
Wouters MW, van Geel AN,
Menke-Pluijmers M, de Kanter AY,
de Bruin HG, Verhoog L, Eggermont AM.
Should internal mammary chain (IMC)
sentinel node biopsy be performed? Breast. 2008
Apr;17:152-8
Wouters MW, van Geel AN, Nieuwenhuis L,
van Tinteren H, Verhoef C, van Coevorden F,
Klomp HM. Outcome after surgical resections of
recurrent chest wall sarcomas. J. Clin Oncol
2008;26:5113-8
Wouters MW, Wijnhoven BP, Karim-Kos HE,
Blaauwgeers HG, Stassen LP, Steup WH,
Tilanus HW, Tollenaar RA. High-volume versus
low-volume for esophageal resections for cancer:
the essential role of case-mix adjustments based
on clinical data. Ann Surg Oncol. 2008
Jan;15:80-7
Zuur CL, Simis YJ, Verkaik RS, Schornagel JH,
Balm AJ, Dreschler WA, Rasch CR. Hearing
loss due to concurrent low-dose cisplatin
chemoradiation for locally advanced head and
neck cancer Radiother Oncol 2008;89:38-43
Zuur JK, Muller SH, Vincent A, Sinaasappel M,
de Jongh FHC, Hilgers FJM. Assessment of
tracheal temperature and humidity in
laryngectomized individuals and the influence of
heat and moisture exchangers on tracheal
climate. Head Neck 2008;30:1072-1082
In press
Ackerstaff AH, Balm AJM, Rasch CRN,
De Boer JP, Wiggenraad R, Rietveld DHF,
Gregor RT, Kröger R, Hilgers FJM. First-year
Quality of Life assessment of an intra-arterial
(RADPLAT) versus intravenous chemoradiation
Phase III trial. Head Neck
Balch CM, Morton DL, 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
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 2008, Sept 2008
De Vreeze RS, Koops W, Haas, RL,
van Coevorden F. An unusual case of
hemosiderotic fibrohistiocytic lipomatous lesion:
correlation of MRI and pathological findings.
Sarcoma

Geurts TW, Balm AJM, Van Velthuysen MLF,
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
Geurts TW, Lohuis PJFM, Pameijer FA,
Van den Brekel MWM. Lipoma of the
nasopharynx. Arch Otolaryngol
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
MA van Rossum, P Jongmans,
CJ van As-Brooks, FJM Hilgers. De
ontwikkeling van een evidence-based
therapieprogramma voor het verbeteren van
spraakverstaanbaarheid bij tracheoesofageale
sprekers. Stem Spraak Taal Pathologie
Nieweg OE, Van der Ploeg IMC, Kroon BBR.
Naar sentinel-nodebiopsie bij melanoom:
antwoorden en nieuwe vragen - interim-verslag
van de Multicenter Selective Lymphadenectomy
Trial. Oncollectie.
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
VB Wreesmann, LM Smeele, FJM Hilgers,
PJFM Lohuis. Closure of tracheoesophageal
fistula with prefabricated revascularized
bilaminar radial forearm free flap. Head Neck
Books
Textbook of Surgical Oncology
Graeme Poston, Daniel Beauchamp,
Theo Ruers
Informa Healthcare, London
ISBN9781841845074
Book chapters
Van den Brekel MWM, Hilgers FJM.
Preoperative workup of the neck in head and
neck squamous cell carcinoma. In: Pearls and
Pittfalls in Head and Neck Surgery;practical
tips to minimize complications. Editor Cernea
CR;associate editors: Dias FL, Fliss D, Lima
RA, Myers EN, Wei WI. Karger Publishers,
Basel, Freiburg, Paris, London, New York,
Bangalore, Bangkok, Shanghai, Singapore,
Tokio, Sydney;2008, pp.34-35. ISBN 978-3-
8055-8425-8. (CATEGORY C)
Hilgers FJM, Van den Brekel MWM. Practical
tips for voice rehabilitation after
pharyngolaryngectomy. In: Pearls and Pittfalls
in Head and Neck Surgery;practical tips to
minimize complications. Editor Cernea
CR;associate editors: Dias FL, Fliss D, Lima
RA, Myers EN, Wei WI. Karger Publishers,
Basel, Freiburg, Paris, London, New York,
Bangalore, Bangkok, Shanghai, Singapore,
Tokio, Sydney;2008, pp 96-97. ISBN 978-3-
8055-8425-8. (CATEGORY C)
Giuliana Mariani, Armando E Giuliano, H.
William Strauss. Radioguided Surgery. A
comprehensive team approach. Contributors:
S. Horenblas. Part II: 11: Dynamic sentinel
lymph node biopsy in penile carcinoma.
Simon Horenblas, Bin K. Kroon, Renato A.
Valdés Olmos, Omgo E. Nieweg
Nieweg OE. Een fellow en zijn PET. Groninger
chirurgische oncologica. Hoekstra HJ,
Wobbes Th, redacteuren. Chirurgische
Oncologie, UMCG, Groningen 2008. ISBN
078-90-9023505-9. pg. 112-114
Vinod H. Nargund, Derek Raghavan, Howard
M. Sandler. Urological Oncology. Part III
Systemic genitourinary oncology, 19: Cancer of
the penis and scrotum. Simon Horenblas and
Bin K. Kroon
T. Ruers. Liver metastases. In Textbook of
Surgical Oncology
Editors: Graeme Poston, Daniel Beauchamp,
Theo Ruers
Informa Healthcare, London
155
SURGICAL ONCOLOGY

156
BIOMETRICS
Department head, group leader Otilia Dalesio
Otilia Dalesio MSc Head
Ninja Antonini MSc Academic staff
Harm Van Tinteren PhD Academic staff
Andrew Vincent PhD Academic staff
Erik Van Werkhoven MSc Academic staff
Danny Baars Technical staff
Frauwkje Bessels Technical staff
Monique Carreno Technical staff
Daphne Daems Technical staff
Gerda De Jong Technical staff
Marjolijn De Waal MSc 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
Joop Jonkman MSc Technical staff
Josien Kant Technical staff
Edwin Klerkx Technical staff
Lies Kolmschate Technical staff
Jenneke Koorn Technical staff
Lissettee Lorist Technical staff
Marianne Mahn MSc Technical staff
Ingrid Mandjes MSc Technical staff
Carla Modder Technical staff
Pietje Muller Technical staff
Lida Oostveen 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
Ria Tilgenkamp Technical staff
Alex Torres Acosta Technical staff
Heleen Vaessen MSc 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
Els Van Oosterwijk Technical staff
Wil Van Waardenberg Technical staff
Marcel Vlaskamp MSc Technical staff
Anneke Wals Technical staff
Lidwina Wever Technical staff
Els Willemse Technical staff
Jeroen Zandbergen MSc Technical staff
Lonny Ziblat Technical staff
BIOMETRICS DEPARTMENT
ICT PROJECTS
We have just reached the 2 year milestone of the 4 years TENALEA Initial Deployment
(TENALEA ID) project. This is a very large project with a total investment of
approximately 16 million euro, with a 3.7 million euro contribution from the eTEN
program of the EC. 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. We also ensure the technical, financial and
organizational coordination of the project. The consortium includes academic
institutes and coordinating centers in Germany, Poland, France, UK and the
Netherlands, including the Academic Medical Centre (AMC) in Amsterdam and the
Clinical Trial Centre Maastricht (CTCM).
Because of our involvement in the development of tools for clinical trials during the
last 20 years, we have currently to our disposal 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 CRF 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 (ALEA eCRFs). The eCRF system allows detection of
inconsistencies at the source resulting in less data queries at later times. This should
help local data managers in terms of immediate feed back on the consistency and
completeness of the data and should improve quality standards and help working
more efficiently. Most importantly, the electronic data is available much earlier and is
of better quality, thereby speeding up the statistical analysis and reporting
dramatically.
ALEA eCRFs is based on generic packages, principally Microsoft Office Forms
Services and InfoPath2007 from Office 2007 Enterprise (Microsoft). These tools
provide a good basis for an electronic case record form system. Figure 1 shows that
InfoPath provides an extensive feature set to define data entry checks in a userfriendly
interface. The final product incorporates an audit trail in compliance with
the requirements of the International Committee for Harmonization and produces
standard based export facilities (CDISC, ODM and SDTM).
The integration of PACS, allowing central review of images (eg for tumor responses
evaluation) the in a clinical setting, is currently being developed in collaboration with
the radiotherapy department within the CTMM project.
During this year, we collaborated with UK partners to design a simulation system
that can be used to evaluate the performance of the different randomization methods
or parameterization of ALEA. Figure 2a and 2b present the results of 1000
simulations of 1000 patients entered into a 3 armed study with 3 stratification factors
of 2 levels each. Figure 2a shows the treatment allocation histogram depicting how
often a treatment was allocated in each simulation. The expectation would be that the
most regularly observed frequency occurs around the number of patients divided by
the number of treatments. Similar to the treatment histogram, Figure 2b shows the
treatment allocation graph that illustrates how often each patient received each
treatment. Assuming the trial is entirely non-deterministic, we would expect to see a
fairly even allocation of each treatment for each patient. If, however, the trial is
deterministic, we would expect to see a more frequent allocation of a certain
treatment to certain patients.
These simulation tools are being included in the new version of the randomization
program (ALEA3) for use of the different clinical trials coordination centers
responsible for the central management, registration and randomization of patients
in clinical trials.

Figure 1: Data Entry checks definition possibilities
In 2008 the number of clinical trial coordination centers using our systems has
increased to 27 and include centers in Europe, UK and also in New Zealand,
Australia and Indonesia. The randomization methods possibilities in ALEA were
extended and were described in the package manual. Our efforts also went to the full
validation of our systems. The Standard Operating Procedures were reviewed and
updated. The systems were audited by an independent auditor and a new
validation cycle was initiated.
Figure 2a: Treatment allocation histogram
Figure 2b: Treatment allocation graph
Publications
157
BIOMETRICS
Zuur JK, Muller SH, Vincent A,
Sinaasappel M, de Jongh FH, Hilgers FJ.
Assessment of tracheal temperature and
humidity in laryngectomized individuals
and the influence of a heat and moisture
exchanger on tracheal climate. Head Neck
2008;30:1072-82
Wouters MW, van Geel AN,
Nieuwenhuis L, van Tinteren H,
Verhoef C, van Coevorden F, Klomp HM.
Outcome after surgical resections of
recurrent chest wall sarcomas. J Clin Oncol
2008;26:5113-8
Verwaal VJ, Bruin S, Boot H,
van Slooten G, van Tinteren H. 8-year
follow-up of randomized trial: cytoreduction
and hyperthermic intraperitoneal
chemotherapy versus systemic chemotherapy
in patients with peritoneal carcinomatosis of
colorectal cancer. Ann Surg Oncol
2008;15:2426-32
van der Weide L, van Sornsen de Koste JR,
Lagerwaard FJ, Vincent A, van Triest B,
Slotman BJ, Senan S. Analysis of carina
position as surrogate marker for delivering
phase-gated radiotherapy. Int J Radiat
Oncol Biol Phys 2008;71:1111-7
van der Ploeg IM, Kroon BB, Antonini N,
Valdes Olmos RA, Rutgers EJ, Nieweg OE.
Axillary and extra-axillary lymph node
recurrences after a tumor-negative sentinel
node biopsy for breast cancer using
intralesional tracer administration. Ann
Surg Oncol 2008;15:1025-31
van den Heuvel-Eibrink MM, Grundy P,
Graf N, Pritchard-Jones K, Bergeron C,
Patte C, van Tinteren H, Rey A,
Langford C, Anderson JR, de Kraker J.
Characteristics and survival of 750 children
diagnosed with a renal tumor in the first
seven months of life: A collaborative study
by the SIOP/GPOH/SFOP, NWTSG, and
UKCCSG Wilms tumor study groups.
Pediatr Blood Cancer 2008;50:1130-4
van den Broek E, Richard W,
van Tinteren H, de Vries N. UPPP
combined with radiofrequency
thermotherapy of the tongue base for the
treatment of obstructive sleep apnea
syndrome. Eur Arch Otorhinolaryngol
2008;265:1361-5

158
BIOMETRICS
Publications (continued)
van den Berg RM, Teertstra HJ,
van Zandwijk N, van Tinteren H, Visser C,
Pasic A, Sutedja TG, Baas P, Golding RP,
Postmus PE, Smit EF. CT detected
indeterminate pulmonary nodules in a
chemoprevention trial of fluticasone. Lung
Cancer 2008;60:57-61
van den Berg JH, Nuijen B, Beijnen JH,
Vincent A, van Tinteren H, Kluge J,
Woerdeman LA, Hennink WE, Storm G,
Schumacher T, Haanen JB. Optimization
of intradermal vaccination by DNA
tattooing in human skin. Hum Gene Ther
2008;
Tol J, Cats A, Mol L, Koopman M, Bos MM,
van der Hoeven JJ, Antonini NF,
van Krieken JH, Punt CJ. Gastrointestinal
ulceration as a possible side effect of
bevacizumab which may herald perforation.
Invest New Drugs 2008;26:393-7
Tol J, Koopman M, Rodenburg CJ, Cats A,
Creemers GJ, Schrama JG, Erdkamp FL,
Vos AH, Mol L, Antonini NF, Punt CJ.
A randomised phase III study on
capecitabine, oxaliplatin and bevacizumab
with or without cetuximab in first-line
advanced colorectal cancer, the CAIRO2
study of the Dutch Colorectal Cancer
Group (DCCG). An interim analysis of
toxicity. Ann Oncol 2008;19:734-8
Stuiver MM, van Wilgen CP, de Boer EM,
de Goede CJ, Koolstra M, van Opzeeland A,
Venema P, Sterken MW, Vincent A,
Dijkstra PU. Impact of shoulder complaints
after neck dissection on shoulder disability
and quality of life. Otolaryngol Head Neck
Surg 2008;139:32-9
Simkens L, Tol J, Koopman M, Mol L,
Antonini N, van Krieken H, Punt C.
Current questions in the treatment of
advanced colorectal cancer: the CAIRO
studies of the Dutch Colorectal Cancer
Group. Clin Colorectal Cancer 2008;7:105-9
Nieuwenhuijzen JA, de Vries RR, Bex A,
van der Poel HG, Meinhardt W, Antonini N,
Horenblas S. Urinary diversions after
cystectomy: the association of clinical
factors, complications and functional results
of four different diversions. Eur Urol
2008;53:834-42
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 systems so obtained are then hosted in our computer
servers.
We collaborate with the Dutch Chest Physician Association (NVALT) in planning
designing and running of clinical trials in lung cancer and mesothelioma. More than
75 hospital in the Netherlands have included in excess of 1600 patients in their 9
randomized studies. We have obtained support from the Dutch Cancer Society for
the management of data of 5 studies (the NVALT 3, 4, 5, 8 and 11).
In 2008 the final reports of the 2 of the NVALT studies has been prepared. The role
of platinum compounds as part of second line therapies in advanced NSCLC was
studied in the NVALT7 study. This was a randomized trial comparing pemetrexed (P)
and pemetrexed-carboplatin (PC) in patients relapsing after platinum-based
chemotherapy. Main eligibility criteria: histological or cytological proof of advanced
NSCLC, relapse > 3 months after (first-line) platinum-based chemotherapy, normal
organ reserve, ECOG PS 0-2, age >18 years and signed informed consent. After
stratification, patients were randomized to (A) P 500 mg/m2 i.v. q 3 wks or (B) C
AUC 5 and P 500 mg/m2 both iv q 3 wks. Response was assessed 6 weekly according
to RECIST and toxicity 3 weekly according to NCI-CTC criteria. The primary endpoint
was time to progression (TTP). Secondary endpoints: objective response rate (ORR),
overall survival (OS), toxicity. From October 2005 to May 2007 151 males and 89
females were enrolled by 24 centers. The median (range) age was 59 (36-84), 33% of
the patients had an ECOG PS score of 0, 58% 1 and 9% had a PS of 2. After a median
follow up of 14.7 months, median TTP for arm A was 2.8 months and for arm B 4.2
months; HR 0.67 (95% CI 0.51 – 0.89, p = 0.005). Median OS: 7.6 months versus
8.0 months. The overall response rate was 4% in arm A and 9% in arm B. Subgroup
analyses suggested that adenocarcinoma is associated with favorable outcome.
Toxicities grade 3/4 was 29% in arm A and 44% in Arm B, mainly hematological
without clinical sequel. It was concluded 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 paper has been recently accepted for
publication in the JCO.
The second final report concerned the NVALT3 study, which is randomized trial in
elderly patients with non-small-cell lung cancer. This study incorporated a battery of
geriatric and quality of life assessments and compared 2 chemotherapy combinations
in terms of quality of life, toxicity and survival and attempt to identify possible
interactions between the assessments and the results of treatment. Patients had to be
70 years or older with cytological or histological confirmation of inoperable stage III
or IV non small cell lung cancer. Using a minimization algorithm, patients were
centrally assigned to receive a maximum of 4 three-week cycles of carboplatingemcitabine
(CG) or carboplatin-paclitaxel (CP). In the CG arm carboplatin was
administered at a dose targeting an area under the concentration-time curve (AUC) of 5,
intravenously (i.v.) at day 1 followed by gemcitabine 1250 mg/m2 i.v. on days 1 and 8.
Before chemotherapy, on day 1, patients received dexamethason i.v. and a serotonin
receptor antagonist. Patients in the CP arm received paclitaxel 175 mg/m2 i.v.
immediately followed by carboplatin AUC 5 mg/ml/min on day 1. Paclitaxel
administration was preceded by routine medication including dexamethasone,
clemastine, ranitidine, and a serotonin receptor antagonist. Between March 2003 and
August 2006 a total of 182 patients were entered into the study by 14 hospitals. The
average age was 75 years (range:70 to 87). The percentage of patients that completed

all 4 cycles was 64% and 65% in the CG and CP arms, respectively. Grade III/IV
toxicities occurred in 75% of the patients in the CG arm and 60% in the CP arm.
Treatment related SAE’s occurred in 19% of the patients in the CG arm and 15% in
the CP arm. As expected, neurologic toxicity ≥ grade II occurred more frequently in
the CP arm than in the CG arm (neuro-sensory 19% vs 5%, p = 0.002; neuro-motory:
10% vs 2%, p = 0.03). The paper has been submitted to the JCO.
Patient accrual to the NVALT4 trial, a randomized placebo-controlled study of docetaxel/
carboplatin with celecoxib or placebo in patients with locally advanced or metastatic
non-small cell lung cancer, has been completed in 2008. Follow-up data is being collected
for the 561 patients entered and final analysis is planned for the first half of 2009.
The NVALT 5 is a phase III trial of the antiangiogenic agent Thalidomide in patients
with malignant pleural mesothelioma after first line chemotherapy. Accrual has
continued in 2008 and it is expected to be completed in 2010. Two studies in
resectable NSCLC low and high risk patients as determined by preoperative PET
SUVmax (NVALT8A and NVALT8B), have been open to patient entry for 1 year.
However, the accrual has been disappointing probably due to the fact that the
organization of the study is rather complex since, it is multidisciplinary involving
surgeons, lung physicians, nuclear medicine experts and pathologists, and requiring
central analysis of PET values and central collection of frozen tissue . For this study
we have developed an e-CRFs application based on the ALEA eCRFS tool developed
within our ICT projects. This should facilitate data capture on the different
departments of the hospitals involved directly into our servers and should improve
communication across professions, speed and quality.
The NVALT 9, a phase II study to establish the efficacy of intravenous loading doses
of Ibandronate 6 mg in patients with lung cancer and skeletal metastases
experiencing moderate to severe bone pain is still slowly accruing patients.
A new study, the NVALT11 was developed during 2008 and is currently accruing
patients. In this study 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).
We are also the coordinating and statistical center for another study of the DLCRG,
the Rosel study, a randomized clinical trial of radiosurgery (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.
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.
After the successfully run CAIRO 1 study, which was published in the Lancet in
2007, the CAIRO 2 study was carried out as rapidly as the first study and in 2008 the
final analysis was completed. This is a randomized study in the same patient group,
advanced colorectal cancer previously untreated patients, studying cetuximab added
to capecitabine, oxaliplatin and bevacizumab. Between June 2005 and December
2006 755 patients were randomized. The publication of the final results has been
accepted in the New England Journal of Medicine.
Multiple studies 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. Accrual started in May 2007 and 232 patients
have already been entered into the study.
Publications (continued)
159
BIOMETRICS
Meijnen P, Peterse JL, Antonini N,
Rutgers EJ, van de Vijver MJ.
Immunohistochemical categorisation of
ductal carcinoma in situ of the breast.
Br J Cancer 2008;98:137-42
Leijte JA, Kirrander P, Antonini N,
Windahl T, Horenblas S. Recurrence
patterns of squamous cell carcinoma of the
penis: recommendations for follow-up based
on a two-centre analysis of 700 patients.
Eur Urol 2008;54:161-8
Leijte JA, Gallee M, Antonini N,
Horenblas S. Evaluation of current TNM
classification of penile carcinoma. J Urol
2008;180:933-8
Kweekel DM, Koopman M, Antonini NF,
Van der Straaten T, Nortier JW,
Gelderblom H, Punt CJ, Guchelaar HJ.
GSTP1 Ile105Val polymorphism correlates
with progression-free survival in MCRC
patients treated with or without irinotecan:
a study of the Dutch Colorectal Cancer
Group. Br J Cancer 2008;99:1316-21
Kweekel DM, Gelderblom H,
Van der Straaten T, Antonini NF, Punt CJ,
Guchelaar HJ. UGT1A1*28 genotype and
irinotecan dosage in patients with
metastatic colorectal cancer: a Dutch
Colorectal Cancer Group study. Br J Cancer
2008;99:275-82
Hoebers FJ, Kartachova M, de Bois J,
van den Brekel MW, van Tinteren H,
van Herk M, Rasch CR, Valdes Olmos RA,
Verheij M. 99mTc Hynic-rh-Annexin V
scintigraphy for in vivo imaging of apoptosis
in patients with head and neck cancer
treated with chemoradiotherapy. Eur J Nucl
Med Mol Imaging 2008;35:509-18
Hilgers FJ, van den Brekel MW,
van Tinteren H. Views on Acton et al’s
“Investigation of tracheoesophageal voice
prosthesis leakage patterns: patient’s selfreport
versus clinician’s confirmation”.
Head Neck 2008;30:1660-1
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 2008;

160
RADIOTHERAPY
Publications (continued)
de Witte MA, Bendle GM, van den Boom MD,
Coccoris M, Schell TD, Tevethia SS,
van Tinteren H, Mesman EM, Song JY,
Schumacher TN. TCR gene therapy of
spontaneous prostate carcinoma requires in
vivo T cell activation. J Immunol
2008;181:2563-71
de Boer JP, Hiddink RF, Raderer M,
Antonini N, Aleman BM, Boot H, de Jong D.
Dissemination patterns in non-gastric
MALT lymphoma. Haematologica
2008;93:201-6
Buitelaar D, Huitink J, Oldenburg H,
Rutgers E, Schutte P, van Tinteren H.
Field block: an additional technique of
potential value for breast surgery under
general anaesthesia. Eur J Anaesthesiol
2008;25:253-5
Buijs C, Rodenhuis S, Seynaeve CM,
van Hoesel QG, van der Wall E, Smit WJ,
Nooij MA, Voest E, Hupperets P,
Ten Vergert EM, van Tinteren H,
Willemse PH, Mourits MJ, Aaronson NK,
Post WJ, de Vries EG. Prospective study of
long-term impact of adjuvant high-dose and
conventional-dose chemotherapy on healthrelated
quality of life. J Clin Oncol
2007;25:5403-9
Bueno-de-Mesquita JM, van Harten WH,
Retel VP, van ‘t Veer LJ, van Dam FS,
Karsenberg K, Douma KF, van Tinteren
H, Peterse JL, Wesseling J, Wu TS,
Atsma D, Rutgers EJ, Brink G, Floore AN,
Glas AM, Roumen RM, Bellot FE,
van Krimpen C, Rodenhuis S,
van de Vijver MJ, Linn SC. Use of 70-gene
signature to predict prognosis of patients
with node-negative breast cancer: a
prospective community-based feasibility
study (RASTER). Lancet Oncol
2007;8:1079-87
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
Van Driel WJ, Dalesio OB, Van Der Donk E.
OVHIPEC-studie in combinatie met het
gebruik van het ALEA eCRF-systeem. Ned
Tijdschr Oncol 2008;5:326-30
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 recently
been modified allowing patients receiving pre-operative chemotherapy, in an attempt
to increase the accrual rate.
We collaborate with medical departments in our Institute in carrying out and
handling the data of large multicenter clinical trials they coordinate, like the
OVHIPEC, which is a study of intraperitoneal chemotherapy and hyperthermia in
ovarian cancer or the Matador study in breast cancer. With the radiotherapy
department we are carrying out a large randomized study in young women with early
breast cancer. More than 1200 patients have been entered by participating centers in
the Netherlands, Belgium and Germany. 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.

CLINICAL TRIALS IN THE
NETHERLANDS CANCER INSTITUTE
161
CLINICAL TRIALS
type of nickname title study coordinator phase activated no pts.
cancer study in NKI-AVL (closed) in NKI-
AVL per
18/12/
ALL SITES
2008
M03D24 Dose-finding and pharmacokinetic Trial of JHM Schellens I ‘02/04/2003 31
Orally Administered D-24851 to patients with (11/01/2008)’
Solid Tumors
M05AZA A phase-I open-label, multicenter study to JHM Schellens I ‘23/05/2005 33
assess the safety, tolerability and pharma- (22/02/2008)’
cokinetics of AZD1152 given as as 2 hour
intravenous infusion on 2 dose schedules in
patients with advanced solid malignancies
M05HOP An open label phase I dose escalation study JHM Schellens I ‘28/06/2005 41
of E7080 (01/08/2008)’
M05PAR A Phase 1, Pharmacokinetic and Biological JHM Schellens I ‘15/11/2005 29
Evaluation of a Small Molecule Inhibitor of (01/03/2008)’
Poly ADP-Ribose Polymerase-1 (PARP-1),
KU-0059436, in patients with advanced tumors
M05PFS A phase 1, open-label, dose escalation study to JHM Schellens I ‘27/09/2005 37
evaluate safety, pharmacokinetics and pharma- (01/10/2008)’
codynamics of 2 dosing schedules of
PF 00299804 in patients with advanced
malignant solid tumors
M05RTB A phase III international randomized trial of M Verheij III 10-1-2006 4
single versus multiple fractions for re-irradiation
of painful bone metastases
M06BBC Phase I study to determine the safety, maximum JHM Schellens I ‘19/05/2006 13
tolerated dose and pharmacokinetics of (01/04/2008)’
BAY57-9352 in combination with bevacizumab
in subjects with advanced solid tumors
M06BCI Phase I study to determine the safety, maximum JHM Schellens I ‘12/10/2006 12
tolerated dose, pharmacokinetics and biomarker (18/04/2008)’
status of BAY 57-9352 in combination with
capecitabine and irinotecan in subjects with
advanced solid tumors
M06OGP Phase I dose escalation study of oral JHM Schellens I 29-8-2006 30
gemcitabine prodrug (LY2334737) alone and in
combination with erlotinib (Tarceva) in patients
with advanced solid tumors
M07CUP CUP-PRINT Molecular profiling as a strategy for the M Kerst other 27-3-2008 10
identification of primary solid tumour in
metastatic cancer patients

162
CLINICAL TRIALS
type of nickname title study coordinator phase activated no pts.
cancer study in NKI-AVL (closed) in NKI-
AVL per
18/12/
2008
M07ERI An open label parallel group study to explore JHM Schellens other 11-3-2008 8
the 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 20-5-2008 *
prevention of chemotherapy-induced hair loss
M07KUC A phase I, open-label, study to assess the safety JHM Schellens I 25-6-2007 32
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
M07MKC A phase I dose escalating study evaluating JHM Schellens I 21-2-2008 20
MK-1775 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 JHM Schellens I 18-2-2008 3
with cancer and impaired renal function
M07PFU Pharmacogenomic and pharmacokinetic JHM Schellens other 16-5-2007 7
safety and cost-saving analysis in patients
treated with fluoropyrimidines
M07POB The relationship between cost-effectiveness WJ Nooijen other 10-1-2007 *
of blood safety measures and the distribution
of patients receiving blood
M08AZD A Phase I, Open-Label Study to Assess the JHM Schellens I ‘01/07/2008 6
Effect of Dosing AZD6244 Hyd Sulfate in the (01/09/2008)’
Presence and Absence of Food in Patients
With Advanced Solid Malignancies
M08EVC A phase I/II, non-randomized, multicenter, JHM Schellens I/II ‘26/08/2008 1
dose-escalating, two-stage efficacy and (suspended)’
feasablity study of the combination of
everolimus and capecitabine in patients with
advanced malignancies
M08HYT A phase I open-label study of the safety, JHM Schellens I 15-9-2008 5
tolerability and pharmacokinetics of two
schedules of oral topotecan in combination
with pazopanib in subjects with advanced
solid tumors
N03JJP Relative bioavailability of Paclitaxel in JHM Schellens Pilot ‘26/01/2004 12
combination with Cyclosporin A. (18/04/2008)’
A pilot investigation.
N05GEN Genotyping as tool for dose individualization A Huitema other ‘14/06/2005 9
in high-dose chemotherapy with cyclo- (29/10/2008)’
phosphamide, thiotepa and carboplatin
* = not registered at trialbureau

163
CLINICAL TRIALS
type of nickname title study coordinator phase activated no pts.
cancer study in NKI-AVL (closed) in NKI-
AVL per
18/12/
2008
N06PFB Pleural fluid bank MM van den Heuvel other 27-9-2006 *
N07DOW Weekly administration of oral Docetaxel in JHM Schellens I 14-11-2007 20
combinaton with Ritonavir
N07NEX Phase I study of gemcitabine and carboplatin JHM Schellens I 29-1-2008 10
plus sorafenib in patients with advanced
solid tumors
N08BPM 24 hour ambulatory blood pressure monitoring JHM Schellens other ‘02/09/2008 0
in patients with malignancies enrolled in (suspended)’
phase I oncological studies
N08CTC Validation of circulating tumor cells (CTC) JHM Schellens other 31-7-2008 8
detection techniques in patients with advanced
solid tumors
N08NPM NVALT Palliative care Protocol on malignant
pleural effusion MM van den Heuvel other 13-3-2008 38
N08TKI An open-label, non-randomized, single center, JHM Schellens I ‘26/03/2008 13
phase I study to determine the absorption, (03/11/2008)’
distribution, metabolism and excretion (ADME)
of TKI258 after a single oral administration of
TKI258 in patients with advanced solid
malignancies
BRAIN / CNS
N05THB Totale hersenbestraling met een eenmalige LGH Dewit other 7-4-2005 35
boost bij patienten met solitaire hersenmetastase
van een epitheliale tumor – een
registratiestudie
BREAST
E10031 SOFT ‘A phase III trial evaluating the role of ovarian GS Sonke III 20-7-2005 4
function suppression and the role of
exemestane as adjuvant therapies for
premenopausal women with endocrine
responsive breast cancer; tamoxifen versus
ovarian function suppression + tamoxifen
versus ovaria’
E10041 MINDACT Micro-array in node-negative disease may Avoid EJTh Rutgers III 2-1-2007 144
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
E10981 AMAROS After Mapping of the Axilla: Radiotherapy EJTh Rutgers III 21-12-2000 419
Or Surgery?
* = not registered at trialbureau

164
CLINICAL TRIALS
type of nickname title study coordinator phase activated no pts.
cancer study in NKI-AVL (closed) in NKI-
AVL per
18/12/
2008
E22051 SUPREMO SUPREMO, an MRC phase III randomised trial NS Russell III 27-2-2007 11
to assess the role of adjuvant chest wall
irradiation in ‘intermediate risk’ operable breast
cancer following mastectomy
E90061 Multicenter parallel phase II trial of BI 2536 JHM Schellens II ‘16/01/2008 0
administered as one hour IV infusion every (01/09/2008)’
3 weeks in defined cohorts of patients with
various solid tumors. A new drug screening
program of the EORTC network of core
institutions (NOCI)
M03RBC YOUNG Radiation dose intensity study in breast cancer GMM Bartelink III 29-3-2004 115
BOOST in young women: a randomized phase III trial
of additional dose to the tumor bed
(young boost)
M04MAT MATADOR Microarray Analysis in breast cancer to Tailor SC Linn III 26-4-2004 49
Adjuvant Drugs Or Regimens (MATADOR)
M05BRI BRIGHT Long term risk of breast cancer following NS Russell other 5-1-2006 89
treatment of Hodgkin’s disease
M05HIR HIRISE Hormonal substitution after prophylactic M van Beurden other 31-5-2005 *
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)
M06DAT DATA A prospective randomised open multicentre SC Linn III 1-5-2007 34
phase III study to assess different Durations
of Anastrozol therapy after 2 to 3 years
Tamoxifen as Adjuvant therapy in postmenopausal
women with breast cancer
M06ERB EGF 103659 An open-label expanded access SC Linn IV ‘11/01/2007 20
study of lapatinib and capecitabine therapy in (30/06/2008)’
subjects with ErbB2 overexpressing locally
advanced or metastatic breast cancer
M06HER CANDY ‘Prospective randomized pharmacological JHM Schellens III 18-6-2007 10
intervention 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’
M06TM2 TEAM II A randomised, multicenter, prospective, SC Linn III 26-9-2006 28
phase III trial investigating (a) Neoadjuvant
horminal therapy with exemestane for three
versus six months and/or (b) The efficacy and
safety of the addition of ibandronate to adjuvant
hormonal therapy
* = not registered at trialbureau

165
CLINICAL TRIALS
type of nickname title study coordinator phase activated no pts.
cancer study in NKI-AVL (closed) in NKI-
AVL per
18/12/
2008
M07ATX ATX Phase II randomized trial of combination SC Linn II 12-12-2007 6
therapy 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 SSB Schagen other 1-7-2008 *
functioning in the elderly
M07FEP A randomized double-blind phase 2 trial of GS Sonke II 12-9-2007 7
Fulvestrant plus Enzastaurin versus Fulvestrant
plus placebo in aromatase inhibitor-resistant
metastatic breast cancer
M07LET IDEAL IDEAL: Investigation on the duration of EJTh Rutgers III 16-11-2007 19
extended 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 sensitiv
M07LTB ALTTO ‘A randomised, multi-centre, open-label, GS Sonke III 27-5-2008 0
phase III study of adjuvant lapatinib,
trastuzumab, their sequence and their
combination in patients with HER2/ErbB2
positive primary breast cancer; ALTTO study’
M08BCP Prospective and Retrospective register study SC Linn other 27-3-2008 27
of 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 25-11-2008 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
M08PTP A phase Ib open-label, two arm study of i.v. and JHM Schellens I 11-12-2008 0
oral panobinostat(LBH589) in combination with
i.v. trastuzumab (Herceptin) and i.v. paclitaxel
as treatment for adult female patients with HER2
overexpressing metastatic breast cancer (MBC)
M08TRA TRAIN Trastuzumab in a neoadjuvant regimen for GS Sonke II 18-9-2008 6
Her2+ breast cancer - the TRAIN study
N04POM Tailored preoperative chemotherapy in stage II S Rodenhuis II 21-3-2005 84
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 Pilot 5-10-2004 70
* = not registered at trialbureau

166
CLINICAL TRIALS
type of nickname title study coordinator phase activated no pts.
cancer study in NKI-AVL (closed) in NKI-
AVL per
18/12/
2008
N05ECP Prospectief, dubbelblind, gerandomiseerde JHM Schellens III 10-10-2005 70
placebo gecontroleerde farmacologisch
interventieonderzoek: evaluatie van de
frequentie, duur en intensiteit van opvliegers
na interventie met Venlafaxine (EfexorÆ) en
Clonidine versus placebo
N05MIB 99mTc-methoxyisobutylisonitrile (MIBI) for RA Valdes Olmos other 7-6-2005 *
imaging of apoptosis and prediction of tumour
respons to chemotherapy and/or radiotherapy
in cancer patients
N05RHN Feasibility study of intensive alkylating S Rodenhuis other ‘14/11/2005 13
chemotherapy in residual HER2/neu-negative (18/04/2008)’
breast cancer after pre-operative chemotherapy
N05TOM a clinical trial to compare the efficacy of the HJ Teertstra other 7-2-2006 *
lorad tomosynthesis mammography system to
conventional mammography
N06GLB Phase I study of gemcitabine plus lapatinib JHM Schellens I 16-5-2007 8
(GW572016) in women with advanced breast
cancer
N06IAA randomized phase II/III study of intensified S Rodenhuis II/II 8-1-2007 9
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 20
kinase activities assessed by microarray derived
phosphorylation profiles
N06VNI Meting van de functie van de arm en de bijdrage JJ Hage other 26-9-2006 *
daaraan van de grote borstspieren voor en na de
tweezijdige implantatie van een endoprothese
ten behoeve van een borstreconstructie die
direct uitgevoerd aansluitend aan de huidsparende
borstoperatie
N07BOS BOSOM Genetic determinants of survival and second EJTh Rutgers other 12-12-2007 *
breast cancer development in premenopausal
breast cancer patients
N07MAN Randomized phase II/III study of second-line S Rodenhuis II/II 3-4-2008 0
endrocrine treatment followed by capecitabine
versus capecitabine followed by endrocrine
treatment in patients with metastatic ER
positive breast cancer
N08AFT AFTER A randomized prospective trial of 2-6 weeks SC Linn II 4-8-2008 7
pre-operative hormonal treatment for hormone
receptor postive breast cancer: Anostrozole +/-
fulvestrant or tamoxifen exposure – response in
molecular profile (AFTER-study)
* = not registered at trialbureau

167
CLINICAL TRIALS
type of nickname title study coordinator phase activated no pts.
cancer study in NKI-AVL (closed) in NKI-
AVL per
18/12/
2008
N08RMB Tumorresponse monitoring in patients with MTFD Vrancken other 23-9-2008 5
breast cancer treated with primary systemic Peeters
therapy: towards predicting response in both
the primary tumor and in axillary lymph nodes
GASTRO INTESTINAL
M05RAX RAX Pre-operative chemoradiotherapy regimen with C Marijnen II 20-3-2006 5
capecitabine and bevacuzimab in locally
advanced rectal cancer. A feasability study (RAX)
M06CRI CRITICS A multicenter randomized phase III trial of M Verheij III 11-1-2007 18
neo-adjuvant chemotherapy followed by surgery
and chemotherapy or by surgery and chemoradiotherapy
in resectable gastric cancer
(CRITICS-study)
M06RCS M1 STUDY A phase II study evaluating short course VJ Verwaal II 22-2-2007 2
radiotherapy, neoadjuvant bevacizumab,
capecitabine and oxaliplatin and radical
resection of primary tumor and metastases
in primary stage IV rectal cancer
M06SCR SCRIPT A multicenter phase III randomised trial A Cats III 9-5-2006 5
comparing total mesorectal excision with
pre-operative radiotherapy with or without
post-operative oral capecitabine in the
treatment of operable primary rectal cancer
M06SUS SUSTENT ‘Surgical gastrojejunostomy or endoscopic A Cats III ‘06/09/2006 0
duodenal stent placement for the palliation (10/05/2008)’
of malignant gastric outlet obstruction;
a randomized study. Surgery versus Stent for
malignant gastro-duodenal obstruction;
SUSTENT study’
M07CBO CAIRO3 Maintenance treatment with capecitabine and A Cats III 4-9-2007 2
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
M07DDO D-DOCCS Development of a docetaxel, oxaliplatin, A Cats I 23-7-2007 9
capecitabine combination schedule in patients
with advanced cancer of the stomach or the
gastro-esophageal junction, a phase I study
M07HBT HerBerT Feasibility study of external beam radiation C Marijnen I/II 25-7-2007 9
therapy followed by high-dose rate endorectal
brachytherapy (HDBRT) in inoperable rectal
cancer patients

168
CLINICAL TRIALS
type of nickname title study coordinator phase activated no pts.
cancer study in NKI-AVL (closed) in NKI-
AVL per
18/12/
2008
M07HEP HEPATICA A randomized two arm phase III study in patients A Cats III 28-4-2008 5
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)
M07NAR NARCIS ‘Neo-Aduvant Radiotherapy-Chemotherapy E Jansen I/II 28-1-2008 0
In Stomach 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 12-2-2008 *
as a tracer of VEGF expression in patients with
colorectal liver metastases.
Selecting patients who may benefit from
anti-VEGF therapy and visualizing the response’
M07RCM SILENT Simultaneous integrated boost radiation A Cats I 31-1-2008 6
therapy with concomitant capecitabine and
mitomycin-C chemotherapy for locally advanced
anal carcinoma
M08ACL Accelerated growth of synchronous colorectal TJM Ruers II 21-2-2008 4
liver metastases: effects of neo-adjuvant therapy
M08DCS Tumor destruction and DC activation in situ: TJM Ruers Pilot 18-9-2008 *
towards in vivo loaded DC vaccines
M08GPO Genetic and protein profiling in patients with JW van Sandick other 1-9-2008 3
oesophageal cancer
N02ECC A single institution phase II study of ECC H Boot II 29-8-2002 133
(Epirubicin, Cisplatin, Capecitabin) in locally
advanced or metastatic gastric cancer and
adenocarcinoma of the oesophago-gastric
junction and distal oesophagus
N02RCA Post operative chemo-radiotherapy after H Boot I/II ‘19/12/2002 70
resection of gastric and esophageal cancer (11/09/2008)’
N05GEA Saving the gastro-epiploic artery at VJ Verwaal III 21-3-2006 37
omentectomy and the effect on post-surgical
recovering gastric function
N05MCT a phase II study of treatment with the ME Smits other 5-12-2005 3
combination of unlabelled (cold) and
radioactive (hot) meta-iodobenzylguanidin
(MIBG) in metastatic carcinoid tumours
N05STP Serum and tissue protein profiling and tumour ME Smits other 19-1-2006 133
genetic analysis in patients with potential
premalignant conditions or colorectal cancer
* = not registered at trialbureau

169
CLINICAL TRIALS
type of nickname title study coordinator phase activated no pts.
cancer study in NKI-AVL (closed) in NKI-
AVL per
18/12/
2008
N06DCM Feasibility of the analysis of platinum-DNA JHM Schellens other 12-12-2006 4
adducts in tumour tissue in cisplatinum treated
with advanced gastric cancer
GYNAECOLOGICAL
E55041 A randomised, multicentre, phase III study of WJ van Driel III ‘25/09/2006 9
Erlotinib vs observation in patients with no (20/02/2008)’
evidence of disease progression after first line,
platinum-based chemotherapy for high-risk
stage I and stage II-IV ovarian epithelial,
primary peritoneal, or fallo
M05HIR HIRISE Hormonal substitution after prophylactic M van Beurden other 31-5-2005 *
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 WJ van Driel other 12-1-2006 *
ovarian cancer patients
M05SNV GROINSS-V II GROningen International study on sentinel WJ van Driel other 26-3-2007 9
nodes in vulvar cancer (GROINSS-V) II
M06HRT NOVARIA The effect of hormonal replacement therapy on CM Korse other 25-9-2006 48
menopausal complaints related to biochemical
changes in surgically and naturally postmenopausal
women. A prospective observational
comparative study
M06OVH OVHIPEC Phase III randomised clinical trial for stage III WJ van Driel III 4-1-2006 15
ovarian carcinoma randomising between
secondary debulking surgery with or without
hyperthermic intraperitoneal chemotherapy
(OVHIPEC-1)
M06RTE PORTEC-3 Randomised phase III trial comparing B van Triest III 28-3-2007 1
concurrent chemoradiation and adjuvant
chemotherapy with pelvic radiation alone in
high risk and advanced stage endometrial
carcinoma: PORTEC-3
M07OVE OVERT-1 A phase II, double blind placebo controlled S Rodenhuis II 1-7-2008 5
multicenter randomised study of AZD0530 in
patients with ovarian cancer sensitive to
platinum based chemotherapy (OVERT-1)
M07RCV Phase II study of definitive radiochemotherapy WJ van Driel II 26-6-2007 0
for locally advanced squamous cell cancer of
the vulva: an efficacy study
* = not registered at trialbureau

170
CLINICAL TRIALS
type of nickname title study coordinator phase activated no pts.
cancer study in NKI-AVL (closed) in NKI-
AVL per
18/12/
2008
N05CGO Randomized clinical pharmacological dose- JHM Schellens I 8-9-2005 18
escalation 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
HEAD AND NECK
E90061 Multicenter parallel phase II trial of BI 2536 JHM Schellens II ‘16/01/2008
administered as one hour IV infusion every (01/09/2008)’ 0
3 weeks in defined cohorts of patients with
various solid tumors. A new drug screening
program of the EORTC network of core
institutions (NOCI)
M02STA The effect of a statin on the progression of the FJP Hoebers III 28-1-2003 79
intima-media thickness induced by radiotherapy
M05PET FDG-PET for avoidance of futile direct M van de Brekel other 11-5-2005 *
laryngoscopies under general anaesthesia with
taking of biopsies in patients with suspicion on
recurrent laryngeal carcinoma after radiotherapy
M07CMD Treatment of myogenic cranio mandibular FJM Hilgers III 29-8-2007 *
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 10-6-2008 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
M07MSH Molecular staging of surgically treated head and M van de Brekel other 12-2-2008 *
neck cancer patients: towards rationalized
postoperative clinical management
M08SNU SNUS Ultrasound guided fine needle aspiration M van de Brekel other 26-6-2008 1
cytology and sentinel node biopsy in the
detection of occult lymph node metastases of
early oral and oropharyngeal cancer
N02SNL Early detection of lymph node metastasis of PJFM Lohuis Pilot 3-4-2002 8
squamous cell carcinoma of the larynx with
lymphatic mapping and sentinel node biopsy
N04LFV Longfunctie na toediening van inhalatie- FJM Hilgers III 7-2-2005 *
medicatie (salbutamol-ipratropiumbromide
dosisaerosol) met en zonder voorzetkamer bij
gelaryngectomeerden
N04RTB Effecten van bestraling op bloedvaten NS Russell Pilot 5-10-2004 70
* = not registered at trialbureau

171
CLINICAL TRIALS
type of nickname title study coordinator phase activated no pts.
cancer study in NKI-AVL (closed) in NKI-
AVL per
18/12/
2008
N05HME De korte termijn invloed van een Heat and JK Zuur Pilot 1-9-2005 *
Moisture Exchanger op de endotracheale
temperatuur en luchtvochtigheid bij
gelaryngectomeerden
N05MIB 99mTc-methoxyisobutylisonitrile (MIBI) for RA Valdes Olmos other 7-6-2005 *
imaging of apoptosis and prediction of tumour
respons to chemotherapy and/or radiotherapy
in cancer patients
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 16-5-2007 *
heat and moisture exchanger with bacteria, virus
and pollen filtration capabilities (HME-F)
N07MCP Microcirculatory changes during photodynamic MP Copper Pilot 21-5-2007 *
therapy (PDT) in squamous cell carcinoma of
the oral cavity and oropharynx
N07SHT Subglottic humidity and temperature in nasal RJ Scheenstra other 14-5-2007 *
breathing and tracheotomy breathing with
upper airway occlusion
N07VEG Development of a next generation indwelling FJM Hilgers I/II ‘28/03/2007 *
Provox voice rehabilitation system (Vega) (25/09/2008)’
N07VIN Transoral resection of stage I-II carcinomas of M van de Brekel II 12-3-2007 5
the 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
M05H65 HOVON 65 A randomized phase III study on the effect of JW Baars III ‘03/10/2005 4
Bortezomib combined with Adriamycin, (16/05/2008)’
Dexamethason (AD) for induction treatment,
followed by High Dose Melphalan and
Bortezomib alone during maintenance in
patients with multiple myeloma
M05H68 HOVON 68 A randomized phase III study in previously M Kerst III 21-3-2006 2
untreated patients with biological high-risk CLL:
fludarabine + cyclophophamide (FC) versus
FC + low dose alemtuzumab
LUNG
E08062 Randomized phase II study of amrubicin as
single agent or in combination with cisplatin
versus etoposide-cisplatin as first-line treatment
in patients with extensive stage SCLC (ES)
P Baas II 28-2-2008 0
* = not registered at trialbureau

172
CLINICAL TRIALS
type of nickname title study coordinator phase activated no pts.
cancer study in NKI-AVL (closed) in NKI-
AVL per
18/12/
2008
M03THA NVALT 5 Phase III trial of the antiangiogenic agent P Baas III 18-2-2004 86
Thalidomide in patients with malignant pleural
mesothelioma after first line chemotherapy
(NVALT5)
M05ATD Accurate target definition of non-small cell lung J Stroom other 5-1-2006 *
tumors using pathology-validated PET and CT
imaging for the optimization of radiation
treatment
M05GCP Pharmacogenomic and pharmacokinetic study JHM Schellens other 13-9-2005 124
in 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 8-11-2006 27
clinical stage I/II non-small cell lung cancer
(NSCLC)
M07CCL RADITUX Open-label, randomised multi-center study MM van den Heuvel I/II 13-3-2008 15
investigating Cetuximab, in combination with
concurrent chemo-radiotherapy in locally
advanced non-small cell lung carcinoma
M07ESU Randomized double blind phase 2 study of P Baas II 29-5-2007 9
erlotinib with or without SU11248 in the treatment
of metastatic non small cell lung cancer
M07OSM A phase III randomized, double blind, placebo P Baas II/II 9-10-2007 22
controlled trial of oral suberoylanilide
hydroxamic acid (L-001079038) in patients with
advanced malignant pleural mesothelioma
previously treated with systemic chemotherapy
M07STN START A multi-center phase III randomized, double- MM van den Heuvel III 13-12-2007 3
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
N00ALF Endoscopic detection op pre-neoplastic lesions P Baas Pilot 30-6-2000 41
and carcinoma in the bronchial tree with deltaaminolevunic
acid fluorescence
N04LSN Feasibility of lymphoscintigraphy and sentinel HM Klomp Pilot 8-9-2004 5
node biopsy in patients with non-small lung
cancer
N05MIB 99mTc-methoxyisobutylisonitrile (MIBI) for RA Valdes Olmos other 7-6-2005 *
imaging of apoptosis and prediction of tumour
respons to chemotherapy and/or radiotherapy
in cancer patients
* = not registered at trialbureau

173
CLINICAL TRIALS
type of nickname title study coordinator phase activated no pts.
cancer study in NKI-AVL (closed) in NKI-
AVL per
18/12/
2008
N07NRA A phase I/II study with NAMI-A, a Novel JHM Schellens I/II 31-7-2008 3
Ruthenium Anticancer Agent, and gemcitabine
combination second-line therapy in NSCLC
patients
LYMPHOMA - HODGKIN’S DISEASE
E20012 BEACOPP (4 cycle’s escalated + 4 cycle’s JW Baars III 29-3-2004 1
baseline) vs 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
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 RLM Haas III 2-2-2004 6
irradiation 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 20-7-2005 1
after induction therapy (R-CHOP versus R-FC)
for elderly patients with mantle cell lymphoma
not suitable for autologous stem cell
transplantation
M05H63 HOVON 63 Randomized phase III study of Rituximab with JW Baars III ‘21/03/2006 1
intensified CHOP chemotherapy (R-iCHOP-14) (25/03/2008)’
versus Rituximab with High-Dose Sequential
Therapy and Autologous Stem Cell Transplantation
(R-HDT+ASCT) in adult patients (18-65years)
M06CMV Cytomegalovirus (CMV) – specific T cell M Kerst other 28-9-2006 *
immunity in B cell malignancies
M06H77 HOVON 77 Efficacy and safety of a single dose of JW Baars II 23-5-2006 0
14.8 MBq/kg 90Y-ibritumomab tiuxetan (Zevalin)
in elderly 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 HOVON 80 Phase II study on the feasability and efficacy of JW Baars II 10-9-2007 1
R-DHAP-MTX combined with i.t. rituximab and
autologous SCT in patients with a recurrent
aggressive B-cell NHL with CNS localisation
M07H84 HOVON 84 Randomized phase III study on the effect of JW Baars III 22-1-2008 0
early intensification of rituximab in combination
with 2-weekly CHOP chemotherapy followed by
rituximab maintenance in elderly patients with
DLBCL
* = not registered at trialbureau

174
CLINICAL TRIALS
type of nickname title study coordinator phase activated no pts.
cancer study in NKI-AVL (closed) in NKI-
AVL per
18/12/
2008
N03OFP ‘A phase II study of eradication therapy with JP de Boer II 6-10-2003 8
additional oral fludarabine in t(11;18) positive
gastric MALT lymphoma’
N03RIM Radioimmunotherapy (131I-antiCD20 monoclonal JW Baars Pilot 25-6-2003 1
antibody) as consolidation treatment after
remission induction for patients with relapsed
or refractory CD20+ B-cell NHL
N05MIB 99mTc-methoxyisobutylisonitrile (MIBI) for RA Valdes Olmos other 7-6-2005 *
imaging of apoptosis and prediction of tumour
respons to chemotherapy and/or radiotherapy
in cancer patients
N07RIT Determination of the pharmacokinet ics of JW Baars 14-4-2008 22
rituximab and Ab to rituximab
N08IRP Iodine-124-rituximab positron emission WV Vogel Pilot 17-12-2008 *
tomography (IR-PET) for molecular imaging of
CD20 expression
MELANOMA / SKIN
M05MSL MSLT-II A phase III multicenter randomized trial of OE Nieweg III 11-9-2006 3
sentinel 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
M06MDX A randomized, double-blind, multicenter study JBAG Haanen III ‘13/09/2006 17
comparing MDX-010 monotherapy, MDX-010 in (22/07/2008)’
combinatio with a melanoma vaccine monotherapy
in HLA-A*0201-positive patients with
previously treated unresectable stage III or IV
melanoma
N03CYL Pilot study of topical application of salicylate in R Oosterkamp Pilot ‘26/06/2003 5
familial cylindromas (30/01/2008)’
N03LAM Longitudinal analysis of melanoma-specific JBAG Haanen II 22-8-2003 3
immunity in stage III and IV melanoma patients
N06TIS ‘Integrated analyses of melanoma-T cell JBAG Haanen other 29-8-2006 11
interactions; relevance for immunotherapy’
MISCELLANEOUS
M06PRO PROTECT The efficacy of therapeutic compression hoses M Stuiver other ‘05/09/2006 59
for prevention of lymphedema after inguinal (11/09/2008)’
lymph node dissection in cancer patients
N01RIT Identifications of molecular mechanisms NS Russell Pilot 7-5-2002 30
involved in radiation-induced telangiectasia
* = not registered at trialbureau

175
CLINICAL TRIALS
type of nickname title study coordinator phase activated no pts.
cancer study in NKI-AVL (closed) in NKI-
AVL per
18/12/
2008
N03THY Therapeutic management of thymoma and LGH Dewit Pilot 25-11-2003 15
thymic carcinoma: – a prospective registration
study based on preoperative risk assessment
of local failure.
N05CHO The efficacy of cordotomies in patients with A Lukas II 17-1-2006 *
chest pain due to primariy localized malignancies
of the chest
SOFT TISSUE / OSTEOSARCOMA
E62012 Randomised trial of single agent doxorubicin S Rodenhuis III 9-7-2003 19
versus doxorubicin plus ifosfamide in the first
line treatment of advanced or metastatic soft
tissue sarcoma
E62024 Intermediate and high risk localized, completely S Rodenhuis III ‘25/11/2004 13
resected, gastrointestinal stromal tumors (GIST) (20/10/2008)’
expressing KIT receptor:a controlled randomized
trial on adjuvant Imatinib mesylate (Glivec)
versus no further therapy after complete surgery
E62061 Randomised phase II study of brostallicin M Kerst II ‘12/03/2007 7
(PNU-166196A) versus doxorubicin as first line (07/08/2008)’
chemotherapy in patients with advanced or
metastatic soft tissue sarcoma
E62072 PALETTE A randomized double blind phase III trial of M Kerst III 18-11-2008 0
Pazopanib versus placebo in patients with soft
tissue sarcoma whose disease has progressed
during or following prior therapy
M01EUR EURO-EWING EURO-E.W.I.N.G. 99. EUROpean Ewing tumor S Rodenhuis III ‘18/09/2001 13
Working Initiative of National Groups (04/12/2008)’
M01ROS Phase II study of rosiglitazone in advanced S Rodenhuis II 24-8-2001 5
liposarcoma
URO-GENITAL
E30983 Randomized phase II/III study of Taxol-BEP M Kerst II/II 7-9-1999 4
versus BEP in patients with intermediate
prognosis germ cell cancer
E30986 Randomized phase II/III study assessing JH Schornagel II/II ‘07/05/2002 21
Gemcitabine/Carboplatin and Methotrexate/ (17/03/2008)’
Carboplatin/Vinblastine in previously untreated
patients with advanced urothelial cancer
ineligible for Cisplatinum based chemotherapy
E30994 Randomized phase III trial comparing immediate M Kerst III ‘28/01/2003 26
versus deferred chemotherapy after radical (25/07/2008)’
cystectomy in patients with pT3-pT4, and/or
N+ M0 transitional cell carcinoma (TCC) of the
bladder.
* = not registered at trialbureau

176
CLINICAL TRIALS
type of nickname title study coordinator phase activated no pts.
cancer study in NKI-AVL (closed) in NKI-
AVL per
18/12/
2008
M00LMT Identification of occult lymph node metastases S Horenblas Pilot 14-11-2000 *
in testicular cancer to select patients for
adjuvant treatment. Feasability of a laparoscopic
selective retroperitoneal lymphadenectomy.
M06HFP HYPRO Hypofractionated irradiation for prostate cancer: F Pos III 8-3-2007 87
a randomized multicenterphase III study
M06LAN Late Adverse effects in Dutch testicular cancer M Kerst other 12-2-2007 *
survivors: a Nationwide case-control study on
Cardiovascular Events
M06SIL Phase I dose-escalation trial for the combination JBAG Haanen I 20-11-2006 3
of sorafenib with interleukin-2 treatment in
patients with clear cell renal carcinoma
M07ILS An open label, dose escalation safety and JBAG Haanen I/II ‘23/07/2007 2
tolerability trial of the combination of s.c. (18/04/2008)’
recombinant human IL-21 (rIL-21) and sunitinib
(phase I) followed by an open label stratified
randomized 2-arm trial of rIL-21 + sunitinib
versus sunitinib alone (phas
M07PGC Phase II trial of paclitaxel, gemcitabine and M Kerst II 8-1-2008 0
cisplatin in patients with relapsing germ cell
cancer after first line chemotherapy
M08ZDP ENTHUSE M1 A phase III randomized double blind study to HG van der Poel III 8-9-2008 4
assess the efficacy and safety of 10 mg ZD4054
versus placebo in patients with hormone
resistant prostate cancer and bone metastasis
who are painfree and mildly symptomatic
M94SAL Salvage regimen incorporating repeated ablative S Rodenhuis II 4-7-1994 24
chemotherapy with autologous PSCT, a phase II
study
N06MPM Samarium-153-EDTMP (QUADRIMET) versus HG van der Poel II 14-3-2007 7
docetaxel for multiple painful oseous
metastases in prostate cancer
N06SNR Feasability of lymphoscintigraphy and sentinel
node biopsy in patients with renal cell carcinoma A Bex other 9-10-2007 *
N06SUN Sunitinib prior to nephrectomy in patients with A Bex II 16-5-2007 10
metastatic renal cell carcinoma and the primary
in situ
N07TAP TAP-block Evaluation of the Transversus Abdominis Plane – DR Buitelaar other 8-1-2008 84
block (TAP-block) in preventing painful urine
bladder spasm in patients undergoing Robot
Assisted Laparoscopic Radical Prostatectomy
under general anesthesia, a prospective
randomized controlled trial
* = not registered at trialbureau

INVITED SPEAKERS 2008
Fabrizio d’Adda di Fagagna, Milan, Italy
Molecular bases of cellular senescence
Bruce Alberts, San Francisco, CA, USA
Biology past and biology future: where have we been and
where are we going?
Haico van Attikum, Leiden, The Netherlands
Chromatin and the DNA damage response
Bradley Bernstein, Boston, MA, USA
Genome-wide maps of chromatin state in pluripotent
and multipotent cells
Eveline Bleiker, Amsterdam, The Netherlands
Psycho-oncogenetics: opportunities for a center of
excellence
Thijn Brummelkamp, Boston, MA, USA
Organ size, stem cells and loss of function genetics in
mammals
Luc Brunsveld, Dortmund, Germany
Chemical biology approaches for the nuclear receptor –
cofactor interaction
Dmitry Bulavin, Singapore
Wip1 phosphatase to trace adult stem cells and wip-out
diseases
Harmen Bussemaker, New York, NY, USA
Predicting expression from sequence using data-driven
models of (post-)transcriptional networks
Anthony Chalmers, Brighton, UK
Radiation plus PARP inhibition in the treatment of high
grade glioma: enhancing the therapeutic ratio
Gerhard Christofori, Basel, Switserland
Distinct mechanisms of tumor cell invasion and
metastasis
Sarah Darby, Oxford, UK
Effects of breast radiotherapy on cardiovascular disease
Heran Darwin, New York, NY, USA
Proteasomes, a new ubiquitin-like protein, and
tuberculosis
Marileen Dogterom, Amsterdam, The Netherlands
Dynamics and force generation of cytoskeletal systems
in vitro and in vivo
Anindya Dutta, Charlottesville, VA, USA
Genomic instability of mammalian cells from disorders
of replication
177
INVITED SPEAKERS
Ger van den Engh, Seattle, WA, USA
Cell sorters for advanced research: how standard flow
cytometers can be adapted for microbiology, large, fragile
cell analysis and single-cell genomics
Ype Elgersma, Rotterdam, The Netherlands
Oncogenes on my mind: the role of the RAS/ERK/
mTOR pathways in neuronal function
Emer S. Ferro, São Paulo, Brazil
Intracellular peptides as natural regulators of cell
signaling
Alan Fersht, Cambridge, UK
The tumour suppressor p53: structure, function-rescue
Pier Paolo di Fiore, Milan, Italy
Integration of EGFR signaling and traffic
Judy Garber, Boston, MA, USA
Triple negative breast cancer: An update
Kenneth Gilhuijs, Amsterdam, The Netherlands
Multi-modality image analysis to guide diagnosis and
therapy of cancer
Ernesto Guccione, Milan, Italy
Histone Methylation: Regulator of chromatin activity
and transcription factor binding
Peter van Haastert, Groningen, The Netherlands
Navigation of amoeboid cells in chemical gradients
William Hahn, Boston, MA, USA
Functional genomics, experimental models and cancer
genes
Benjamin Haibe-Kains, Brussels, Belgium
Gene modules, breast cancer subtypes and prognosis
Samir Hanash, Seattle, WA, USA
Mining the cancer proteome for diagnostic markers and
therapeutic targets
Kristian Helin, Copenhagen, Denmark
Epigenetics, stem cells and cancer
Thomas Helleday, Oxford, UK
Homologous recombination in mammalian cells
Jeroen den Hertog, Utrecht, The Netherlands
Phosphotyrosine signaling in gastrulation cell
movements: modeling Noonan syndrome in zebrafish
Ian Hickson, Oxford, UK
Genome instability and cancer: lessons from analysis of
Bloom’s syndrome

178
INVITED SPEAKERS
Tan Ince, Boston, MA, USA
Cell of origin and neoplastic phenotype
Metello Innocenti, Frankfurt, Germany
Actin dynamics in membrane protrusion and
endocytosis
Stefan Jakobs, Goettingen, Germany
Fluorescence nanoscopy: Taking a closer look at cellular
structures
Stefan Jentsch, Martinsried, Germany
Role of Ubiquitin and Sumo in DNA transactions
Joseph Jiricny, Zürich, Switzerland
When DNA repair kills: cytotoxic processing of
methylation damage and 5-fluorouracil
Jagath Reddy Junutula, San Francisco, CA, USA
Targeted cancer chemotherapy: new insights into
antibody drug conjugates
Sander van Kasteren, Dundee, Scotland
Imaging inflammation – novel probes for detecting
vascular selectin upregulation
Patrick Kemmeren, Utrecht, The Netherlands
An accurate physical interactome map of saccharomyces
cerevisiae
Batsheva Kerem, Jerusalem, Israel
Regulation of chromosomal instability in response to
replication stress
Nevan Krogan, San Francisco, CA, USA
Biology without bias: Functional insights from genetic
and physical interaction maps
Peter Kuhn, La Jolla, CA, USA
Circulating tumor cells in human blood – a point of
entry for cancer diagnosis, prognosis and therapy
management
Alan Lehmann, Brighton, UK
Replication of DNA damage and translesion synthesis
Susanne Lens, Utrecht, The Netherlands
The chromosomal passenger complex and the control of
chromosomal stability
Daniel Low, St. Louis, MO, USA
Modeling human breathing motion for radiation
therapy and dose response assessments
Umar Mahmood, Charlestown, MA, USA
Oncological applications of optical molecular imaging
Tomi Mäkelä, Helsinki, Finland
Mesenchymal LKB1 is required for suppression of
polyposis and TGFß signaling
Elisabetta Marangoni, Paris, France
Cancer stem cells and residual disease
Eric May, Baden-Baden, Germany
Communicating difficult topics effectively
Martin McMahon, San Francisco, CA, USA
BRaf-induced tumor initiation, progression and
senescence in mouse models of lung cancer and
melanoma
Tom Muir, New York, NY, USA
The histone code explored through protein
semisynthesis
Alexander van Oudenaarden, Cambridge, MA, USA
Signal processing, perfect adaptation and evolvability in
the yeast osmo-sensing network
KJ Patel, University of Cambridge, UK
The FA pathway DNA repair pathway in model
organisms
Jan-Michael Peters, Vienna, Austria
How chromosomes are segregated in mitosis
Richard Peto, Oxford, UK
Changing cancer mortality
Ruth Pfeiffer, Bethesda, MA, USA
Probability of detecting disease-associated Single
Nucleotide Polymorphisms (SNPs) in single stage or
multi-stage case-control genome-wide association studies
Hidde Ploegh, Cambridge, MA, USA
Ubiquitin and a ubiquitin-like modifier that targets RNA
Marie France Poupon, Paris, France
Patient derived breast cancer xenografts a tool for drugefficacy
evaluation
Oliver Rando, Worcester, MA, USA
Static and dynamics genome-wide views of yeast
chromatin
Caetano Reis e Sousa, London, UK
Innate regulation of dendritic cell unction
Chris Reutelingsperger, Maastricht, The Netherlands
Molecular imaging of the biomarker phosphatidylserine
with Annexin A5. Shifting from a diagnostic towards a
therapeutic arena
Sjoerd Rodenhuis, Amsterdam, The Netherlands
Response prediction and response-monitoring in the
neoadjuvant treatment of breast cancer
Rinat Rotem-Jehudar, Yavne, Israel
CT-011, a humanized PD-1-interacting antibody, for the
treatment of hematological malignancies and solid
tumors

Sanne Schagen, Amsterdam, The Netherlands
Cognitive problems following cancer and cancer
treatment
Ben Scheres, Utrecht, The Netherlands
Stem cells: tales from another kingdom
Eran Segal, Rehovot, Israel
Cracking the code of gene regulation
David Sherwood, Durham, NC, USA
Breaching the basement membrane: Anchor cell
invasion in C. elegans
Jan Jakob Sonke, Amsterdam, The Netherlands
Sharpening the soft-knife
Hergen Spits, San Francisco, CA, USA
The role of STAT5 in development of normal and
malignant human B cells
John Sundberg, Bar Harbor, MA, USA
Getting old at the Jackson Laboratory: an overview of the
Jackson Aging Center
Jack Szostak, Boston, MA, USA
What can we learn about the origin of life from efforts to
design an artificial cell
Stepen Taylor, Manchester, UK
How do anti-mitotic drugs kill cancer cells?
Heiddis Valdimarsdottir, New York, NY
Emotion, cognition, and biology in cancer: Theories and
interventions
Richard Vallee, New York, NY, USA
Live in vivo imaging of molecular motor-driven brain
stem cell division and migration
Lidia Vasilieva, Boston, MA, USA
Role of transcription-coupled RNA degradation in
heterochromatic silencing
Karin de Visser, Amsterdam, The Netherlands
The inflammatory tumor microenvironment:
tumor-protective or tumor-promoting?
Robert Weinberg, Cambridge, MA, USA
Mechanisms of malignant progression
Katarina Wolf, Nijmegen, The Netherlands
Molecular determinants of tumor cell invasion and
plasticity
179
INVITED SPEAKERS

180
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 1-10-2003 1-12-2009
Aaronson, Neil KWF 2006-3470 Cognitive behavioral therapy (CBT) and physical exercise for
climacteric symptoms in breast cancer patients experiencing
treatment-indiced menopause
1-9-2006 1-9-2010
Agami, Reuven KWF 2005-3387 Identification and characterization of human tumor suppressor
genes in the p53 pathway
1-9-2005 1-9-2009
Agami, Reuven KWF 2006-3558 Genetic screens to identify cancer related functions of human
microRNAs
1-11-2006 1-11-2010
Agami, Reuven KWF 2007-3881 Role of miRNA genes in etiology of malignant germ cell tumors 1-3-2007 1-3-2011
Agami, Reuven KWF 2007-3908 In vivo functional screens for oncogenic and tumorsuppressive
microRNAs
1-8-2008 1-8-2012
Begg, Adrian KWF 2005-3420 Expression profiling to predict outcome after combined radiotherapy 1-3-2005
and chemotherapy in head and neck tumors
1-5-2008
Bernards, Rene KWF 2005-3375 The Role of PRAME in oncogenesis 1-8-2005 31-7-2009
Bernards, Rene KWF 2008-4042 Identification of enzymes involved in regulatory ubiquitination in
TNF a--stimulated signalling by loss-of-function screens
1-1-2008 1-1-2012
Berns, Ton KWF 2004-3086 Dissecting the role of the pp21 INK4a genes in development and
tumor suppression in mouse models
1-12-2004 1-12-2008
Berns, Ton KWF 2005-3390 Mouse models for Small Cell Lung Cancer. Genotype-phenotype
correlations as a basis to design better therapeutic intervention
strategies
1-5-2005 30-4-2009
Berns, Katrien KWF 2008-4027 Understanding resistance to HER2-targeting therapy in human
breast cancer through functional genetics
1-1-2008 1-1-2012
Blank, Christian KWF2008-3988 Blockade of PD-1/D-L1 interaction to improve T cell mediated
immunotherapy of cancer
1-1-2008 1-1-2012
Bleiker, Eveline KWF 2004-2987 Long-term physosocial impact of genetic testing among familial
adenomatous polyposis(fap) families
1-11-2004 1-11-2008
Bleiker, Eveline KWF 2005-3209 Psychosocial aspects of genetic testing in families at high risk of
multiple tumors at various sites and ages
1-11-2005 1-11-2009
Bleiker, Eveline KWF 2008-4016 Identification of psychosocial problems and perceived need for
support in cancer genetics
1-1-2008 1-1-2012
Blitterswijk, Wim van KWF 2005-3377 Lipid rafts as novel targets for anti-cancer therapy and induction
of apoptosis
1-1-2005 1-1-2009
Borst, Jannie KWF 2003-2859 Improving anti-tumor immunity via TNF receptor family member
CD27 and its ligand
1-8-2003 1-3-2008
PROJECTS

181 PROJECTS
Principal Number of Title Started Ended/Ends
investigator project
Borst, Jannie KWF 2004-3087 Manipulation of the CD70 locus to monitor and modulate the
anti-tumor immune response and tumor development
14-6-2004 13-6-2008
Borst, Jannie KWF 2004-3079 Tumor therapy with ionizing radiation and death ligand Trail:
efficacy and key molecular determinants
1-7-2004 14-5-2009
Borst, Jannie KWF2008-4028 T cell programming at the dendritic cell interface: impact on
anti-tumor immunity
1-1-2008 1-1-2012
Borst, Jannie KWF2008-4110 Impact of TRAIL death receptor trafficking on pro-apoptotic signaling 1-3-2008 1-3-2012
Borst, Piet KWF 2005-3379 A study of drug resistance mechanisms using large scale RNA
interference screens
13-6-2005 13-6-2009
Borst, Piet KWF 2006-3566 Mechanisms of chemotherapy resistance in spontaneous tumors
of genetically modified mice
21-6-2006 21-6-2010
Brekel, Michiel van den KWF 2007-3941 Prediction of local control after radiotherapy in head and neck cancer 1-11-2007 1-11-2009
Collard, John KWF 2004-3062 The role of Tiam1 and Rac in Wnt/beta-catenin-induced tumorigenicity 1-12-2004 1-12-2008
Collard, John KWF 2007-3753 The par complex in cell polarity and tumor progression 1-7-2007 1-7-2011
Dalesio, Otilia KWF Datamanagement KWF Datamanagement 1-1-1982 1-1-2009
Dannenberg, KWF 2007-3978 Genetic analysis of class I HDACs in development and treatment 1-9-2007 1-9-2011
Jan-Hermen of cancer
Driel, Willemien van KWF CKTO 2006-16 Phase III randomised clinical trial for stage III ovarian carcinoma
randomising between secondary debulking surgery with or without
hyperthermic intraperiotoneal chemotherapy (OVHIPEC-1)
14-3-2007 14-3-2010
Fornerod, Maarten KWF 2004-3080 The role of RanBP3 in beta-catenin dependent nuclear signalling and 1-9-2004
oncogenesis
1-9-2008
Gilhuijs, Kenneth KWF 2004-3082 Application of Pathology-correlated Imaging to Improve the Accuracy 18-10-2004
of Surgery and Radiotherapy in Breast-conserving Therapy
17-10-2008
Haanen, John KWF 2007-3943 HPV 16 E6/E7 DNA prime and E6/E7 long peptide boost vaccination 1-1-2008
for multifocal Vulvar Intraepithelial Neoplasia (VIN)
1-1-2010
Herk, Marcel van KWF 2007-3895 Probability based treatment planning with biological objectives for
high-dose high-precision radiotherapy of prostate cancer
1-11-2007 1-11-2011
Herk, Marcel van KWF 2007-3751 High precision image-guided radiotherapy for bladder cancer 1-1-2008 1-1-2012
Hilkens, John KWF 2001-2489 Cloning of novel mammary tumor progression and metastasis genes 1-7-2001 1-8-2008
Jacobs, Heinz KWF 2008-4112 A cancer genome atlas of the activation-induced cytidine deaminase: 1-3-2008
novel insights into the pathogenesis and prognosis of b cell lymphoma
1-3-2012
Jacobs, Jacqueline KWF 2005-3458 Novel factors involved in chromosome end protection by telomeres 1-1-2006 1-1-2010
Jacobs, Jacqueline KWF 2007-3907 Dissecting the telomere damage response 1-5-2008 1-5-2010
Jalink, Kees KWF 2007-3733 TRMP7, a novel regulator of cytoskeletal tension: implications for
cancer progression, invasion and metastasis
1-9-2007 1-9-2011
Jonkers, Jos KWF 2006-3486 Dissection of the role of E-cadherin loss-of-function in breast cancer 13-2-2006
development and metastasis
12-2-2010

182
PROJECTS
Principal Number of Title Started Ended/Ends
investigator project
Jonkers, Jos KWF 2006-3715 Functional assessment of innate and adaptive immunity in breast
cancer development
1-1-2007 1-1-2011
Jonkers, Jos KWF 2007-3772 Preclinical validation of chemical inhibitors of poly (ADP-ribose)
polymerase (PARP) in conditional mouse models for BRCAassociated
breast cancer
1-2-2007 1-2-2011
Jonkers, Jos KWF2008-4116 Impact of BRCA muntations on breast tumorigenesis and treatment 1-1-2008
response: functional analysist of defined truncation mutants and
unclassified variants
1-1-2012
Leeuwen, Floor van KWF 2004-3068 Long-term risk of second cancers and cardiovascular disease
following treatment of Hodgkiâ€s disease
1-11-2004 1-11-2008
Leeuwen, Floor van KWF 2006-3631 Long-term risk of cancer after ovarian stimulation for in vitro
fertilization
1-4-2007 1-4-2011
Leeuwen, Floor van KWF 2008-3994 Cardiovascular morbidity and mortality in breast cancer survivors 1-10-2008 1-10-2012
Linn, Sabine KWF 2006-3706 Towards patient-tailored systemic therapy in breast cancer:
a combined approached of translational research and mouse
model systems
1-1-2007 1-1-2013
Lohuizen, Maarten van KWF 2005-3376 Investigating the role of the Polycomb- group gene BMi in
medullosblastomas and in neural cancer stem cell maintenance
1-7-2005 1-7-2009
Lohuizen, Maarten van KWF 2007-3877 Epigenetic regulation by the deubiquitinating enzyme USP3:
impact on genome stability and tumorgenesis
1-3-2007 1-3-2010
Lohuizen, Maarten van KWF 2007-3803 Cancer stem cells and breast cancer: a polycomb connection 1-4-2007 1-4-2011
Marijnen, Corrie KWF 2007-3945 Avoidance of overtreatment with radiotherapy in rectal cancer patients 15-9-2007 15-9-2009
Medema, Rene KWF 2004-3063 Structure/function analysis of the role of polo-like kinase-1 in
cellular transformation
1-4-2004 31-3-2008
Michalides, Rob KWF 2005-3388 Molecular basis of resistance to anti-estrogens in estrogen-receptor
positive breast cancer
1-10-2005 1-10-2009
Moolenaar, Wouter KWF 2005-3392 Autotaxin, a secreted lysophospholipase D: role in tumor progression 1-1-2005 1-1-2009
Moolenaar, Wouter KWF 2005-3383 PIP4K-beta/PtdIns5P/p53: A cellular stress regulated pathway and its 1-11-2005
implication in oncogenesis
31-10-2009
Nederlof, Petra KWF 2007-3749 Specific chromosomal imbalance in breast carcinomas as a marker
for breast cancer susceptibility
15-8-2007 15-8-2011
Neefjes, Jacques KWF 2005-3391 TPPII and the role in antigen presentation by MHC Class I molecules 1-10-2005 1-10-2009
Neefjes, Jacques KWF 2007-3883 Anti-cancer drugs and their effects on the ubiquitin cycle 1-4-2007 1-4-2011
Ovaa, Huib KWF 2005-3368 Activity and distribution of proteasome inhibitors 1-7-2005 30-6-2009
Peeper, Daniel KWF 2007-3957 Towards improved melanoma treatment: gene identifcation, in vivo
modeling and drug target discovery
15-4-2008 15-4-2014
PROJECTS
Peeper, Daniel KWF 2005-3382 The role of the neurotrophic tyrosine kinase receptor TrkB in anoikis 11-7-2005
resistance, inavsion and metastasis of human tumors
10-7-2009

183 PROJECTS
Principal Number of Title Started Ended/Ends
investigator project
Peeper, Daniel KWF 2006-3505 Human melanocytic nevi: oncogene-induced senescence as a
potential tumor-suppressing mechanism
1-8-2006 1-8-2010
Remeijer, Peter KWF 2008-4024 Image guided radiotherapy for breast cancer 20-10-2008 20-10-2012
Riele, Hein te KWF 2004-3084 Subtle gene modification to study the role of the mismatch repair
complex MSH2/MSH6 in mutation avoidance and tumor suppression
15-11-2004 14-11-2008
Riele, Hein te KWF 2006-3589 Significance of the Fanconi anemia caretaker pathway in development 1-1-2007
and treatment of cancer
1-1-2011
Riele, Hein te KWF 2007-3790 Role of the G2 restriction point in tumor development and behavior 1-4-2007 1-4-2011
Rookus, Matti KWF 2004-3088 Gene-environment interactions in BRCA1/2 breast/ovarian cancer
families, a project of the Netherlands Collaborative Group on
Hereditary Breast Cancer
6-9-2004 5-9-2008
Rookus, Matti KWF 2007-3756 Heterogeneity of risk of breast and ovarian cancer in BRCA1 and
BRCA2 mutayion carriers
15-11-2007 15-11-2009
Schagen, Sanne KWF 2002-2771 The incidence, nature and etiology of cognitive problems following
chemotherapy for cancer
18-11-2002 17-11-2008
Schagen, Sanne KWF 2007-3797 Late effects of chemotherapy on brain functioning in the elderly 1-1-2007 1-1-2010
Schinkel, Alfred KWF 2003-2940 Compound Mrp2(Abcc2) and other drug transporter knock-out mice 1-11-2003
to study determinants of pharmacokinetics, toxicity risks and possible
therapy optimization for anticancer drugs
1-4-2008
Schinkel, Alfred KWF 2007-3764 ‘OATP1A/1B (SLCO1A/1B) drug uptake transporters in anticancer
drug pharmacokinetics and toxicity risks; possible strategies for
therapy optimization’
1-12-2007 1-12-2011
Schmidt, Marjanka KWF 2007-3839 Genetics determinants of survival and second breast cancer
development in premenopausal breast cancer patients
1-1-2007 1-1-2011
Schumacher, Ton KWF 2003-2860 Feasibility and limitations of T cell receptor gene transfer 1-8-2004 1-1-2009
Schumacher, Ton KWF 2006-3530 Generation of human tumor specific T cells with high affinity
T cell receptors
1-12-2006 1-12-2010
Schumacher, Ton KWF 2007-3825 SNP-based genome-wide identification of hematopoiesis-restricted
minor histocompatibility antigens
1-7-2007 1-7-2011
Sixma, Titia KWF 2006-3476 Structural and functional analysis of a novel E3 ubiquitin ligase
consisting of the polycomb proteins BMI1 and Ring1b/RNF2
1-8-2006 1-8-2010
Sixma, Titia KWF 2008-4014 Comparative study of Ubiqutin-specific proteases 1-2-2008 1-2-2012
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 of
radiotherapy
1-8-2006 1-8-2010
Sonnenberg, Arnoud KWF 2006-3714 Tumor metastatis: the role of acto-myosin contraction in the
downregulation of E-cadherinmediated cell-cell adhesion
15-2-2007 1-2-2008
Sonnenberg, Arnoud KWF 2004-3067 Function of integrin-CD151 complexes in tumor invasion
and metastasis
1-11-2004 31-10-2008

184
PROJECTS
Principal Number of Title Started Ended/Ends
investigator project
Sonnenberg, Arnoud KWF 2008-3996 A mouse stem cell model for skin cancer: Dual role of the integrin
a6B4 in tumor initiation and progression
1-8-2008 1-8-2012
Stewart, Fiona KWF 2005-3370 The role of TGFb and Notch signaling in the development of
radiation-induced telangiectasia
1-4-2005 1-4-2009
Stewart, Fiona KWF 2005-3373 KWF 2005-3373 Radiation-induced atherosclerosis: mechanisms and 1-6-2005
preventive intervention strategies
1-6-2009
Stewart, Fiona KWF 2008-3993 The role of endoglin in induction and repair of cardiovascular damage 1-2-2008 1-2-2012
after radiation alone or in combinatione with trastuzumab (Herceptin)
Tan, Bing KWF Ontwikkelingssamenwerkingsprogramma
KWF Ontwikkelingssamenwerkingsprogramma 1-1-2008 1-1-2012
Tan, Bing KWF 2008-4233 Early detection of primary and recurrent nasopharyngeal carcinoma
(NPC) using (anti-)EBV based tumor markers and options for using
photodynamic therapy (PDT) in the treatment of local disease
1-9-2008 1-9-2013
Verheij, Marcel KWF 2007-3939 Improvement of chemoradiation response in head and neck cancer
by (-)Gossypol (AT-101), a small molecule inhibitor of Bcl-XL/Bcl-2
1-2-2008 1-2-2010
Verheij, Marcel KWF 2008-4113 Short-chain sphingolipids for enhanced cellular uptake of
amphiphilic anti-cancer drugs
1-1-2008 1-1-2012
Vijver, Marc van de KWF 2002-2575 Microarray analysis of breast cancer as a diagnostic tool to guide
optimal treatment
1-2-2002 1-11-2009
Wolthuis, Rob KWF 2007-3789 Targeting Mitotic Protein Destruction as an approach for cancer
therapy
1-9-2007 1-9-2011
Wolthuis, Rob KWF 2008-4135 Divergent Control of Cyclin B1-Cdk1 in cancer cells: a key role in
therapy
1-9-2008 1-9-2012

MAJOR PROJECTS
SUPPORTED BY OTHER ORGANIZATIONS
185 PROJECTS
Principal Granting agency Title Started Ended/Ends
investigator
Agami, Reuven Dr. Josef Steiner
Krebsstiftung
Dr Josef Steiner Cancer Award 2007 1-5-2007 1-5-2011
Agami, Reuven EEG-CEC / EU Mutant p53 as target for improved cancer treatment (mutp53) 1-2-2004 1-2-2009
Agami, Reuven EEG-CEC / EU Indentifying novel regulatory mechanisms of miRNA functions 1-10-2008 1-10-2013
Agami, Reuven NWO Genome wide search for DNA damage checkpoint functions in human
cells (Euryi-award) 15-2-2005 14-2-2010
Agami, Reuven NWO Combined miRNA profiling and genetic screens to identify and
characterize cancer-related miRNAs
1-10-2007 1-10-2012
Balm, Fons Maurits en Anna
de Kock Stichting
Acute en late ototoxiciteit na chemoradiatie van hoofd-halstumoren 1-7-2007 31-12-2008
Bartelink, Harry EEG-CEC / EU Molecular imaging for Biologically Optimized Cancer Therapy (BioCare) 1-3-2004 1-9-2009
Beijersbergen, Roderick Johnson Johnson en Johnson 1-12-2006 1-12-2008
Bernards, Rene Astra Identification of novel activity modulators of small molecule mTOR
kinase inhibitos in breast cancer
7-12-2007 7-12-2009
Bernards, Rene CBG CBG 2e fase: Identification of cancer-relevant genes using a functional
genetic approach 1-1-2004 31-12-2008
Bernards, Rene CGC CGC 2008-2012 1-1-2008 1-1-2013
Bernards, Rene EEG-CEC / EU Identification of Novel Targets for Cancer Therapy (INTACT) 1-1-2004 1-7-2008
Bernards, Rene EEG-CEC / EU Translational and Functional Onco-Genomics: from cancer-oriented
genomic screenings to new diagnostic tools and improved cancer
treatment. (Transfog)
1-6-2004 1-12-2008
Bernards, Rene EEG-CEC / EU Breast Cancer Biomarkers and Functional mediators : harnessing the
New Wealth of Omic Data (Target Breast)
13-2-2006 12-2-2010
Bernards, Rene EEG-CEC / EU EG Rubicon Annex I EC 1-1-2006 1-1-2011
Bernards, Rene NIH System-based predications of responses to cancer therapy 1-9-2005 1-9-2009
Bernards, Rene NWO Spinozapremie 26-9-2005 26-9-2009
Bernards, Rene TI Pharma Kinases in cancer (TI Pharma) 1-7-2007 1-7-2011
Berns, Ton EEG-CEC / EU “Lung stem cells and small-cell carcinoma; Marie Curie International
Incoming Fellowships”
1-1-2007 1-1-2009
Berns, Ton EEG-CEC / EU MUGEN: Identification of Novel Targets for Cancer Therapy.
Translating basic knowledge of functional oncogenomics into cancer
diagnosis and treatment
1-1-2005 1-1-2010

186
PROJECTS
Principal Granting agency Title Started Ended/Ends
investigator
Berns, Ton Merck Merck Res Coll LKR 25006 1-2-2006 1-2-2009
Berns, Ton TI Pharma Kinases in Cancer (TI Pharma) 1-11-2007 1-11-2011
Bleiker, Eveline Dutch Gastr Res
Foundation
Pancreas project (Rotterdam) 1-11-2008 1-11-2009
Boersma, Menno Maastro Accurate target definition of non-small cell lung tumors using
pathology-validated PET and CT imaging for the optimization of
radiotion treatment
1-9-2006 1-9-2010
Borst, Jannie NWO A novel type of ubiquitination regulates apoptosis signaling by
BH3-only protein Bid
1-11-2008 1-11-2012
Borst, Jannie SFN SFN OIO Hendriks SFR 2.1.25 1-11-2002 1-1-2040
Borst, Jannie TI Pharma TNF ligands in cancer 1-3-2006 1-3-2010
Borst, Jannie ZonMw The role of CD27/CD70-regulated cell survival in shaping effector and
memory T cell responses
1-5-2004 1-5-2008
Borst, Piet EEG-CEC / EU Molecular mechanisms underlying chemotherapy resistance,
therapeutic escape, efficacy and toxicity (CHEMORES)
1-2-2007 1-2-2012
Borst, Piet ZonMw Characterization of the physiological roles of Multidrug Resistance
Protein (MRP) 1-6 by in vivo screening for their substrates (TOP subsidie)
1-1-2008 1-1-2012
Cats, Annemieke Roche Ondersteuning junior medical trials coordinator. CRITICS-studie en
rectum-carcinoom projecten. ML20482
1-10-2007 1-10-2009
Coevorden, Frits van SFN Liposarcoom project 1-1-2007 1-12-2008
Cohen, Serge NWO NWO Veni 700,55,405 1-2-2006 31-1-2009
Collard, John EEG-CEC / EU An integrated concept of tumor metastasis: implications for therapy 1-4-2008 1-4-2011
Dalesio, Otilia EEG-CEC / EU ‘eTEN’ Programme - Call identifier: 05/1 ETEN-029334 - ‘TENALEA-ID’ 1-11-2006 1-5-2010
Dalesio, Otilia IKA IKA kankerregistratie 1-9-1990 1-1-2009
Dalesio, Otilia NVALT Statistical Center NVALT Clinical Trials in oncology 1-5-2004 1-1-2010
Dalesio, Otilia VIKC Verdeling voor CKTO projecten VIKC 1-7-2007 1-1-2011
Dam, Frits van Vereniging tegen
de Kwakzalverij
Vereniging tegen de Kwakzalverij 1-1-2007 1-1-2009
Dannenberg, Jan-Hermen NWO-ALW The role of class I HDACs in normal physiology, tumorigenesis and
transcriptional regulation
1-8-2007 1-8-2012
Elsakkers, Peter AMC Poli Familiare Tumoren (vervolg PFT) 1-1-2008 1-1-2009
Filippo, Ronald VU medisch
centrum
Cytofys (EVA project) 1-9-2007 1-9-2011
Fornerod, Maarten NWO-ALW Connecting chromatin to the plasma membrane 1-1-2007 1-4-2010
Gilhuijs, Kenneth EEG-CEC / EU HYPERImage: Hybrid PET-MR system for concurrent
ultra-sensitive imaging
1-4-2008 1-4-2011

Principal
investigator
Granting agency Title Started 187 PROJECTS
Ended/Ends
Haanen, John NWO ZonMW 432-00-001 1-7-2006 1-7-2009
Haramis, Anna Pavlina NWO NWO VIDI Haramis 917-56-322 1-1-2005 31-12-2009
Harten, Wim van CvZ Operations/Lean Management 1-1-2002 1-12-2009
Harten, Wim van CvZ CvZ Array Raster 3 (fase 2) 1-1-2006 31-12-2009
Herk, Marcel van CTMM Personalized chemo-radiation of lung and head and neck cancer
(aiming at the relief of suffering of patients with tumors of the upper
aero-digestive tract using innovative navigation tools in high precision
tumor targeting by chemo-radiation
1-10-2008 1-10-2013
Herk, Marcel van EOS /
voorheen Philips
EOS 1-5-2000 1-5-2010
Hilgers, Frans ATOS Development and evaluation of a third generation ProvoxTM voice
prosthesis
1-6-2003 1-2-2009
Hilgers, Frans ATOS ATOS Medical Collaboration 30-6-2003 1-1-2009
Hofland, Linda Biolitec PDT Biolitec: Photodynamic Therapy 1-1-2005 1-1-2009
Holtkamp, Christine EEG-CEC / EU ESF (Europees Sociaal Fonds) 1-6-2007 1-6-2009
Jacobs, Heinz ZonMw Role of translesion DNA synthesis in immunity and cancer development 1-1-2005 1-1-2010
Jacobs, Jacqueline NWO NWO Vidi Jacobs 1-2-2007 1-2-2012
Jalink, Kees ZonMw ZonMW 9120.6110 1-6-2006 1-6-2010
Jonkers, Jos AICR Genome-wide insertional mutagenesis screens to identify cancer genes
that collaborate with E-cadherin loss in mammary tumourigenesis
1-10-2007 1-10-2010
Jonkers, Jos EEG-CEC / EU Eurosystem 1-3-2008 1-3-2011
Jonkers, Jos Kudos NKI-Kudos Collaborative Research 1-7-2006 1-7-2009
Jonkers, Jos Merck Merck Mouse Model License 1-12-2006 1-12-2008
Jonkers, Jos Organon NKI-Organon 1-8-2006 1-8-2008
Jonkers, Jos TI Pharma Kinases in cancer (TI Pharma) 1-11-2007 1-11-2011
Klomp, Houke Roche Roche HQ Neo adjuvant Tarceva: Tumor remission rate with neoadjuvant 1-7-2006
erlotinib in operable patients with clinical stage I/II non-small cell lung
cancer (NSCLC) (M06NEL)
1-1-2009
Klomp, Houke Vrolijk Aanstelling voor arts-onderzoeker 4-8-2006 1-7-2009
Leeuwen, Fijs van SFN Multifunctional labels for translational in vitro - in vivo annexin V
diagnostics
1-3-2008 1-3-2010
Leeuwen, Flora van AZG Cardiovascular morbidity in testicular cancer survivors: case-control
study of risk factors and assessment of pharmacogenomic determinants
of toxicity
18-2-2005 31-12-2009
Leeuwen, Flora van CGC The benefits and risks of cancer genomics for society 1-1-2006 1-3-2008

188
PROJECTS
Principal Granting agency Title Started Ended/Ends
investigator
Leeuwen, Flora van EEG-CEC / EU EG GENE-Rad-Risk: Radiation exposures at an early age: impact of
genotype on breast cancer risk
1-6-2005 1-6-2009
Leeuwen, Flora van NIH Risk and prognosis of uterine corpus cancer after tamoxifen treatment
for breast cancer
25-9-2007 1-9-2009
Leeuwen, Flora van Pink Ribbon Invloed van lichaamsbeweging en andere leefstijlaspecten op kwaliteit
van leven en prognose bij borstkanker
1-8-2007 1-1-2009
Leeuwen, Flora van SFN SFN 2.4.54. NO 1-3-2008 1-9-2008
Leeuwen, Flora van SFN Ondersteuning epidemiologisch onderzoek 1-7-2007 1-1-2009
Leeuwen, Fred van NPC Chromatin dynamics and epigenetic changes 1-1-2009 1-1-2013
Leeuwen, Fred van NWO The role of histone methylation in silencing and transcriptional memory 1-2-2005 1-2-2009
Leeuwen, Fred van NWO A multifunctional genome-wide screen to unravel the roles of the
conserved epigenetic regulator Dot1 in chromosome organization
(ECHO subsidieronde 2006-2007)
1-7-2007 1-1-2010
Lohuizen, Maarten van Dijkzigt Stem cells in Development and Disease 1-1-2004 31-12-2008
Lohuizen, Maarten van EEG-CEC / EU Platforms for biomedical discovery with human ES cells 1-8-2006 1-8-2010
Lohuizen, Maarten van EEG-CEC / EU Eurosystem 1-3-2008 1-3-2012
Lohuizen, Maarten van Ernst Schering Regulation of histone H2A monoubiquitination byRing1b/Bmi1 and 1-9-2007 1-9-2009
Foundation USP3 in DNA damage response and oncogenic transformation
Lohuizen, Maarten van Merck Merck LKR #40778 PO X21000363 2-6-2006 31-1-2010
Lohuizen, Maarten van NWO SFN 2.2.33 Startgeld 1-1-2005 31-12-2009
Lohuizen, Maarten van NWO-ALW Genome-wide Spatial Profiling of Polycomb Domains in
Drosophila melanogaster
1-12-2007 1-12-2010
Lohuizen, Maarten van NWO-ALW Maintenance of genome integrity by histone (de)ubiquitinating enzymes 1-11-2008 1-11-2013
Luenen, Henri van NWO L1 and Alu elements: Structural and functional analysis of human
non-LTR retrotransposons
1-10-2004 1-10-2009
Luenen, Henri van Roche AvL-prijs 2006 Voorhoeve 1-1-2007 1-1-2009
Luenen, Henri van Roche AvL-prijs 2007 Bruggeman 1-1-2008 1-1-2010
Michalides, Rob TI Pharma Nuclear receptors in targeted cancer therapy: improved methods for
candidate selection
1-2-2007 1-2-2011
Neefjes, Jacques EEG-CEC / EU High throughput development of drugs for immunotherapy of
autoimmune diseases. (Drugs for Therapy)
1-11-2004 1-11-2008
Neefjes, Jacques EEG-CEC / EU The role of the intracellular peptidases in the immune system and in the
cellular metabolism
1-12-2006 1-12-2008
Neefjes, Jacques EEG-CEC / EU Microban 1-11-2005 1-11-2009
Neefjes, Jacques EEG-CEC / EU Systems biology of phagosome formation and maturation - modulation
by intracellular pathogens
1-12-2008 1-12-2012

189 PROJECTS
Principal Granting agency Title Started Ended/Ends
investigator
Neefjes, Jacques KNAW Tissue responses to radiotherapy and development of immunotherapy
for successful radioimmunotherapy of human melanoma
15-11-2007 15-11-2011
Neefjes, Jacques NWO Manipulating the MHC class II system to control immune responses in
autoimmunity 1-10-2007 1-10-2012
Neefjes, Jacques NWO-ALW Defining the MHC class II loading microdomain to understand
immune responses
1-11-2005 1-11-2009
Nieweg, Omgo SFN SFN 2.5.6 IKR Lymphatic mapping 1-5-2004 1-12-2008
Oldenburg, Hester Pink Ribbon Werkhervatting en seksualiteit na borstkanker 1-9-2008 1-9-2011
Ovaa, Huib NWO Modulation of autotaxin activity by small molecules 1-11-2006 1-11-2009
Ovaa, Huib NWO Chemical biology of ubiquitin-like modifications 1-9-2005 1-9-2010
Ovaa, Huib NWO “Development of conditional protein-ligand exchange applied to
immune technology; Onderzoeksprogramma: Integration of Biosynthesis
& Organic Synthesis (IBOS)”
1-9-2008 1-9-2012
Ovaa, Huib SFN SFN: 2.4.58 Cye Dye 11-8-2008 11-8-2009
Ovaa, Huib ZonMw Immunoproteomics: antigen mapping through MHC epitope
exchange reactions
1-11-2008 1-6-2010
Peeper, Daniel EEG-CEC / EU ‘EU FP6 Grant No.037758 ‘ ‘The anoikis suppressor TrkB as a target for
novel anti-cancer agents’
1-1-2007 1-1-2010
Peeper, Daniel EMBO EMBO Young Investigators 1-9-2006 1-9-2009
Peeper, Daniel NWO Novel functional genomic screens to identify pathways that protect
mouse and human cells against oncogenic transformation by mutant RAS
2-12-2002 5-1-2008
Peeper, Daniel NWO Synthetic lethality: a novel tactic for selective anticancer drug target
discovery
1-6-2007 1-6-2012
Peeper, Daniel NWO-ALW Genome-wide identification and functional characterization of novel
tumor suppressor genes
1-1-2007 31-12-2009
Perrakis, Anastassis EEG-CEC / EU EG Biocrystallography Bioxhit 1-1-2004 1-7-2008
Perrakis, Anastassis EEG-CEC / EU BIOXHIT Management 1-1-2004 1-7-2008
Perrakis, Anastassis EEG-CEC / EU Integrating and strengthening the European Research Area FP6-2005
Lifescihealth 6 Proposal NO 037198
1-1-2007 1-7-2009
Perrakis, Anastassis EEG-CEC / EU A multidisciplinary approach to determine the structures of protein
complexes in a model organism
1-2-2005 31-1-2010
Perrakis, Anastassis EEG-CEC / EU From Receptor to Gene: structures of complexes from signalling
pathways linking immunology, neurobiology and cancer (SPINE 2)
1-7-2006 1-7-2010
Perrakis, Anastassis NIH NIH grant 2R01 GM062612-05, EMBL Heidelberg 1-5-2006 1-5-2010
Peters, Peter Aeras Global TB Public Private Partnership for research into and development of 1-5-2007 1-5-2011
Vaccine Foundation medicines, vaccines and diagnostic aids in the domain of AIDS,
tuberculosis and malaria
Peters, Peter EEG-CEC / EU EG AntePrion CT2006-019090 1-6-2006 1-6-2009

190
PROJECTS
Principal
investigator
Granting agency Title Started Ended/Ends
Peters, Peter EEG-CEC / EU EG AntePrion CT2006-019090 (M) Development of a pre-clinical blood
test for prion diseases Coordiantie
1-6-2006 1-6-2009
Peters, Peter EEG-CEC / EU EG ImmunoPrion Immunological and structural studies of prion diversity 1-6-2006 1-6-2009
Peters, Peter EEG-CEC / EU Understanding prion strains and species barriers and devising novel
diagnostic approaches (strainbarrier)
1-11-2006 1-5-2010
Peters, Peter MIN OCW ‘E-cadhering and cadhering-11 and their role in cancer migration’
‘binnen het SmartMix programma NIMIC’
1-2-2008 1-2-2012
Peters, Peter NIH Pathways of antigen presentation by CD 1 a, b and c 1-1-2005 31-12-2009
Riele, Hein te AFM Cellular factors that determine somatic (CTG)n hypermutability in DM1:
targets for therapy
1-7-2008 1-7-2009
Riele, Hein te NWO Horizon 050-71-051 1-12-2006 1-12-2011
Rodenhuis, Sjoerd CTMM Neoadjuvant drug treatment for breast cancer-response-prediction and
response monitoring
1-10-2008 1-10-2013
Rookus, Matti DES ‘DES Schadefonds; Projectleiderschap: Matti Rookus en
Floor van Leeuwen’
15-6-2006 1-1-2010
Rookus, Matti SFN ‘startgeld’ ‘setting up an IBCCS coordination centre at NKI’ 1-1-2009 1-1-2010
Roos, Ed Johnson Sponsorcontract Johnson & Johnson 1-11-2008 1-11-2011
Roos, Ed SFN SFN nieuwe ontwikkelingen 2008 2.4.52 1-3-2008 1-3-2009
Rutgers, Emiel EORTC EORTC breast group 1-1-2000 1-1-2010
Rutgers, Emiel EORTC EORTC Breast Group werkvergadering 1-3-2002 1-1-2010
Sandick, Johanna van Vrolijk Slokdarmkankeronderzoek Stichting Vrolijk 1-1-2008 31-12-2009
Schagen, Sanne Pfizer Assessment of neuropsychological sequelae of tamoxifen and
exemestane in postmenopausal women with early breast cancer.
TEAM-neuropsychologiestudie
1-6-2003 1-1-2009
Schagen, Sanne SFN Nature and mechanism of cognitive deficits following chemotherapy:
an (f)MRI study SFN 2.2.40
1-4-2007 1-1-2009
Schellens, Jan aCBG Conv. NKI-aCBG (Geneesm.) 1-8-2006 1-1-2009
Schellens, Jan Astra AstraZeneca Affinity of AZD1152 24-3-2005 1-1-2009
Schellens, Jan Astra Preclinical in vivo testing of AZD1152 to determine the effect of the
ABC-drug transporters P-glycoprotein (Pgp, Mdr1) and BCRP (Bcrp1,
Abcg2) on tissue distribution and clearance of AZD1152
1-9-2007 1-1-2009
Schellens, Jan Fonds NutsOhra Veiligheid- & kostenbesparingstudie voor DPYD2A genotypering bij
fluoropyrimidinetherapie
1-1-2008 1-1-2010
Schellens, Jan Novartis Novartis evaluation gimatecan 1-3-2005 1-3-2008
Schinkel, Alfred GLAXO An evaluation of organic anion transporters on the pharmacokinetics
and hepatic disposition of drugs using in vitro and in vivo techniques
13-12-2004 12-12-2008

191 PROJECTS
Principal Granting agency Title Started Ended/Ends
investigator
Schinkel, Alfred SLVRT Imapct of the multidrug transportes P-glycoprotein, MRP2 and BCRP on
the in vivo behaviour of experimental anticancer drugs
1-10-2004 1-10-2008
Schinkel, Alfred STW Generation and validation of cyctochrome P450 3A knockout and
transgenic mice as tools for improved drug development and research
1-6-2004 1-10-2009
Schumacher, Ton ACTS (NWO) Development of conditional protein-ligand exchange applied to immune 1-9-2008
technology
1-9-2012
Schumacher, Ton EEG-CEC / EU MIF1-CT-2006-039477 (naik) Determining the relationships between
haematopoletic lineages using genetic barcoding
1-12-2006 1-12-2008
Schumacher, Ton EEG-CEC / EU Attack: Adoptive engineered T-cell targeting to activate cancer killing 1-11-2005 1-11-2010
Schumacher, Ton Landsteiner Stichting MHC multimer-based adoptive T cell therapy 1-5-2006 1-1-2009
Schumacher, Ton Landsteiner Stichting Graft versus Host disease by TCR-engineered T cells: mechanisms and
means for prevention
1-11-2008 1-11-2011
Schumacher, Ton Melanoma
Research Alliance
Platform For MHC-Exchange Based T-Cell therapy For Melanoma 1-9-2008 1-9-2010
Schumacher, Ton University College LRF New Grant Award 06037 toegekend aan Dr Gavin Bendle: 1-3-2007 1-3-2009
London Adjuvant strategies in T cell receptor gene therapy
Schumacher, Ton ZonMw Molecular dissection of AIRE-driven ectopic antigen expression 1-6-2005 1-6-2008
Schumacher, Ton ZonMw T cell receptor gene therapy of metastatic melanoma: a phase I clinical trial 1-4-2005 31-3-2010
Sixma, Titia AZVU Onderzoekschool Oncologie 1-1-2000 1-1-2009
Sixma, Titia EEG-CEC / EU MCRTN Ubiregulators: Signal Transduction by Ubiquitination, a Matter
of Location
1-1-2007 31-1-2011
Sixma, Titia EEG-CEC / EU Neurotransmitter Cys-loop recptors: structure, function and disease 1-2-2008 1-2-2012
Sixma, Titia EEG-CEC / EU Mismatch2model-Characterization and quantitative modeling of DNA 1-10-2008 1-10-2012
mismatch repair and its role in the maintenance of genomic stability and
cancer avoidance
Sixma, Titia INTAS Selective ligands of nicotinic acetylcholine receptor subtypes designed on 1-6-2006
the basis of X -ray structures of complexes of acetylcholine-binding
proteins with agonists and antagonists
31-12-2008
Sixma, Titia NWO NWO CW PIO 700.51.401 1-2-2003 1-1-2009
Sixma, Titia SFN Protein purification facility 1-11-2007 1-11-2011
Sixma, Titia STW Exploring new uses of ChhBP as model for ligand-gated ion channel
research
1-12-2003 1-5-2008
Sixma, Titia TI Pharma New approaches for Ligand-Gated Ion Channel (LGIC) drug discovery 1-9-2007 1-9-2011
Smit, Linda AICR Uncovering the molecular pathways that regulate the self-renewal of
breast cancer initiating cells
1-4-2008 1-5-2008
Sonnenberg, Arnoud AICR AICR Grant 06-528 1-10-2006 1-2-2008
Sonnenberg, Arnoud DEBRA Generation of a mouse model for EBS-MD 1-12-2005 1-12-2009

192
PROJECTS
Sonnenberg, Arnoud nierstichting Function of tetraspanin-integrin complex Cd151- a3B1 in development and 1-10-2008
maintenance of the glomerular filtration barrier
1-10-2012
Sonnenberg, Arnoud NWO NWO 815.02.002 1-11-2005 1-11-2009
Sonnenberg, Arnoud NWO-ALW Controlling hemidesmosome dynamics by phosphorylation of residues
on the integrin b4 subunit
1-3-2007 1-4-2011
Steensel, Bas van EEG-CEC / EU NET member of The Epigenome Network of Excellence: Epigenome,
epigenetic plasticity of the genome
1-6-2005 1-6-2008
Steensel, Bas van NWO Whole-genome analysis of the regulation of chromatin structure and
gene expression
1-3-2005 1-3-2010
Steensel, Bas van NWO Multi-gene chromatin domains 1-12-2006 1-12-2010
Steensel, Bas van NWO Chromatin Protein Discovery Project 1-2-2008 1-2-2012
Stewart, Fiona EEG-CEC / EU Cardiorisk - The mechanisms of cardiovascular risks after low
radiation doses
1-2-2008 1-2-2011
Valdes Olmes, Renato ONCO Vision Dection of Deep Located Sentinel Nodes using Preoperative SPECT-CT
with an Hybrid System and Peroperative Real Time Imaging with a
Portable Small Camera
1-5-2008 1-5-2010
Valdes Olmes, Renato EEG-CEC / EU Mammography with molecular imaging (MAMMI) Specific Targeted
project
1-1-2007 1-1-2011
Veer, Laura van ‘t EEG-CEC / EU Molecular signatures as diagnostic and therapeutic targets for
disseminated epithelial malignancies
1-11-2005 1-5-2009
Veer, Laura van ‘t EEG-CEC / EU Translating molecular knowlegde into early breast cancer management:
building on the BIG (Breast International Group) network for improved
treatment tailoring. TRANSBIG
1-3-2004 1-3-2011
Veer, Laura van ‘t EEG-CEC / EU Collaborative Oncological Gene-environment Study 1-10-2008 1-10-2012
Veer, Laura van ‘t TI Pharma Predicting drug responses in breast cancer. TI Pharma T3-108 1-7-2007 1-7-2011
Vijver, Marc van de Astra ASTRA/Kudos/Vijver/Linn 1-1-2006 1-1-2009
Visser, Karin de ZonMw The inflammatory tumor microenvironment and its impact on
breast cancer development and therapy
1-1-2009 1-1-2014
Vyth-Dreese, Florry AMC MR UVA 2006-3606 1-7-2006 1-7-2010
Vyth-Dreese, Florry LUMC ‘KWF 2003-2804 (UL) Ex vivo in situ analyses of Graft versus Host
mechanisms by Minor Histocompatibility antigen specific T cells;
relevance for stem cell transplantation’
1-12-2003 31-5-2008
Wessels, Lodewyk MIN OCW NBIC Biorange 1-1-2004 1-1-2010
Wessels, Lodewyk NBIC NBIC / BioAssist / HTHC Programmeur 1-1-2008 1-1-2011
Wolthuis, Rob NWO-ALW Coordination of cell division by regulated protein destruction 1-1-2007 31-12-2011

PERSONNEL INDEX
Aaronson, Neil 90
Aarts, Marieke 62
Achachah, Mohamed 101
Ackerstaff, Annemieke 143
Adriaansz, Sandra 117
Agami, Reuven 46
Ahmed, Kalam 144
Ait Moha, Daoud 101
Ajouaou, Abderrahim 101
Albers, Harald 34
Alderden, Carolien 117
Alderliesten, Maaike 30
Alderliesten, Tanja 101
Aleman, Berthe 95, 126
Alendar, Andrej 82
Alfieri, Andrea 18
Anirudhan, Gireesh 84
Annabel, Zwaagstra 86
Antonini, Ninja 156
Ariotti, Silvia 57
Armstrong, Nicola 72
Atsma, Douwe 100
Aukema, Tjeerd 100
Axwijk, Priscilla 101
Baank, Saskia 100
Baars, Danny 156
Baars, Joke 116
Baars, Philippe 100
Baas, Paul 116
Baas-Vrancken Peeters, Marie-Jeanne 143
Bakker, Martine 100
Bakker, Sietske 62
Balague Ponz, Olga 100
Balm, Alfons 143
Bartelink, Harry 126
Batteau, Lukas 101
Beaudry, Jean Bernard 80
Beelen, Karin 116
Beers-Bauhuis, Carolien 100
Begg, Adrian 40
Beijersbergen, Roderick 78
Beijnen, Jos 44, 116
Bejaoui, Inès 87
Belderbos, Jose 126
Bendle, Gavin 55
Bentin Toaldo, Cristiane 33
Bergman, André 116
Berkers, Celia 34
Bernards, René 76
Berns, Anton 82
Berns, Katrien 76
Besnard, Peter 101
Bessels, Frauwkje 156
Betgen, Anja 127
Bex, Axel 144
Bin Ali, Rahmen 82
Blank, Christian 61, 116
Bleiker, Eveline 94, 101
Blitz, Johanna 82
Bloemers, Monique 126
Blom, Marleen 80
Blom, Marie-José 95
Boekel, Naomi 95
Boekhout, Annelies 117
Boerrigter-Barendsen, Lucie 100
Bonfrer, Hans 100
Boogerd, Willem 92, 116
Boot, Henk 116
Borkner, Lisa 61
Borst, Jannie 53
Borst, Gerben 126
Borst, Piet 64
Bos, Arnold 46
Bosma, Astrid 38
Boss, David 116
Boutmy-de Lange, Majella 101
Bouwman, Peter 66
Braaf, Linde 38
Brandsma, Dieta 92, 116
Brandwijk, Kim 38
Braumuller, Tanya 66
Braunschweig, Ulrich 48
Broeks, Annegien 38, 95
Brohet, Richard 95
Brouwers, Elke 116
Bruggeman, Sophia 80
Bruin, Sjoerd 38, 143
Bruin, Natascha 100
Buckle, Tessa 100, 101
Bueno de Mesquita, Jolien 68, 99,100
Buikhuisen, Wieneke 116
Buitelaar, Dirk 144
Bulut, Tomas 144
Buning-Kager, Marian 100
Burgers, Sjaak 116
Calbo, Joaquim 82
Cardous-Ubbink, Mathilde 95
Carreno, Monique 156
Cats, Annemieke 116
Cavalli, Silvia 34
Celie, Patrick 20
Chin, Patrick 101
Christodoulou, Evangelos 20
Citterio, Elisabetta 80
Clijsters, Linda 74
Cohen, Serge 20
Collard, John 26
Copper, Marcel 143
Coquet, Jonathan 53
Cornelissen, Sten 38
Cornelissen, Paulien 80
Courrech Staal, Ewout 143
Daems, Daphne 156
Dahmen, Rutger 99
Dalesio, Otilia 156
Damen, Carola 116
Damen, Eugène 126
Dannenberg, Jan-Hermen 87
De Boer, Jan Paul 116
De Boer, Johan 127
De Boer, Roel 126
De Bois, Josine 127
De Bruin, Michiel 66
De Bruin, Chiel 68
De Bruin, Marieke 95
De Castro Genebra de Jesus, Inês 48
De Haas, Marcel 64
De Jong, Annemieke 34
De Jong, Monique 40
De Jong, Daphne 100
De Jong, Gerda 156
De Leeuw, Renée 33
De Marco, Valeria 20
De Punder, Karin 36
De Ridder, Jeroen 72
De Ronde, Jorma 72
De Ruiter, Michiel 92
De Visser, Karin 66
De Vreeze, Ronald 143
De Vries, Diederick 20
De Vries, Evert 53
De Vries, Hilda 82
De Vries, Sandra 62
De Waal, Marjolijn 156
De Widt, John 30
Deenen, Maarten 44, 116
Deetman, Joost 99
Dekker, Marleen 62
Dekker, Rob 62
Dellemijn, Trees 57
Delzenne-Goette, Elly 62
Desmet, Christophe 84
Devriese, Lot 44
Dewit, Luc 126
Dirac, Annette 76
Doodeman, Petra 100
Doodeman, Valerie 44
Dorlo, Thomas 116
Douma, Sirith 84
Douma, Kirsten 90, 94
Doumont, Gilles 66
Drost, Brigit 144
Drost, Jarno 46
Drost, Rinske 66
Dubbelman, Anne Charlotte 116
Dubbelman, Ria 117
Dufournij, Brigitte 156
Duijts, Saskia 90
Duppen, Joop 127
Eenink, Martijn 126
Efthymiou, Katina 144
Egan, David 78
Eijzenga, Willem 94
El Oualid, Farid 34
Elkon, Rani 46
Elkuizen, Paula 126
Ellenbroek, Saskia 26
193
PERSONNEL INDEX

194
PERSONNEL INDEX
Elouarrat, Dalila 24
Engelsma, Dieuwke 52
Epping, Mirjam 76
Evers, Bastiaan 66
Fabius, Armida 78
Faesen, Alex 18
Fazalalikhan, Arifa 100
Feddema, Wouter 64
Feenstra, Christel 100
Fernandez Salcedo, Ernesto 156
Filion, Guillaume 48
Fish, Alex 18
Fles, Renske 38
Floot, Ben 40, 42
Foekema, Joke 117
Fornerod, Maarten 52
Frederiks, Floor 50
Frijns, Evelyne 22
Gadiot, Jules 61
Gallenne, Tristan 84
Gast, Marie-Christine 116
Gebretensae, Abadi 117
Gehring, Karin 90
Geiger, Thomas 84
Genest, Paul-André 88
Gerard, Audrey 26
Gerlach, Carmen 55
Gerritsen, Bram 72, 78
Gerritsma, Miranda 90
Geurts, Tom 143
Geutjes, Ernst-Jan 76
Gilhuijs, Kenneth 101
Gilles, Rozan 99
Godsave, Sue 36
Gomez, Raquel 57
Gomez Benito, Maria 46
Gonggrijp, Maaike 64
Graafland, Niels 144
Gundy, Chad 90, 92, 94
Haanen, John 57, 116
Haas, Rick 126
Haenen, Inge 99
Hage, Joris 144
Hagenaars, Christiane 156
Hahn, Daniela 94, 101
Hajdo-Milasinovic, Amra 26
Hamming-Vrieze, Olga 126
Haramis, Anna-Pavlina 89
Harms, Emmy 117
Hart, Guus 126
Hau, Song-Hieng 156
Hauptmann, Michael 72
Hausmann, Jens 20
He, Youji 38
Heemsbergen, Wilma 126
Heemskerk, Bianca 55, 57
Heessen, Stijn 52
Heidebrecht, Tatjana 20
Heideman, Marinus 59
Heideman, Wieke 95
Heijens, Claudia 116
Hengeveld, Trudi 24
Hesselink, Gijs 99
Heynen, Guus 76
Hibbert, Rick 18
Hiemstra, Annelies 156
Hijmans, Marielle 76
Hilarius, Doranne 90
Hilgers, Frans 143
Hilkmann, Henk 34
Hillebrand, Michel 117
Hoebers, Frank 126
Hoefnagel, Cornelis 100
Hoekstra, Paula 156
Hofland, Ingrid 40
Hogenbirk, Marc 59
Hogervorst, Frans 38, 100, 101
Hollmann, Birgit 126
Holstege, Henne 66
Holtkamp, Marjo 117
Hölzel, Michael 76
Hömig, Cornelia 84
Hoopman, Rianne 90
Hoppes, Rieuwert 34
Houben, Anna 24
Houben, Diane 36
Hoving, Saske 42
Huang, Sidong 76
Huitema, Alwin 116
Hulsman, Danielle 80
Hummel, Marian 99
Iden, Sandra 26
Iri-Kiraz, Hasibe 38
Iusuf, Dilek 70
Ivanov, Eduard 95
Iyer, Jayasree 64
Jacobs, Heinz 59
Jacobs, Jacqueline 88
Jalink, Kees 29
Jansen, Edwin 126
Jansen, Hans 36
Jansen, Robert 116
Janssen, Esther 95
Janssen, Lennert 31
Jaspers, Janneke 66
Jeanson, Kiki 95
John, Hilkens 86
Jongmans, Petra 143
Jongsma, Maikel 24
Jongsma, Marlieke 31
Jongsma, Johan 82
Jonkers, Jos 66
Jonkman, Joop 156
Joosse, Simon 38
Joosten, Krista 20
Jorritsma, Annelies 57
Joshi, Niel 144
Kaiser, Andrew 55
Kalisvaart, Robin 127
Kalverda, Bernike 52
Kant, Josien 156
Kaplon, Joanna 84
Kappers, Ingrid 143
Kasiem, Mobien 101
Kedde, Martijn 46
Keessen, Marianne 117
Keijzer, Christiaan 144
Keizer, Ron 116
Keller, Anna 53
Kerkdijk, Desiree 143
Kersbergen, Ariena 64
Kerst, Martijn 116
Kerver, Emile 116
Ketema, Mirjam 22
Kim, Yeung-Hyen 57
Kimmings, Nikola 143
Kind, Jop 48
Klarenbeek, Jeffrey 29, 30
Klarenbeek, Sjoerd 66
Klerkx, Edwin 156
Klijn, Christiaan 66, 72
Klokman, Willem 95
Klomp, Houke 143
Klop, Martin 143
Klous, Marjolein 116
Kluijt, Irma 94, 101
Knauer, Michael 68, 100
Knegjens, Joost 126
Knols, Ruud 90
Kok, Marleen 38, 68
Kolmschate, Lies 156
Kolodziej, Katy 48
Kool, Jaap 80, 82
Koolen, Stijn 116
Koopman-Kroon, Ciska 117
Koops, Wim 101
Koorn, Jenneke 156
Koornstra, Rutger 68
Koppelmans, Vincent 92
Korse, Tiny 100
Kortlever, Roderik 76
Kortmann, Jan 144
Koseoglu, Murat 46
Koudijs, Marco 66
Kranenburg, Andor 82
Krap, Menno 143
Kreeft, A 143
Kreft, Maaike 22
Kregel, Eva 66
Kreike, Bas 126
Kremmler, Lukas 61
Krewinkel, Han 126
Krijger, Peter 59
Krimpenfort, Paul 82
Kröger, Robert 101
Kross, Kenneth 143
Kuenen, Marianne 90, 95
Kuijl, Coen 31
Kuiken, Johan 78
Kuikman, Ingrid 22
Kuilman, Thomas 84
Kuiper, Maria 117
Kujala, Pekka 36

Kunst, Peter 116
Kwint, Margriet 127
Lagas, Jurjen 70
Lambooij, Jan Paul 82
Lammens, Chantal 90, 94
Lange, Charlotte 101
Lange, Jan 144
Langerak, Petra 59
Langeslag, Michiel 29
Lankheet, Nienke 117
Le Duc, Quint 22
Le Sage, Carlos 46
Lebesque, Joos 126
Leijen, Suzanne 44
Leijte, Joris 144
Lenain, Christelle 84
Léveillé, Nicolas 46
Lieftink, Cor 78
Linders, Dorothé 100
Linn, Sabine 38, 68, 92, 116
Linnemann, Carsten 55
Littler, Dene 20
Liu, Xiaoling 66
Lohuis, Peter 143
Loo, Claudette 101
Lorist, Lissette 156
Lubsen–Brandsma, Lotti 144
Lucas, Luc 117
Lukas, Anne 116
Maas, Chiel 53
Maas, Marnix 126
Madiredjo, Mandy 76
Mager, Alet 116
Mahn, Marianne 156
Mallo, Henk 117
Manders, Peggy 95
Mandjes, Ingrid 156
Mandy, Boer 86
Mans, Anton 127
Marchetti, Serena 44
Marees, Tamara 95
Margadant, Coert 22
Marijnen, Corrie 126
Masselink, Harry 126
Mattiroli, Francesca 18
Mazgani, Mona 144
McDermott, Leah 127
Meijer, Joost 28
Meijerman, Irma 44
Meijnen, Philip 126
Meinhardt, Wim 144
Meissl, Katrin 84
Mencarelli, Angelo 127
Menendez, Victoria 31
Mertens, Sander 26
Meuleman, Wouter 72
Meulenaar, Jelte 117
Mexner, Vanessa 127
Michalak, Ewa 66
Michalides, Rob 33
Michaloglou, Chrysiis 84
Middendorp, Sabine 53
Mijnheer, Ben 126
Mikolajewska, Izabela 38
Minderhoud, Tom 127
Mitsiki, Eirini 20
Modder, Carla 156
Moerman, Esther 144
Moes, Johannes 117
Molloy, Tim 38
Monserrat, Veronica 31
Mooij, Wijnand 20
Mooij, Thea 95
Mook, Stella 38, 101
Moolenaar, Wouter 24
Moonen, Luc 126
Mulder, Ina 95
Mulder, Lennart 38
Mulder, Renée 95
Mullenders, Jasper 76
Muller, Saar 100, 101
Muller, Pietje 156
Muusers, Rick 100
Nacerdine, Karim 80
Nagel, Remco 46
Nagtegaal, Tanja 90, 94
Naik, Shalin 55
Nan-Offeringa, Lianda 117
Nederlof, Petra 38, 95, 100, 101
Neefjes, Jacques 31
Neijenhuis, Sari 40
Nieuweboer-Krobotova, Inka 144
Nieuwenhuis, Lotte 143
Nieweg, Omgo 143
Nijholt, Juup 127
Nijkamp, Jasper 127
Nijkamp, Wouter 78
Nijwening, Jeroen 78
Nol, Annemarie 117
Nooijen, Willem 100
Nota, Ingrid 99
Numan, Rachel 143
Nuyen, Bastiaan 116
Nuyten, Dimitry 126
Nyst, Heike 143
Ogink, Janneke 74
Oldenburg, Hester 90, 143
Olijve, Albert 117
Olsthoorn-Ooms, Marije 101
Olszewska, Agnieszka 127
Ong, Nico 36
Oostendorp, Roos 44, 117
Oosterhuis, Koen 57
Oosterkamp, Rianne 76
Oostveen, Lida 156
Oude Vrielink, Joachim 46
Ouwens, Gabey 95
Ovaa, Huib 34
Paape, Anita 101
Pagie, Ludo 48
Pajic, Marina 64
Pameijer, Frank 101
Pang, Baoxu 31
Panneman, Carmen 127
Paul, Petra 31
Paulus van Pauwvliet, Cecile 156
Pawlitzky, Inka 80
Peeper, Daniel 84
Pengel, Kenneth 101, 127
Peperzak, Victor 53
Peric-Hupkes, Daniel 48
Perrakis, Anastassis 20
Peters, Carolien 127
Peters, Peter 36
Peuscher, Marieke 88
Piek-den Hartog, Marianne 143
Pierson, Jason 36
Pieterse, Mark 66
Pietersen, Alexandra 80
Pijpe, Anouk 95
Pindyurin, Alexey 48
Ploeger, Lennert 127
Plug, Rob 101
Pluim, Dick 44
Ponsioen, Bas 24, 29
Pool, Bert 100
Pos, Floris 126
Postel, Ruben 22
Pramana, Jimmy 143
Prevoo, Warner 101
Prieur, Alexandre 84
Pritchard, Colin 82
Pronk, Loes 156
Proost, Natalie 82
Pruntel, Roelof 101
Puppe, Julian 80
Qiao, Xiaohang 31
Quaak, Susanne 117
Rasch, Coen 126
Rehorst, Harriet 156
Reichgelt, Babs 126
Reinders, Anneke 156
Reker Hadrup, Sine 55
Remeijer, Peter 126
Remmelzwaal, Jolanda 156
Retel, Valesca 99
Reumer, Annet 18
Richard, Wietske 143
Rijkhorst, Erik-Jan 127
Rinia, Bas 143
Rit, Simon 127
Roberts, Daniel 156
Rodenhuis, Sjoerd 116
Rodenko, Boris 34
Roelvink, Marja 117
Rondaij, Mariska 33
Ronday, May 144
Rookus, Matti 95
Roos, Ed 28
Roosjen, Edwin 127
Rooswinkel, Rogier 53
Rooze, Lyandra 100
Rosado, Aranzazu 84
195
PERSONNEL INDEX

196
PERSONNEL INDEX
Rosenberg, Efraim 100, 101
Rosing, Hilde 116
Roskam, Marielle 156
Rossi, Maddalena 127
Rottenberg, Sven 64
Rubio, Miguel 76
Rucktooa, Prakash 18
Ruers, Theo 143
Ruijter-Schippers, Henrique 101
Russell, Nicola 42, 95, 126
Rutgers, Emiel 90, 95, 143
Rzadkowski, Adrian 24
Sachs, Norman 22
Salomon, Izhar 31
Salverda, Govert 126
Sander, Saskia Amra 26
Sani, Musa 36
Scanu, Tiziana 31
Schaap, Jeffrey 143
Schaefer, Henning 46
Schagen, Sanne 92
Scharpfenecker, Marion 42
Scheenstra, Renske 143
Scheerman, Esther 101
Schellens, Jan 44, 116
Schilder, Christien 92
Schinkel, Alfred 70
Schippers-Gillissen, Carla 101
Schmidt, Marjanka 38, 95, 101
Schmitz, Alexander 101
Schmitz, Annemarie 101
Schneider, Christoph 126
Schol, Joke 117
Schornagel, Jan 116
Schot, Margaret 117
Schotte, Remko 55
Schrier, Mariette 46
Schumacher, Ton 55
Schuster, Kerstin 61
Schutte, Peter 144
Schuurman, Karianne 34
Secades, Pablo 22
Seemann, Ingar 42
Seigers, Riejanne 92
Sein, Johan 57
Shanmugham, Anitha 34
Shu, Jenny 55
Siedschlag, Christian 101
Simon, Horenblas 144
Sinaasappel, Michiel 100, 101
Sivro Prndelj, Ferida 100
Sixma, Titia 18
Smeele, Ludi 143
Smit, Judith 18
Smit, Linda 76
Smit, Marjon 84
Smits, Marianne 116
Smitsmans, Monique 127
Snoek, Margriet 82
Sonke, Gabe 116
Sonke, Jan-Jakob 127
Sonneborn, Mariska 100
Sonnenberg, Arnoud 22
Sparmann, Anke 80
Speijer, Gabriele 126
Speksnijder, Ewoud 66
Srámek, Michael 144
Sriram, Jincey 101
Steenbakkers, Roel 126
Stewart, Fiona 42
Stoppa, Tino 144
Storm, Dea 156
Straver, Marieke 143
Stroom, Joep 126
Stulemeijer, Iris 50
Sutherland, Kate 82
Swart, Erwin 55
Taal, Babs 116
Taghavi, Panthea 80
Talhout, Wendy 48
Tan, Bing 143
Tanger, Ellen 80
Te Poele, Johannes 42
Te Riele, Hein 62
Te Velde, Lisette 143
Teertstra, Jelle 101
Temurhan, Mine 127
Ten Bokkel Huinink, Wim 116
Ten Cate, Julia 144
Ter Brugge, Petra 66
Ter Heine, Rob 117
Ter Riet, Bas 64, 74
Tesselaar, Margot 116
Teunissen, Bas 117
Teunissen, Hans 80
Teunissen, Jaap 100
Thijssen, Bas 117
Tibben, Matthijs 117
Tick, Lidwine 116
Tielen, Ivon 101
Tielenburg, René 127
Tilgenkamp, Ria 156
Timmers, Adriaan 144
Tjin, Esther 57
Toebes, Mireille 55
Tolhuis, Bas 80
Topolnjak, Rajko 127
Torres Acosta, Alex 156
Tran, Ly 117
Tromp-van Driel, Catrien 116
Udo, Renate 95
Urbanus, Jos 55
Uren, Anthony 82
Uyterlinde, Wilma 117
Vaessen, Heleen 156
Vainchtein, Liia 117
Vainio, Saara 64
Valdés Olmos, Renato 100
Valkenet, Ludy 156
Van ’t Veer, Laura 38, 95, 100, 101
Van As-Brooks, Corina 144
Van Bemmel, Joke 48
Van Berkel, Peter 99
Van Beurden, Marc 90, 143, 144
Van Blitterswijk, Wim 30
Van Boven, Hester 100
Van Bunningen, Bart 126
Van Coevorden, Frits 143
Van Dam, Frits 92
Van de Ahé, Fina 82
Van de Kasteele, Willeke 57
Van de Meij, Suzan 143
Van de Poel, Henk 144
Van de Steeg, Evita 70
Van de Velde, Tony 156
Van de Vijver, Marc 100
Van de Wetering, Koen 64
Van Deemter, Liesbeth 64
Van den Belt-Dusebout, Sandra 95
Van den Berg, Joost 57, 117
Van den Berk, Paul 59
Van den Bongard, Desiree 126
Van den Brekel, Michiel 143, 144
Van den Brink-de Vries, Nienke 100
Van den Haak, Marjolijn 156
Van den Heuvel, Michel 116
Van den Hoorn, Tineke 31
Van den Hoven, Jolanda 117
Van der Berg, Marieke 144
Van der Burg, Eline 66
Van der Donk, Emile 156
Van der Gulden, Hanneke 66
Van der Heijden, Ingrid 66, 68
Van der Horst, Gerda 53
Van der Kammen, Rob 26
Van der Kant, Rik 31
Van der Kruijssen, Corina 70
Van der Maas, Martin 57
Van der Molen, Lisette 143
Van der Ploeg, Iris 143
Van der Pot, Wouter 144
Van der Sar, Jana 117
Van der Torre, Jaco 88
Van der Veen, Wietze 144
Van der Velde, Hella 52
Van der Velden, Yme 89
Van der Velden, Sophie 101
Van der Wal, Anja 62
Van der Weide, Marchien 116
Van der Wel, Nicole 36
Van Dijk, Pim 18
Van Dongen, Miranda 76
Van Driel, Willemien 144
Van Duijn, Miranda 99
Van Esch, Anita 70
Van Gijn, Roel 116
Van Haaften, Gijs 46
Van Harten, Wim 99
Van Heijst, Jeroen 55
Van Hell, Albert 30
Van Herk, Marcel 126
Van Hien, Richard 38
Van Huizum, Martine 144

Van Kouwenhove, Marieke 46
Van Kranen, Simon 127
Van Leeuwen, Flora 38, 95
Van Leeuwen, Fred 50
Van Leeuwen, Fijs 42, 100, 101
Van Leeuwen, Anne 101
Van Lent, Wineke 99
Van Lohuizen, Maarten 80
Van Luenen, Henri 64
Van Meer, Oene 126
Van Montfort, Erwin 82
Van Mourik, Anke 127
Van Netten, Gabry 156
Van Noord, Janet 88
Van Oosterwijk, Els 156
Van Pel, Renée 100
Van Rens, Anja 101
Van Sandick, Johanna 143
Van Seters, Manon 144
Van Steensel, Bas 48
Van Tellingen, Olaf 100
Van Tinteren, Harm 156
Van Tongeren, Joost 144
Van Triest, Baukelien 126
Van Velthuysen, Loes 100
Van Vliet, Martin 72
Van Vliet-Vroegindeweij, Corine 126
Van Vugt, Huub 80
Van Waardenberg, Wil 156
Van Waterschoot, Robert 70
Van Welsem, Tibor 50
Van Werkhoven, Erik 156
Van Winden, Annemieke 117
Van Zandwijk, Nico 116
Van Zeijl, Leonie 24
Van Zon, Maaike 36
Van Zon, Wouter 74
Van Zwienen, Marieke 126
Vanneste, Ben 126
Vargas, Mark 18
Veltkamp, Sander 44
Vens, Conchita 40
Veraar, Elise 53
Verbrugge, Inge 53
Vergouwe, Ingeborg 144
Verhagen, Caroline 40
Verhagen, C 144
Verheij, Marcel 30, 126
Verhoef, Senno 38, 90, 94, 95, 101
Verhoeven, Els 80
Verloop, Janneke 95
Vermeeren, Lenka 100
Vermeulen, Eric 90, 95
Verschueren, Karijn 126
Verwaal, Vic 143
Verwijs, Manon 40
Verwoerd, Désirée 33
Verzijlbergen, Kitty 50
Vincent, Andrew 156
Visser, Daan 29
Visser, Nils 42
Vissers, Joep 80
Vlaming, Marijn 70
Vlaskamp, Marcel 156
Voets, Erik 74
Vogel, Maartje 48
Vogel, Celia 84
Vogel, Wouter 100
Vollebergh, Marieke 68
Voncken, Francine 126
Vormer, Tinke 62
Vos, Matthijn 36
Vos, Sanne 144
Voskuil, Dorien 95
Vredeveld, Liesbeth 84
Vroonland, Colinda 100
Vuist, Ilona 29
Vyth-Dreese, Florry 57
Wagenaar, Els 70
Wals, Anneke 156
Wang, Liqin 89
Wendling, Markus 127
Wesseling, Jelle 100
Wessels, Lodewyk 72
Westerman, Bart 80
Wever, Lidwina 156
Wevers, Marijke 90
Wielders, Camiel 62
Wielders, Eva 62
Wientjens, Ellen 66
Wieringa-Ariaens, Aafke 101
Wigbout, Gea 101
Wijnands, Yvonne 28
Willemse, Els 156
Winterwerp, Herrie 18
Wit, Niek 59
Witte, Marnix 127
Wittkämper, Frits 126
Woerdeman, Leonie 94, 144
Wolthaus, Jochem 127
Wolthuis, Rob 74
Wouters, Michel 143
Xiao, Yanling 53
Xylourgidis, Nikos 52
Yang, Jonathan 127
Yanover, Eva 87
Yüksel, Gül 127
Zandbergen, Jeroen 156
Zander, Serge 64
Zerp, Shuraila 30
Zevenhoven, John 82
Ziblat, Lonny 156
Zijp, Lambert 127
Zlotorynski, Eitan 46
Zupan-Kajcovski, Biljana 144
Zuur, Karel 144
Zwart, Wilbert 33
197
PERSONNEL INDEX

THE
NETHERLANDS
CANCER
INSTITUTE
SCIENTIFIC
ANNUAL
REPORT 2008
The Netherlands Cancer Institute
Antoni van Leeuwenhoek Hospital
Plesmanlaan 121
1066 CX Amsterdam
www.nki.nl
p26.1
p25.3
p24.3
p24.2
p24.1
p22.3
p22.1
p21.31
p14.3
p14.2
p14.1
p13
p12.3
p12.2
p12.1
q11.2
q13.11
q13.13
q13.31
q21.3
q22.1
q23
q24
q26.1
q26.2
q26.31
q26.32
p26.33
p28
p29
p15.33
p15.31
p15.2
p15.1
p14.3
p14.1
p13.3
p13.2
p13.1
p12
q11.2
q12.1
q12.3
q13.2
q13.3
q14.1
q14.3
q15
q21.1
q21.3
q23.1
q23.2
q23.3
q31.1
q31.3
q32
q33.1
q33.2
q33.3
q34
q35.1
q35.2
q35.3