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Half the Story - Discovery Education

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The Netherlands Cancer Institute
Antoni van Leeuwenhoek Hospital
Plesmanlaan 121
1066 CX Amsterdam
www.nki.nl
T HE NE T HE RLA NDS C A NC ER INSTITU TE SCIENTIFIC ANNU AL R E PO R T 2 0 06
T HE
N E T H E R LANDS
C A NCE R
I N ST I T UT E
S C IE NT I FI C
A N NUA L
R E PO R T 2 0 06
THE
NETHERLAND S
CANCER
I NSTI TUTE
SCI ENTI FI C
ANNUAL
REP ORT 20 0 6
3
S C IE NT I FI C ANNU AL REP OR T 2 0 0 6
4
Patron HM The Queen Beatrix
5
SC I E NT I FI C ANNU AL REPO R T 2 0 0 6
THE NE T HERL ANDS CANCER INS TITU TE
CANCER RESEARCH LABORATORY AND CANCER HOSPITAL
www.nki.nl
6
COPY R IGHT
Scientific Annual Report 2006
Illustrations and unpublished data in these reports may not be used without permission of the author.
Copyright В©:
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
7
CONT E NTS
Board Members
Research Divisions
Introduction
Education in Oncology
I
Division of Cell Biology
II
Division of Molecular Carcinogenesis
III
Division of Cellular Biochemistry
IV
Division of Immunology
V
Division of Molecular Biology
VI
Division of Tumor Biology
VII
Division of Molecular Genetics
VIII Division of Experimental Therapy
IX
Division of Radiotherapy
X
Division of Medical Oncology
XI
Division of Surgical Oncology
XII
Division of Psychosocial Research and Epidemiology
XIII Division of Diagnostic Oncology
Biometrics Department
Clinical Trials
Invited Speakers
Projects
Personnel Index
8
9
11
16
17
24
32
41
49
60
69
76
88
102
1 14
124
132
143
147
161
164
176
8
B OARD MEMBERS
President of Board of Governors W Kok
BO A R D MEMB ERS
INTERNATIONAL SCIENTIFIC ADVISORY BOARD
T De Lange, Leon Hess Professor, Rockefeller
University, New York, USA
RA Flavell, Professor of Immunobiology, Yale University
School of Medicine, New Haven, USA
S Hellman, Pritzker Distinguished Service Professor,
The University of Chicago, Chicago, USA
WGJ Hol, Professor of Biochemistry and Biological
Structure, University of Washington, Seattle, USA
J Mendelsohn, President, MD Anderson Cancer Center,
The University of Texas, Houston, USA
P Nurse, President, The Rockefeller University, New
York, USA
R Nusse, Professor of Developmental Biology, Stanford
University, USA
HL Ploegh, Professor of Biology MIT, Whitehead
Institute of Biomedical Research, Cambridge, USA
K Vousden, Director, Beatson Institute for Cancer
Research, Glasgow, UK
NATIONAL SCIENTIFIC ADVISORY BOARD
LA Aarden, Professor of Molecular Immunology,
Amsterdam
SWJ Lamberts, Professor of Internal Medicine,
Rotterdam
B Löwenberg, Professor of Hematology, Rotterdam
CJLM Meijer, Professor of Pathology, Amsterdam
CJM Melief, Professor of Immunohematology, Leiden
HM Pinedo, Professor of Clinical Oncology, Amsterdam
GNJ Tytgat, Professor of Gastroenterology, Amsterdam
AJ Van der Eb, Professor of Fundamental Virology,
Leiden
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
PJ Kalff, treasurer
FH Schröder
D Sinninghe DamstГ©
MC Smeets
PF Van der Heijden
J Van der Meer
PC Van der Vliet
GP Vooijs
SCIENTIFIC ADVISORY COUNCIL
AJM Berns, chairman
W Moolenaar, secretary
S Rodenhuis
T Schumacher
T Sixma
M Van de Vijver
9
RESEARCH DIVISIONS
R E S E A R CH DIVISIONS
I Cell Biology
Arnoud Sonnenberg (head)
John Collard
Kees Jalink
Ed Roos
Natasja Borsje-Maeyer (office manager)
II Molecular Carcinogenesis
RenГ© Bernards (head)
Roderick Beijersbergen
Anastassis Perrakis
Titia Sixma
Suellen Boonen (office manager)
III Cellular Biochemistry
Wouter Moolenaar (head)
Nullin Divecha
Huib Ovaa
Wim Van Blitterswijk
Fred Van Leeuwen
Marcel Verheij
Suzanne Corsetto (office manager)
IV Immunology
Jannie Borst (head)
John Haanen
Heinz Jacobs
Ton Schumacher
Florry Vyth-Dreese
JosГ© Overwater (office manager)
V Molecular Biology
Hein Te Riele (head)
Piet Borst (honorary staff member)
Jos Jonkers
Sabine Linn
Henri Van Luenen
Bas Van Steensel
Lodewyk Wessels
Tom De Knegt (office manager)
Linda Römer (secretary)
VI Tumor Biology
Jacques Neefjes (head)
Reuven Agami
Maarten Fornerod
Rob Michalides
Peter Peters
Marieke Van der Velde (office manager)
VII Molecular Genetics
Maarten Van Lohuizen (head)
Anton Berns
John Hilkens
Paul Krimpenfort
Daniel Peeper
Margriet Snoek
Marion Rustenberg (office manager)
Marie Anne Van Halem (secretary)
VIII Experimental Therapy
Alfred Schinkel (head)
Adrian Begg
Petra Nederlof
Jan Schellens
Fiona Stewart
Marc Van de Vijver
Laura Van ’t Veer
Thea Eggenhuizen (office manager)
IX Radiotherapy
Harry Bartelink (head)
Berthe Aleman
JosГ© Belderbos
IaГЇn Bruinvis
Eugene Damen
Roel De Boer
Luc Dewit
Rick Haas
Guus Hart
Wilma Heemsbergen
Frank Hoebers
Edwin Jansen
Joos Lebesque
Corrie Marijnen
Harry Masselink
Ben Mijnheer
Luc Moonen
Floris Pos
Coen Rasch
Peter Remeijer
Nicola Russell
Govert Salverda
Christoph Schneider
Joep Stroom
Jacqueline Theuws
Bart Van Bunningen
Marcel Van Herk
Corine Van Vliet
Marcel Verheij
Conny Vrieling
Frits Wittkämper
Patricia Haye-Fewer (management assistent)
X Medical Oncology
Sjoerd Rodenhuis (head)
Joke Baars
Paul Baas
Evert Bais
Jos Beijnen
Willem Boogerd
Henk Boot
Sjaak Burgers
Annemieke Cats
Jan Paul De Boer
Bert De Gast
Leo Gualtherie van Weezel
10
RESEARCH DIVISIONS
John Haanen
Alwin Huitema
Martijn Kerst
Sabine Linn
Anne Lukas
Herman Neering
Jan Schellens
Jan Schornagel
Marianne Smits
Babs Taal
Wim Ten Bokkel Huinink
Jaap Van der Sande
Marchien Van der Weide
Nico Van Zandwijk
MariГ«lle De Kwant (secretary)
XI Surgical Oncology
Bin Kroon (board; head)
Fons Balm (board)
Omgo Nieweg (board)
Axel Bex
Dick Buitelaar
MatthГ© Burger
Marcel Copper
Menno De Rie
Guus Fons
Steven Gonggrijp
Joris Hage
Frans Hilgers
Simon Horenblas
Hans Huitink
Christiaan Keijzer
Houke Klomp
Frans Kroon
Peter Lohuis
Lottie Lubsen
Wim Meinhardt
Hester Oldenburg
May Ronday
Emiel Rutgers
Peter Schutte
Michael ScrГЎmek
Marian Slot
Ludi Smeele
Tino Stoppa
Bing Tan
Julia Ten Cate
Adriaan Timmers
Marc Van Beurden
Frits Van Coevorden
Michiel Van den Brekel
Wietze Van der Veen
Henk Van der Poel
Willemien Van Driel
Arjen Van Turnhout
Vic Verwaal
Marie-Jeanne Vrancken Peeters
Leonie Woerdeman
Frans Zoetmulder
Joke Van der Veen (office manager)
XII Psycosocial Research and Epidemiology
Neil Aaronson (head)
Matti Rookus
Frits Van Dam
Wim Van Harten
Flora Van Leeuwen
Marian Chin-A-Kwie (office manager)
XIII Diagnostic Oncology
Marc Van de Vijver (head)
Tanja Alderliesten
Philippe Baars
Peter Besnard
Hans Bonfrer
Daphne De Jong
Kenneth Gilhuijs
Cees Hoefnagel
Frans Hogervorst
Irma Kluijt
Wim Koops
Robert Kröger
Claudette Loo
Saar Muller
Petra Nederlof
Willem Nooijen
Frank Pameijer
RenГ©e Van Pel
Hans Peterse
Warner Prevoo
Michiel Sinaasappel
Ferida Sivro
Jelle Teertstra
Renato Valdes Olmos
Hester Van Boven
Fijs Van Leeuwen
Olaf Van Tellingen
Laura Van ’t Veer
Loes Van Velthuysen
Senno Verhoef
Jelle Wesseling
Christine Arkes (secretary)
Carla Van Tiggelen (secretary)
Biometrics Department
Otilia Dalesio (head)
Harm Van Tinteren
Financial Administration
Vacancy
General Facilities
Vacancy
Personnel Department
Eric De Wilde
General Research Coordination
Wouter Moolenaar, laboratory research coordinator
Henri Van Luenen, laboratory research manager
11
INTRODUCTION
Our clinical activities showed a steady expansion, with
further growth occurring in the Divisions of Surgery
and Radiotherapy. A major upgrading of equipment
took place in our Radiology department, with the
installation of a 3 Tesla MRI and a PET-CT scanner. In
the Radiotherapy department two advanced linear
accelerators were put into action permitting imageguided radiotherapy (IGRT) as a regular treatment
modality. One of our operating theaters was equipped
with a Da Vinci robot allowing highly refined endoscopic
surgical interventions, such as prostate resections.
On December 8, the Division of Surgical Oncology
opened the Photodynamic Therapy Center for function
preserving treatment of early staged carcinomas of the
head and neck, skin and cervix.
Director of Research Ton Berns
I N TR O DUCTION
I am pleased to present our Scientific Annual Report
2006. 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 more lucid
description of our activities with illustrations can be
found in our new brochure that came out in November
2006.
In 2006 the hospital budget was under even more
strain than in the previous year, due to only partial
compensation of rising expenses and additional budget
cuts by the Ministry of Health. Our research also had
great difficulty to balance the budget. The effects of the
earlier 10% budget cut by the Ministry of Health, which
is funding roughly half of our infrastructure including
the salaries of our permanent staff, were made worse by
a lower contribution of the Dutch Cancer Society as a
result of lower revenues from legacies. Fortunately, the
contribution of the Dutch Cancer Society will increase
again in 2007, and this effect is expected to become more
substantial in 2008. We hope that a new government will
appreciate the important contributions of the NKI-AVL
to the battle against cancer and agree on a funding
arrangement that does justice to the contribution of the
NKI-AVL to the public cause. In November 2006 we held
our first institute-wide two-day long retreat in which staff
and postdoctoral fellows participated. This meeting
stimulated discussions among staff and investigators of
both clinical and basic research divisions that do not
regularly interact in person. The meeting was a success
and we envision that this will become a bi-annual event.
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
cell-based screens, mouse tumor models, cell biology,
structural biology, immunology and translational
research. There is close 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 Report.
2006 brought a number of major renovations and
expansions of the research and clinical activities of the
NKI-AVL. Our renovated auditorium was put into use.
The auditorium with its 300 seats compares with the best
auditoria elsewhere providing the NKI-AVL with a
state-of-the-art facility for seminars, teaching courses
and scientific meetings. The old hospital building has
been completely stripped and will be transformed into
a modern laboratory facility in the course of 2007.
Highlights
The scientific output of the Institute is again well above
par. 2006 was a productive year with important new
findings and scientific publications that received wide
recognition in top scientific journals.
Table 1
Research funding NKI-AVL from the different sources in period 1990-2006 in million Euro’s.
Year
Cancer Society
Ministry of Health
Project Grants
Other income
Total
1990
1993
1996
1999
2001
2002
2003
2004
2005
2006
2.5
7.3
6.4
2.6
4.1
7.3
8.1
2.5
5.6
8.1
8.6
2.9
7.0
8.8
12.0
2.4
8.7
9.4
12.6
3.8
8.7
9.9
14.4
3.3
9.1
10.1
18.4
1.0
8.9
9.1
18.0
0.8
8.6
9.6
17.4
1.7
8.3
9.4
21.9
1.2
18.8
22.0
25.2
30.2
34.0
36.3
38.6
36.8
36.8
40.8
An additional 2.5 million Euro/year is received from the �Stichting Fondsen’ of the NKI for specific investments in equipment.
12
INTRODUCTION
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
interesting 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).
Norman Sachs and colleagues in the group of Arnoud
Sonnenberg (Division of Cell Biology) showed that mice
lacking the cell surface molecule known as tetraspanin
Cd151 develop a similar kidney failure as mice with a
podocyte specific knockout of the integrin a3 subunit,
indicating the existence of functional integrin-tetraspanin
complexes in vivo. This supports the notion that CD151
plays a key role in strengthening integrin-mediated
adhesion, a feature that makes these molecules relevant
for keeping cells properly positioned in their microenvironment.
John Collard and co-workers (Division of Cell Biology)
found that the Rac activator Tiam 1, in conjunction with
the Par polarity complex, regulates the polarization
and directional migration of epithelial cells as well as
chemokine-induced polarization and migration of
normal T cells.
Rianne Oosterkamp in the group of Rene Bernards
(Division of Molecular Carcinogenesis) tested the efficacy
of salicylic acid for the treatment for cylindromatosis,
a rare genetic disorder causing neoplasms of the skin
appendages. This clinical study was based on previous
RNA interference studies in which his group had shown
that loss of the cylindromatosis tumor suppressor gene
leads to activation of NF-kappaB, a transcription factor
having anti-apoptotic activity. Cell-based assays indicated
that salicylate can prevent NF-kappaB activation caused
by loss of the cylindromatosis gene. Salicylate might then
also benefit patients with cylindromatosis, a prediction
that was supported by the pilot clinical study by
Oosterkamp. This work nicely illustrates how basic
research can sometimes be quickly translated into
tangible clinical results and in general highlights why
NKI-AVL has both research and patient care under one
roof.
Roderik Kortlever from the group of Rene Bernards
identified the p53 target gene Plasminogen Activator
Inhibitor-1 (PAI-1) as a critical mediator of the
p53-dependent senescence response. His data indicate
that PAI-1 is not merely a marker of senescence, but
both necessary and sufficient for the induction of
replicative senescence downstream of p53.
The groups of Huib Ovaa and Wouter Moolenaar
(both Division of Cellular Biochemistry) have evaluated
Autotaxin (ATX) as a therapeutic target. ATX is a secreted
phospholipase that generates the lipid growth factor LPA,
which has an important signaling function in tumor
progression and angiogenesis. In collaboration with
David Egan (Division of Molecular Carcinogenesis), they
identified three distinct classes of potent small-molecule
inhibitors of ATX, which will be further optimized for
evaluation in mouse models.
Defective cell-cell communication is a hallmark of
malignancy. The groups of Wouter Moolenaar, Kees
Jalink (Division of Cell Biology) and Nullin Divecha
(Division of Cellular Biochemistry) discovered that cellcell communication through gap junctions is tightly
regulated by the levels of phosphatidylinositol
bisphosphate (PIP2) in the plasma membrane. Using
state-of-the-art live-cell imaging techniques, they showed
that a fall in PIP2 is necessary and sufficient to shut
down gap junctional communication. This new finding
may suggest strategies as to how to restore defective
cell-cell communication in tumor cells.
In an approach combining organic synthesis and
immune technology, the groups of Huib Ovaa and Ton
Schumacher (Division of Immunology) have developed
conditional MHC ligands that can be cleaved by UV
irradiation thereby allowing the production of �empty’
MHC complexes. They have used this strategy for the
high-throughput production of MHC complexes and
MHC tetramers for T cell detection. This approach has
led to the identification of highly immunogenic avian flu
epitopes and is now being applied to identify T cell
epitopes relevant for immunotherapy.
Wim van Blitterswijk (Division of Cellular Biochemistry)
and Marcel Verheij (Division of Radiotherapy) and
collaborators modified the liposomal formulation of the
anti-cancer drug doxorubicin (CaelyxВ®) by incorporating
N-octanoyl-glucosylceramide in the liposomal bilayer.
This sphingolipid significantly facilitated the uptake of
doxorubicin in tumor cells xenografted into nude mice.
This new delivery method looks promising for clinical
application.
Matthijs Voorhoeve and Carlos Le Sage in the group of
Reuven Agami (Division of Tumor Biology) have
set up systems to evaluate the function of individual
microRNA’s and identified two as being involved in
formation of testicular germ cell tumors through
targeting the tumor-suppressor LATS2.
Jacques Neefjes (Division of Tumor Biology), in
collaboration with Eric Reits (AMC, Amsterdam), and
James Hodge and Jeffrey Schlom (NCI, Washington)
identified a novel anti-cancer immunotherapy combining
the specificity of adaptive immunotherapy and the spatial
application of (low dose) radio-immunotherapy. This
combined radio-immunotherapy proved superior over
the individual therapies in eliminating local tumors and
is currently further developed for application.
Helen Pickersgill in the group of Maarten Fornerod
(Division of Tumor Biology) in collaboration with Bas
Van Steensel (Division of Molecular Biology) defined the
DNA segments interacting with the nuclear lamina
coating the nuclear envelop. These appear to be
transcriptionally silent areas and indicate that DNA is
packaged in the nucleus in functionally different areas.
Nico Dantuma and Tom Groothuis in the group of
Jacques Neefjes have studied the Ubiquitin equilibrium
in living cells through a series of biochemical and
biophysical experiments. Ubiquitin is a small protein
modifying others and shared in many cellular processes.
Ubiquitin appears to be present in limiting amounts
13
INTRODUCTION
and removed from histones for polyubiquitin under
conditions of acute stress. Also anti-cancer drugs induce
this, and the mechanisms and consequences are further
studied.
Sven Rottenberg, a postdoc working in the groups of Piet
Borst and Jos Jonkers (Division of Molecular Biology),
showed that the Brac1-/-; p53-/- mouse model for
mammary cancer is suitable for studying chemotherapy
resistance. He was able to generate resistance against
natural product drugs, such as doxorubicin and
paclitaxel, which had never been obtained before in an
animal tumor model. The tumors also respond to
cisplatin, but they do not become resistant and can be
treated successfully over and over again. The �remnants’
from which tumors regrow after an initial response are
of great interest, as these have never been accessible in
patient samples or tumor models before. Characterization
of these �remnants’ may offer new possibilities for
characterizing and tackling the tumor stem cell fraction,
held responsible for relapse after chemotherapy.
Bas van Steensel’s group (Division of Molecular Biology)
co-authored three papers in Nature Genetics, each
reporting important new insights into chromatin
structure and composition. Together with the labs of
Maarten Van Lohuizen (Division of Molecular Genetics),
Maarten Fornerod (Division of Tumor Biology), and
Wouter De Laat (Erasmus Medical Center, Rotterdam),
they uncovered genome-wide patterns of respectively
Polycomb protein binding, nuclear lamina interactions,
and three-dimensional chromosome folding.
Paul Krimpenfort and Annemieke IJpenberg in the
group of Ton Berns (Division of Molecular Genetics)
uncovered an unexpected backup function of the
Cdkn2b tumor suppressor for Cdkn2a, the latter being
the most frequently inactivated tumor suppressor after
p53. Whereas loss of one of these two suppressors in
mouse models gives rise to only a slightly enhanced
tumor predisposition, co-deletion shows a dramatic
tumor-prone phenotype. Both genes map close together
on the genome and are co-deleted in many human
tumors. Since both suppressors appear to preferentially
inhibit one specific cyclin-dependent kinase, this offers
interesting perspectives for treating tumors in which this
locus is deleted or silenced.
In search of genes that preserve the senescence growth
arrest of human nevi, Daniel Peeper’s group (Division of
Molecular Genetics) has identified an interleukin
network as a critical mediator of senescence.
John Hilkens and coworkers (Division of Molecular
Genetics) finished the first large scale MMTV insertional
mutagenesis screen for mammary cancer genes, which
resulted in the identification of 17 novel candidate
oncogenes. Two of them belong to a novel family of
secreted proteins, R-spondins, that stimulate the
expression of a unique set of downstream genes in
addition to the Wnt pathway.
Stephen Tait in the group of Jannie Borst (Division of
Immunology) discovered a novel mode of ubiquitination
that is essential for mitochondrial permeabilization by
the BH3-only protein Bid, which is a key mediator in
apoptosis induction by death receptors and DNA
damaging anti-cancer regimens. Chiel Maas in the same
group has performed a successful RNA interference
screen for Trail-induced apoptosis, using the entire NKI
RNAi library. This resulted in the identification of a
dozen, both known and unknown, mediators of the Trail
apoptosis pathway.
The image-guided radiotherapy (IGRT) developments
based upon the Cone-Beam CT as explored in the
Division of Radiotherapy have already led to better
results: for example in prostate cancer and breast cancer
our own trials demonstrated that by delivering a higher
radiation dose, a better local control is achieved.
At the same time it was shown that in prostate cancer
the toxicity could be reduced with this sophisticated
radiotherapeutic approach.
The Epidemiology group (Division of Psychosocial
Research and Epidemiology) coordinated a nationwide
collaboration of Clinical Genetic Centers and brought
together 139 families, comprising 1811 family members,
with pathogenic mutations in the BRCA2 gene. They
assessed the risk of cancers other than breast or ovarian
cancer in this database and observed that risk compared
to the general population was increased for four cancer
sites: pancreas (Relative risk (RR)=5.9), prostate
(RR=2.5), bone (RR=14.4) and pharynx (RR=7.3).
Families with mutations outside the previously defined
ovarian cancer cluster regions tended to have a higher
cancer risk.
The group of Neil Aaronson reported the results of a
nationwide observational study of 846 women at
increased risk of hereditary ovarian cancer, 44% of
whom had opted for prophylactic bilateral salpingooophorectomy (PBSO) and 56% for periodic gynecologic
screening (GS). Women in the PBSO and GS groups
reported similar levels of generic quality of life.
Compared with GS, PBSO was associated with fewer
breast and ovarian cancer worries (p < 0.001) and more
favorable cancer risk perceptions (p < 0.05). However,
the PBSO group reported significantly more endocrine
symptoms (p < 0.001) and worse sexual functioning
(p < 0.05). The favorable effects of PBSO in terms of
reduced cancer worries and risk perception need to be
weighed against the increase in endocrine and sexual
symptoms. Balanced information will help clinicians
and high-risk women in making informed decisions
about the optimal preventive health strategy.
The Radiology department (Division of Diagnostic
Oncology) has extended its work on evaluating the added
value of MRI to guide optimal breast conserving therapy
for breast cancer patients. MRI led to a change in
treatment in approximately 20% of the patients, showing
that MRI is a valuable addition to the diagnostic work-up
of breast cancer patients.
14
INTRODUCTION
Work of the Molecular Pathology group (Division of
Diagnostic Oncology) has previously led to the
identification of a 70 gene prognosis profile in tumors
from patients with lymph node negative breast cancer.
To assess whether this 70-gene MA test can be
implemented in daily clinical practice, a multi-center
study (“RASTER” study) was performed successfully in
17 Dutch community hospitals. The results of this study
have been very helpful in setting up the MINDACT trial,
that will prospectively evaluate the prognostic value of the
70 gene prognosis profile.
The pharmacology group of the Division of Medical
Oncology has performed a phase I study with the PARP1
(poly(ADP-ribose)polymerase-1) inhibitor KU36-92, in
which the agent was administered to cancer patients for
the first time. Theoretically, PARP1 inhibitors should
represent �targeted therapy’ for tumors that lack certain
DNA-repair mechanisms, such as in patients with
hereditary breast cancer syndromes. Major clinical
responses were indeed observed in 2 patients known to
be BRCA2 mutation carriers.
Quality of research
The quality of research can be monitored in several ways.
Our scientific productivity as based on 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,
although in recent years the number of citations and
impact has shown some fluctuation (Table 2). It is
satisfying to note that the Institute has acquired an
internationally prominent position in cancer research.
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, and from
our success in obtaining competitive grant awards.
We score highly on all those items. We are fairly
successful in securing grant support, scoring on average
twofold better than our colleagues in The Netherlands.
Quality is also measured by external site visits.
International leaders in a particular field of research
review the work of a division or research groups with a
similar theme, on a quinquennial basis. These site visits
require that group leaders prepare a report and reflect on
their research activities. This by itself is a useful exercise
that helps to focus the research. Early 2006 the work of
the Division of Molecular Carcinogenesis was evaluated
in a one-day site visit by a site visit team consisting of
Bob Weinberg (Whitehead Institute, Cambridge), Ray
Stevens (Scripps, La Jolla), Bill Kaelin (Dana Farber,
Boston), and John Kuriyan (UC Berkeley). In November
2006, the Division of Psychosocial Research and
Epidemiology was evaluated by Patricia Ganz
(UC Los Angeles), Bruce Ponder (Hutchison/MRC
Research Centre, Cambridge UK), and Christoffer
Johansen (Research Institute of Cancer Epidemiology,
Copenhagen). The site visitors were overall positive about
the activities of both divisions, but also made a number
of recommendations to be considered by some of the
individual groups. These recommendations will be
discussed and, when applicable, implemented to further
improve science at the NKI-AVL.
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 facilitates awarding degrees
to graduate students receiving their training at The
Netherlands Cancer Institute. In 2006 27 staff members
had professorships at one of the Dutch Universities.
No new professorships were granted in 2006.
Daniel Peeper, appointed to permanent staff member in
2005, obtained a prestigious VICI award, providing him
with 1.2 million euros of funding. VIDI awards were
given to Jacqueline Jacobs and Rob Wolthuis, and of the
5 fellowships made available by the Dutch Cancer Society
Table 2
Short-term citations and impact of scientific articles
published by the NKI research staff 1982 - 2006
Publication year
1982
1983
1984
1985
1986
1987
1988
1989*
1990
1991
1992
1993
1994
1995
1996
1997***
1998
1999
2000
2001
2002
2003
2004
2005
2006
Citations*
560
779
1340
1286
1366
1839
1775
1273
2127
2199
2074
2221
3455
2896
3324
3617
3240
3727
3551
3991
7432
5094
4884****
Impact**
295
365
616
549
650
765
742
764
854
910
911
958
1292
1415
1520
1811
1392
1766
1664
1573
2455
2122
1882
2461
2913
* Citations in the two years after publication,
excluding self-citations. Starting 1989 citation
analysis has been carried out on line. This allows
detection (and elimination) of all self-citations.
Before 1989 this pruning was limited to 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.
**** Preliminary data available on Jan 5, 2007
15
to young investigators, 4 were awarded to NKI scientists.
There were also prizes awarded to our staff. Piet Borst
became Commander of the British Empire and Harry
Bartelink was recipient of the bi-annual Regaud Medal
Award of the European Society of Therapeutic Radiation
and Oncology (ESTRO). He also received the Kilroe
Award at the meeting of the Coloprotology Section of
Royal Medicine Society. I received the first Massachusetts
General Hospital Award in Cancer Research. Jacques
Neefjes was elected to EMBO member. The AvL-prize
2006 for young, promising NKI-scientists was awarded
to Matthijs Voorhoeve for his work on the role of specific
microRNAs in cancer.
There are also changes to report in our scientific staff.
Bas Van Steensel, AvL fellow in the institute was
promoted to associate professor. Rob Ten Berg, director
of our animal facility from 1985 to 2002, retired after
having served us during the last 4 years as supervising
veterinarian and secretary of the Animal Experiment
Committee.
Staff of the NKI-AVL fulfilled numerous functions in
national and international organizations, on scientific
boards of scientific journals, as members of study
sections, and as organizers or co-organizers of scientific
meetings, workshops and congresses.
Outlook and acknowledgements
I am confident that research at the NKI-AVL will
continue to flourish, especially given the perspectives
that our funding will again increase in the years to come.
This increase will come from a number of sources,
including the participation in large nation-wide initiatives
that will stimulate joint projects between academia and
industry. Contracts for one of these initiatives, the
Top Institute Pharma, have been signed in December
and will provide funding for several projects that will
be executed together with Dutch industries. A new
substantial initiative, the Centre for Translational
Molecular Medicine, will stimulate development of
biomarkers and imaging. We will submit proposals in the
course of 2007. We will continue to participate in the
Top Research School CBG and the Cancer Genomics
Centre CGC. In addition, we hope to increase our income
from intellectual property. We have now an effective
Technology Transfer Office in place and this will
undoubtedly contribute to our income. A range of other
initiatives, some of which I have outlined above should
provide us with a sound basic funding with a healthy
yearly growth. Wise and cost/effective spending of the
funds given to us is a recurrent priority. In addition,
it is of critical importance that we retain the capacity to
swiftly respond to new developments and opportunities,
required to perform internationally recognized cuttingedge research. Research is the only way to improve cure
rates and the quality of life of cancer patients. I am
convinced that breakthroughs in cancer diagnosis and
treatment can be realized with approaches that are now
broadly applied in the NKI.
We are particularly grateful to 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, last but not least, the many individuals
who support us directly with their gifts and also with
their 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
Theoretical and practical training in research are regular
activities in the institute. Several senior staff members
have joint appointments as professors at Dutch
universities and contribute to the regular curriculum at
these universities. In addition, many staff members
teach in courses for undergraduate and graduate
students, either within the institute or at universities.
The research departments attract Master students from
universities throughout the country, who contribute to
ongoing scientific projects and receive in house
theoretical training. The NKI has a formal connection
with the Science faculty of the University of Amsterdam
(UvA) and is committed to contribute to courses
for Master students of this university. The institute
participates in the Oncology Graduate School
Amsterdam, together with the UvA and the Free
University (VU). The medical staff is actively involved in
medical and surgical oncology fellowship programs,
teaching programs for undergraduate medical students
and (inter)national postgraduate courses.
The departments of Surgical Oncology and Head and
Neck Oncology participate in the residency training
program of the Academic Medical Center. Lectures are
read by staff members for nurses in training for their
oncology certificate. All educational activities are
supervised by the Teaching Committee, which consists
of J Borst (president and dean undergraduate school),
H Te Riele (general affairs), R Beijersbergen
(undergraduate courses), J Hilkens and J Collard
(HLO students and publicity), T Sixma (dean graduate
students) and F Balm (clinical teaching). Specialized
tasks include organization of the lecture course, guided
tours to research departments, contact with technician
training schools, promotional activities at universities.
UNDERGRADUATE STUDENTS
The undergraduate program in Experimental Oncology
attracts students of all national universities in partial
fulfillment of the obligations of their Master’s
curriculum. Students generally have a background in
(Medical) Biology, Health Sciences, Chemistry,
Pharmacology, Medicine, or Psychology. The
undergraduate program offers combined practical and
Table 1 – Courses in Experimental Oncology
theoretical training in various aspects of experimental
oncology. Practical training includes participation in
ongoing research projects for a minimum of 4 months,
after which the student delivers oral and written
accounts of the results obtained. The majority of the
undergraduate students came from the Amsterdam
Universities (UvA and VU), others from Wageningen,
Utrecht, Leiden and Nijmegen. The institute also
provides practical training opportunities to trainee
technicians.
The core element of theoretical training is the course in
Experimental Oncology, given twice a year by staff
members of the institute (Table 1). This course includes
lectures and tutorials. The book Cancer Biology by
RJ King is recommended for background reading.
GRADUATE STUDENTS
For the graduate students the NKI-AVL participates in
the Oncology Graduate school Amsterdam (OOA)
together with the oncology departments of the VUmc
and the AMC. The institute has ~130 graduate students
registered for the OOA graduate school. Students
participate in the research of their group and in the
interdepartmental work discussions. In addition they
participate in the OOA training program, which consists
of courses (Table 2) and an annual retreat in Texel. This
retreat focuses entirely on the research of the students
themselves. At this retreat students not only present
their work in the form of a poster in the first year and an
oral presentation in subsequent years, they are also in
charge of chairing sessions, discussions and in recent
years also in peer review, giving a prize for the best
poster and best presentation. In this manner the retreat
trains important skills in presentation and interaction
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, where the students themselves
can contribute significantly to the interaction between
different groups.
In 2006, 21 graduate students received a PhD degree at
a Dutch university.
Table 2 – OOA graduate student courses
Jan 30 – Feb 10
Feb
Mar 21-24
Will we cure cancer?
Epidemiology
Surgery
Pathology*
Gene array
Molecular diagnostics
Conventional sustemic cancer therapy
Radiotherapy*
DNA damage response
Apoptosis
Cancer therapy and apoptosis
Cell adhesion
Angiogenesis
Cell senescence
Cell cycle
Genomic instability
J Borst
D Voskuijl
F Zoetmulder
D De Jong
R Kerkhoven
L Van �t Veer
J Schornagel
M Verheij
A Begg
J Borst
J Borst
E Roos
E Boven
R Beijersbergen
R Bernards
H Te Riele
June 6-16
Sep 25-Oct 6
Oct 11-13
Nov 6-11
Nov 8, 15, 22, 25
Nov 20, 27, Dec 4
Dec 11,12
* including tour
Discovery of cancer genes and networks in model
organisms.
F Van Leeuwen, J Jonkers
Stam cell differentiation
GJ Schuurhuis, F van Millegen
The Macroscopic, microscopic and pathologic
anatomy of the mouse
R Van Noorden, W Lamers
In the footsteps of Antoni van Leeuwenhoek
R Van Noorden, G Meijer, P Peters, E Reits, J Belien,
K Jalink
Protein structure and function
T Sixma, A Perrakis
Annual retreat
T Sixma, M Van der Velde, S Boonen
Oncogenomics: A sampler dish of genomic
techniques and applications
B Ylstra
Writing & presenting scientific English
A Bless
Introduction in the histopathology of tumors
N Van Grieken en E Thunissen
Biology of colorectal cancer risk
R Fijneman
17
CELL BIOLOGY
I DI VI S I O N OF CEL L B IOL O G Y
RECEPTORS FOR MATRIX ADHESION
Integrins connect the extracellular matrix (ECM) with the cell interior and transduce
signals by interactions of their cytoplasmic domain with cytoskeletal and signaling
molecules. In our group we study the mechanisms by which integrins provide
signals for the regulation of important cellular processes such as cell migration and
proliferation.
Division head, Group leader Arnoud Sonnenberg
Arnoud Sonnenberg PhD Group leader
Johan De Rooij PhD Post-doc
Kevin Wilhelmsen Post-doc
Role of the integrin a6b4 and plectin in hemidesmosome formation
Hemidesmosomes (HDs) are multiprotein adhesion complexes that promote
attachment of epithelial cells to the underlying basement membrane. Modulation of
their function is of crucial importance in a variety of biological processes, such as
differentiation and migration of keratinocytes during wound healing and carcinoma
invasion, in which cells become detached from the substrate and acquire a motile
phenotype. The binding of a6b4 to plectin plays a central role in HD assembly
and it is becoming increasingly clear that disrupting this association through
phosphorylation events is necessary to disassemble HDs. We have defined two
regions on b4 that harbor all the serine/threonine phosphorylation sites. These areas
are not directly involved in the primary interaction with plectin, which is mediated
through its actin-binding domain (ABD), but rather enforce the interaction between
the two proteins. Interestingly, however, we have identified three serines (S1356,
S1360 and S1364) within the b4 connecting segment that regulate the interaction
with the plectin-ABD when they are phosphoylated, suggesting that these
phosphoresidues somehow act remotely, possibly through an intramolecular folding
event, to block binding. We have also recently established that activation of the EGF
receptor, which is known to function upstream of HD disassembly, results solely in
the phosphorylation of these three residues in vivo and that S1360 and S1364 are the
only PKC and PKA phosphorylation sites, respectively. However, other kinases may
also phosphorylate these sites and, in fact, we have evidence that GSK-3b may be
involved. Taken together, our studies indicate that multiple kinases act in concert to
abrogate the structural integrity of HDs in keratinocytes and that phosphorylation of
the b4 subunit at residues S1356, S1360 and/or S1364 is the primary mechanism by
which this is achieved.
The localization of nesprin-3 at the nuclear envelope depends on an interaction with
SUN proteins The position of the nucleus is important for several cellular processes
such as mitosis, meiosis, cell migration and polarization. Although the general
mechanisms of nuclear positioning are known to require a connection between the
nucleus and actin filaments or microtubules, the proteins involved in this connection
have only recently been identified. Nesprin-1 and -2, members of the nesprin protein
family, are responsible for connecting the nucleus to actin filaments, while nesprin-3
connects the nucleus with intermediate filaments via the cytoskeletal linker protein
plectin. Previous studies by other investigators have demonstrated that the
localization of nesprin-1 and -2 at the outer nuclear membrane, is dependent on an
interaction with SUN proteins present at the inner nuclear membrane. We have now
provided evidence that nesprin-3 is retained at the outer nuclear membrane via a
similar mechanism. The C-terminal KASH domain of nesprin-3 interacts with SUN
proteins and the last four amino acids of nesprin-3 are important for this interaction.
In addition, nesprin-3 can associate with itself, forming dimers. Dimers of plectin
will interact with dimers of nesprin-3. Since SUN proteins can interact with nuclear
lamins and plectin can interact with intermediate filaments, there is a direct
connection between the nucleoskeleton and the cytoskeleton, which is potentially
important for signaling and cell integrity.
Iman Van den Bout Graduate student
Mirjam Ketema Graduate student
Coert Margadant Graduate student
Norman Sachs Graduate student
Maaike Kreft Technical staff
Ingrid Kuikman Technical staff
Figure I.1: Model indicating an indirect
connection between the nuclear lamina and
the intermediate filament (IF) system. The
interaction between nesprin-3 and SUN
New insight into integrin-regulated proteins Previously, we have identified
MacMARCKS (MRP) as a protein that is downregulated by overexpression of the b3
subunit. We found that this downregulation is strongly correlated with increased cell
proteins in the perinuclear space (PNS)
links nuclear lamins to cytoplasmic plectin,
which in turn can bind IFs.
18
CELL BIOLOGY
Publications
Bajanca F, Luz M, Raymond K,
Martins GG, Sonnenberg A, Tajbakhsh S,
Buckingham M, ThorsteinsdГіttir S.
Integrin a6b1-laminin interactions regulate
early myotome formation in the mouse
embryo. Development 2006; 133:1635-1644
Gawlik KI, Mayer U, Blomberg K,
Sonnenberg A, Ekblom P, Durbeej M.
Laminin a1 chain mediated reduction
of laminin a2 chain deficient muscular
dystrophy involves integrin a7b1 and
dystroglycan. FEBS Lett
2006; 580:1759-1765
Lebbink RJ, De Ruiter T, Adelmeijer J,
Brenkman AB, Van Helvoort JM, Koch M,
Farndale RW, Lisman T, Sonnenberg A,
Lenting PJ, Meyaard L. Collagens are
functional, high affinity ligands for
the inhibitory immune receptor LAIR-1.
J Exp Med 2006; 203:1419-1425
Litjens SHM, De Pereda JM,
Sonnenberg A. Current insights into
the formation and breakdown of
hemidesmosomes. Trends Cell Biol
2006; 16:376-383
Sachs N, Kreft M, Bergh Weerman MA,
Beynon AJ, Peters TA, Weening JJ,
Sonnenberg A. Kidney failure in mice
lacking the tetraspanin CD151. J Cell Biol
2006; 175:33-39
Wilhelmsen K, Litjens SHM,
Sonnenberg A. Multiple functions of the
integrin a6b4 in epidermal homeostasis
and tumorigenesis. Mol Cell Biol
2006; 26:2877-2886
Wilhelmsen K, Ketema M, Truong H,
spreading and the reorganization of the actin cytoskeleton. Furthermore, we showed
that the Ras/MAPK pathway is directly involved in the regulation of MRP in normal
cells but b3 overexpression inhibits the ability of this pathway to regulate MRP. Since
a precise function for MRP is unknown we continued our investigations into the
mechanism that controls MRP localization. Our studies show that the attachment of
MRP to membranes depends on the myristoyl moiety present at the N-terminus of
the protein. Interestingly, in contrast to other reports, we show that the positively
charged central effector domain (ED) only targets the protein for binding to PIP2
in the plasma membrane but is not essential for membrane binding per se.
Phosphorylation of the ED inhibits this binding and results in the loss of MRP from
the plasma membrane. We further show that the overexpression of MRP increases
dextran uptake, suggesting that MRP plays a role in endocytosis. This is underlined
by our observations that MRP becomes localized to endocytotic vesicle/lysosomes as
well as the transgolgi network when it is phosphorylated in the ED.s directly
responsible for the downregulation of cell-cell contacts in our model cell line.
Regulation of cell-cell adhesion during metastasis Recently, we have shown that the
disruption of cell-cell adhesion by HGF, a notorious metastasis-promoting growth
factor, depends on actomyosin-induced tension. E-cadherin, the main cell-cell
adhesion receptor in these cells, is not affected at the protein level, nor is its ability to
form homotypic interactions impaired. We concluded that E-cadherin is regulated by
cytoskeletal tension, that there must be a direct link between E-cadherin and the
cytoskeleton and that modification of the complex of proteins maintaining this link
mediates this regulation. In a recent publication it was, however, questioned whether
a direct link between E-cadherin and the actin cytoskeleton really exists. To clarify this
discrepancy and to understand the mechanism of E-cadherin disruption, we set out
to study the link between E-cadherin and the cytoskeleton and its regulation by
tension in detail. We find that both the formation and the turnover of E-cadherin
adhesions depend on cytoskeletal tension. During both phases of adhesion, there is
no detectable change in the presence of the members of the core E-cadherin complex,
a- and b-catenin. We find, however, a change in the presence of several proteins,
including vinculin and zyxin, that have been less clearly associated with E-cadherin
adhesions and whose precise functions have remained largely unclear. In time-lapse
microscopy experiments, we find that these proteins localize to junctions during
their formation but are lost during their maturation. Induction of junction-disruption
by HGF results in re-localization of these proteins to E-cadherin adhesions.
Furthermore, in cells depleted of vinculin the formation of E-cadherin adhesions is
strongly impaired. From these data, we hypothesize that a complex of proteins
including vinculin and zyxin, mediates a transient link between E-cadherin and the
cytoskeleton, which is regulated by cytoskeletal tension and is important for the
formation and turnover of E-cadherin-based cell-cell junctions. This unifies the
different observations in the literature and explains how tension results in the
disruption of cell-cell adhesion needed for tumor metastasis.
Sonnenberg A. KASH-domain proteins in
nuclear migration, anchorage and other
processes. J Cell Sci 2006; 119:5021-5029
Deciphering the functions of Cd151 The overexpression of the tetraspanin Cd151 in
tumors of epithelial origin is strongly correlated with enhanced metastasis and poor
prognosis. In order to investigate the contribution of Cd151 to tumor formation and
metastasis, we have generated conditional Cd151 knockout mice on a p53 null
background. Keratinocyte cell lines, established from these mice, are currently being
used to determine the role of Cd151 in cell adhesion, migration, proliferation and
invasion. Furthermore, we are in the process of generating a mouse model for skin
cancer using the conditional Cd151 knockout mice. The physiological role of Cd151 in
the kidney was studied using Cd151-null mice. It was shown that these mice develop
the same renal pathology of human patients, i.e., with age they develop massive
proteinuria caused by focal glomerulosclerosis, disorganization of the glomerular
basement membrane, and tubular cystic dilation. Notably, these abnormalities are
strikingly similar to those seen in mice with a conditionally inactivated integrin a3
subunit in podocytes, thereby supporting the existence of functional integrin-Cd151
complexes in vivo.
19
CELL BIOLOGY
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 signaling pathways involved in the formation and progression of
tumors may lead to the development of novel diagnostic tools or improved
therapeutic strategies.
Group leader John Collard
Function-based screens for invasion-inducing genes In earlier studies we have
identified the invasion-inducing Tiam 1 gene by retroviral insertional mutagenesis
in combination with in vitro selection of invasive T-lymphoma cells. Additional
functional screens for genes involved in T-lymphoma invasion revealed a number of
novel genes, including the a5- subunit of the a5b1 integrin, the LPA2 receptor and
Rap1, which have been implicated in cell-matrix adhesion and/or signaling pathways
mediated by Rho GTPases. For these novel screens, retroviral cDNA libraries were
used in combination with in vitro selection of invasive T-lymphoma cells. Currently,
the molecular mechanisms are being studied how these genes are involved in the
acquisition of an invasive phenotype.
Rho family proteins are controlled by various regulatory proteins The invasioninducing Tiam 1 gene encodes an activator or guanine nucleotide exchange factor
(GEF) for the Rho-like GTPase Rac. Rho GTPases, which include Cdc42, Rac and
RhoA, control a wide range of biological processes such as the adhesion, polarity,
and motility of cells. In particular, they act in signaling pathways that regulate the
reorganization of the actin cytoskeleton in response to receptor stimulation.
The principal regulators of the activities of Rho proteins are the GEFs and
GTPase activating proteins (GAPs). GEFs induce activation of the small GTPases
by exchanging GDP for GTP, whereas GAPs inactivate the small GTPases by
enhancing the intrinsic rate of hydrolysis of bound GTP to GDP. Guanine nucleotide
dissociation inhibitors (GDIs) control the activity of Rho proteins. In the cytoplasma,
Rac is kept in its GDP-bound form by association with RhoGDI. RhoGDI binds and
masks the hydrophobic C-terminal region of Rac, which is responsible for targeting
Rac to the plasma membrane. We found that increased intracellular [Ca2+] and the
activation of PKC induces phosphorylation of RhoGDI leading to its dissociation
from Rac. Cytosolic Rac translocates to the plasma membrane where it can be
activated by membrane associated GEFs such as Tiam 1.
John Collard PhD Group leader
Audrey Gerard PhD Post-doc
Michiel Pegtel PhD Post-doc
Kristin Strumane PhD Post-doc
Saskia Ellenbroek MSc Graduate student
Amra Hajdo-Milasinovic MSc Graduate student
Sander Mertens MSc Graduate student
Tomasz Rygiel MSc Graduate student
Rob Van der Kammen Technical staff
Publications
Engers R, Mueller M, Walter A,
Collard JG, Willers R, Gabbert HE.
Prognostic relevance of Tiam 1 protein
expression in prostate carcinomas.
Br J Cancer 2006; 95:1081-1086
Hajdo-Milasinovic A, Mertens AE,
Hamelers IHL, Collard JG. Rho GTPases
in cell motility and tumorigenesis. In: Wells
A, editor. Cell Motility in Cancer Invasion
and Metastasis. Berlin [etc]: Springer,
2006: 189-220
Malliri A, Rygiel TP, Van der Kammen RA,
Song JY, Engers R, Hurlstone AFL,
GEFs and downstream signaling GEFs are regulated at four different levels in the cell,
i.e. by intra-molecular inhibition, changes in intracellular localization, posttranslational modifications and interaction with other proteins. All of these regulatory
mechanisms seem to be involved in Tiam 1 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 Tiam 1. Tiam 1 may also
determine the nature of the signaling downstream of Rac by interacting with various
effector or scaffold proteins. We found that Tiam 1 is indispensable for a3b1-mediated
Rac activation. In contrast to wild-type keratinocytes, Tiam 1-deficient cells do not
adhere to and spread on a glass substrate, because they are unable to deposit their
own laminin-5 substrate. Tiam 1-deficient keratinocytes are impaired in activation
of Rac upon adhesion to an exogenous laminin-5 substrate, whereas other integrinmediated signaling pathways are intact. Tiam 1-deficiency thereby prevents
keratinocyte migration in vitro and re-epithelialization of excision wounds in mouse
skin in vivo.
It is well known that Rho proteins regulate cell adhesion and migration through their
control of actin cytoskeleton dynamics mediated by the Arp2/3 complex. We found
that Tiam 1 interacts with the p21-Arc subunit of the Arp2/3 complex. As a result,
Tiam 1 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 Tiam 1 and thereby Rac-activity, which both are required for
activation of the Arp2/3 complex. The Arp2/3 complex drives actin polymerization
preliminary at the leading edge of cells.
Clevers H, Collard JG. The Rac activator
Tiam 1 is a Wnt-responsive gene that
modifies intestinal tumor development.
J Biol Chem 2006; 281:543-548
Mertens AEE, Pegtel DM, Collard JG.
Tiam 1 takes PARt in cell polarity. Trends
Cell Biol 2006; 16:308-316
Strumane K, Rygiel TP, Collard JG. The
Rac Activator Tiam 1 and Ras-Induced
Oncogenesis. In: Balch WE, Der CJ, Hall
A, editors. Regulators and Effectors of Small
GTPases: Ras Family. Academic Press,
2006: 269-281
Ten Kooster JP, Evers EE, Janssen L,
Machesky LM, Michiels F, Hordijk P,
Collard JG. Interaction between Tiam 1 and
the Arp2/3 complex links activation of Rac
to actin polymerization. Biochem J
2006; 397:39-45
20
CELL BIOLOGY
Figure I.2: Primary hippocampal neuron
isolated from a Tiam 1-deficient mouse.
Dendrites are visualized by anti-tyrosine
tubulin mAb.
Tiam 1-Rac signaling and cell polarity Tiam 1 also associates with components of the
Par polarity complex and thereby regulates polarity processes. Tiam 1 localizes to
adherens junctions in epithelial cells and ectopic expression of Tiam 1 promotes Ecadherin-based cell-cell adhesions. Conversely, knock down of Tiam 1 leads to loss of
cell-cell adhesions and epithelial-mesenchymal transition. Tiam 1 also controls tight
junction (TJ) formation, which is required for the establishment of apical-basal cell
polarity in epithelial cells. We found that TJ maturation is severely impaired in
Tiam 1-deficient keratinocytes. Interestingly, Tiam 1 interacts with two components of
the Par polarity complex (Par3 and atypical PKCz) and Tiam 1-mediated Rac activation
controls TJ biogenesis by activation of the Par polarity complex. In the absence of
cell-cell adhesions, keratinocytes develop front-rear polarization and migrate in a
directional persistent fashion. Tiam 1 and the Par complex also function in directional
migration. Knock down of Par3, lack of Tiam 1, or reduced PKCz activity impairs
front-rear polarization, persistent migration and chemotaxis of keratinocytes.
Apparently, in the absence of cell-cell contacts, epithelial cells divert Par polarity
signaling and Tiam 1 to promote persistent cell migration. Tiam 1 in conjunction with
the Par complex also affects directional protrusion outgrowth in astrocytes and
controls Rap1- and chemokine-induced polarization and chemotactic migration of
T-cells.
Tumorigenicity in Tiam 1 mutant mice Although Tiam 1 is expressed during embryonic
development, Tiam 1-deficient (Tiam 1-/-) mice develop, grow, and reproduce normally.
In mouse skin, Tiam 1 is present in basal and suprabasal keratinocytes of the
epidermis and in hair follicles. We found that Tiam 1-/- mice display resistance to
DMBA/TPA induced skin tumor formation. Although the number and growth of
tumors is strongly reduced in these Tiam 1-/- mice, Tiam 1-deficiency increases the
frequency of malignant conversion of the skin tumors that do develop. Currently we
are studying the role of Tiam 1 in tumorigenesis induced by different oncogenic
signaling pathways i.e. Ras, Wnt, Myc, Neu and PI3-kinase. We found that Tiam 1 is a
Wnt-responsive gene and that Tiam 1 protein levels are increased in intestinal tumors
produced in APC min mice as well as in human colon tumors. In addition, initiation
and growth of intestinal tumors was reduced in Tiam 1-deficient APC min mutant
mice, suggesting that Tiam 1 is required for b-catenin/TCF-induced 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 Tiam 1-deficient mice.
Mammary tumors induced by MMTV-c-neu appeared dependent on Tiam 1 whereas
mammary tumors induced by MMTV-myc were not. Together, these studies so far
indicate that Tiam 1 may contribute to the formation and progression of tumors
induced by various -but not all-oncogenic signaling pathways.
Opposing functions of Rac1 and Rac3 Rac1 and Rac3 are highly homologous proteins
that share 92% of the amino acid sequences. Most differences are found in the
carboxy-terminus of these proteins. Rac1 is ubiquitously expressed and regulates
adhesion, migration and differentiation in various cell types. 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 contrast, knock down of Rac3 induces stronger cell adhesions and
increases the outgrowth of neurites in neuronal cells. The intracellular localization of
Rac1 and Rac3 is different, which is determined by the different sequences in the
carboxy-terminus. The Rac1-opposing function of Rac3 is not mediated by or
dependent on components of the RhoA signaling pathway. Our data indicate that
Rac3 opposes Rac1 in the regulation of cell-matrix adhesions and differentiation of
neuronal cells.
Figure I.3: The organization of the
microtubule cytoskeleton is disturbed is
Tiam 1-deficient astrocytes (bottom)
compared to wild type astrocytes (top).
21
CELL BIOLOGY
MEC HANISMS IN METASTASIS
Metastasis, the spread of tumor cells to distant sites in the body, is the main cause of
cancer lethality. We study mechanisms of metastasis of lymphomas and carcinomas.
Role of 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, surprisingly, not involved in invasion. We next
observed that CXCR4 is required for continuation of growth when micrometastases
are 500-1000 cells in size. The same was found for dispersed single cells in a s.c.
Matrigel plug. The cells had no CXCR4 in vitro but it was upregulated in vivo. The
CXCL12 is supposed to be derived from the tumor microenvironment but it was not
clear whether it is present in the plugs. To demonstrate involvement of CXCL12, we
transfected the dominant-negative K1R-CXCL12 mutant that should inhibit CXCL12induced signals. Surprisingly, however, these cells as well as control cells transfected
with intact CXCL12 proliferated much faster, even though they did not express
CXCR4. We assume that this is due to the recently discovered receptor CXCR7 that is
expressed by the cells and also binds CXCL12. The method we used to block CXCR4
function in vivo should also affect CXCR7. We are currently investigating which of
the two is involved in the in vivo phenomena.
The role of chemokine receptor CXCR5 in carcinoma metastasis to the liver We
detected CXCR5 in many carcinoma cell lines and in 30% of human pancreatic
carcinomas. This was surprising since CXCR5 was thought to be only expressed by
B- and some T-lymphocytes. We found that the ligand, BCA-1 (CXCL13) enhanced
proliferation of CXCR5-positive cells. CXCL13 is present in the liver. Inhibition of
CXCR5 reduced growth of liver metastases and after some time led to growth arrest.
Preliminary results with a pancreatic carcinoma in CXCL13 knockout mice indicate
that CXCL13 is required to resist an anti-tumor response of probably NKT cells in the
liver. In the knockout mice the liver metastases grow for seven days and then regress,
whereas in +/- litter mates growth is only transiently impeded.
Synaptotagmin: role in chemotaxis? Our original hypothesis was that rapid reactions
of lymphoid (and lymphoma) cells to chemokines involved rapid fusion of vesicles
with the plasma membrane. We thought that this was controlled, as in nerve cells, by
synaptotagmins (Syts), and indeed found that inhibition of Syts impaired migration.
Syt3 was next shown to be involved, and to be required for CXCL12-induced
migration signals. Unexpectedly, however, rapid reaction to chemokines, in particular
the induction of adhesion in flow, did not require Syt3. Furthermore, Syt3 was found
intracellularly, in multivesicular bodies (MVB), and not at the plasma membrane.
Figure 1.4: Regression of pancreatic carcinoma liver metastases in CXCL13 knockout mice. Panc02
pancreatic carcinoma cells, expressing luciferase, were injected into the spleen, which was subsequently
removed. This led to the formation of liver metastases, the growth of which was measured by
bioluminescence upon injection of luciferin. Note logarithmic scale. Metastases grew rapidly for 7 days
in CXCL13-/- knockout mice and heterozygous CXCL13+/- litter mates. After that, growth stopped for a
week in CXCL13+/- mice and resumed thereafter. In contrast, liver metastases regressed completely in
CXCL13-/- mice. This shows that CXCL13, the ligand of CXCR5, allows the cells to resist a host response,
probably of liver NKT cells.
Group leader Ed Roos
Ed Roos PhD Group leader
Agnieszka Masztalerz PhD Post-doc
Joost Meijer MSc Graduate student
Yvonne Wijnands Technical staff
Janneke Ogink Technical staff
22
CELL BIOLOGY
Publications
Masztalerz A, Zeelenberg IS,
Wijnands YM, De Bruijn R, Drager AM,
Janssen H, Roos E. Synaptotagmin
3 deficiency in T cells impairs recycling of
the chemokine receptor CXCR4 and thereby
inhibits CXCL12 chemokine-induced
migration. J Cell Sci 2007; 120:219-228
Meijer J, Zeelenberg IS, Sipos B, Roos E.
The CXCR5 chemokine receptor is expressed
by carcinoma cells and promotes growth of
colon carcinoma in the liver. Cancer Res
2006; 66:9576-9582
Stroeken PJM, Alvarez B, Van Rheenen J,
Wijnands YM, Geerts D, Jalink K, Roos E.
Integrin cytoplasmic domain-associated
protein-1 (ICAP-1) interacts with the
ROCK-I kinase at the plasma membrane.
J Cell Physiol 2006; 208:620-628
This argued against our hypothesis and suggested that Syt3 has another function.
CXCR4 surface levels were much lower in cells in which Syt3 function was blocked,
whereas the total amount of CXCR4 protein was not different. Furthermore,
recycling of CXCR4 after CXCL12-induced endocytosis was impaired. Finally,
overexpression of CXCR4, resulting in increased surface levels, restored migration.
Thus, Syt3 is required for recycling of CXCR4 in these cells, and when recycling is
abrogated, this leads to CXCR4 surface levels that are insufficient for a migratory
response but apparently high enough for arrest of cells in flow. A possibility being
considered is that Syt3 controls, in a calcium-dependent fashion, the recruitment of a
highly active CXCR4 receptor from MVB to the surface. This might have higher
affinity or be associated with particular signaling molecules, and rapidly lose activity.
Interestingly, we found that Syt3 is expressed in many carcinomas whereas it is
apparently absent in the corresponding normal epithelial cells. We are presently
investigating whether this Syt3 is required for the function of chemokine receptors in
carcinomas, as described above.
23
CELL BIOLOGY
BIOPHYSICS IN CELL SIGNALING
Biophysical techniques are exquisitely suited to study cell signaling events with high
spatial and temporal resolution. We employ advanced electrophysiological (e.g. patch
clamping) and bio-photonic techniques (e.g. Fluorescence Resonance Energy
Transfer (FRET), Fluorescence Lifetime Imaging (FLIM), Fluorescence Cross
Correlation Spectroscopy (FCCS) and photorelease of caged compounds) 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 hardware, software and FRET
sensors for various intracellular messengers.
Group leader Kees Jalink
Kees Jalink PhD Group leader
Michiel Langeslag PhD Post-doc
Spatiotemporal aspects of phosphatidylinositol bisphosphate (PIP2) as a membranedelimited messenger Phosphoinositides in the plasma membrane regulate a wide
variety of cellular responses. We developed FRET-based assays for the lipids PIP2 and
PIP3, and use these to study the regulation of agonist-induced PIP2 breakdown and
its subsequent resynthesis by PIPkinases. In collaboration with members from the
division of Cellular Biochemistry we have addressed how the concentration of PIP2 in
the plasma membrane regulates gap junction communication, and involvement of
this lipid in protecting cells against apoptosis.
Bas Ponsioen MSc Graduate student
Gerard Van der Krogt PhD Technical staff
Janneke Ogink Technical staff
Publications
Altelaar AFM, Klinkert I, Jalink K,
De Lange RPJ, Adan RAH, Heeren RMA,
Mechanism of activation of TRPM7, a cat ion channel that regulates cell adhesion
TRPM7 is a non-selective cation channel with inherent kinase activity that is involved
in sensing mechanical forces. TRPM7 mediates agonist-induced cell adhesion
through Ca2+ influx and myosin heavy chain phosphorylation. We discovered that
PLC-activating agonists strongly stimulate TRPM7 channels. Currently, we study
TRPM7 activation in detail by combining biophysical readout techniques with
mutational analysis of the C terminus. Our results indicate involvement of a feedback
loop that incorporates Ca2+ influx-mediated PLC activation in TRPM7 gating. We also
study regulation of the closely related channel TRPM6, which is involved in Mg2+
homeostasis.
Piersma SR. Gold-enhanced biomolecular
surface imaging of cells and tissue by SIMS
and MALDI mass spectrometry. Anal Chem
2006; 78:734-742
Clark K, Langeslag M, Van Leeuwen B,
Ran L, Ryazanov AG, Figdor CG,
Moolenaar WH, Jalink K,
Van Leeuwen FN. TRPM7, a novel
regulator of actomyosin contractility and
cell adhesion. EMBO J 2006; 25:290-301
Scope of cyclic nucleotide messengers in coupled cells The cyclic nucleotide cAMP is
an important messenger with numerous biological effects. Using live cell imaging
with FRET sensors for cAMP we have shown that cAMP readily permeates through
Gap Junctions to influence the fate of neighboring cells. In addition, we have
generated an improved family of cAMP FRET sensors with superior signal-to-noise
ratio and cellular inertia.
Halstead JR, Van Rheenen J, Snel M,
Meeuws S, Mohammed S, D’Santos CS,
Heck A, Jalink K, Divecha N. A Role for
PtdIns(4,5)P2 and PIP5Ka in Regulating
Stress-Induced Apoptosis. Curr Biol
2006; 16:1850-1856
Jalink K. Spying on cGMP with FRET.
Nature Methods 2006; 3:11-12
Langeslag M, Clark K, Moolenaar WH,
Van Leeuwen FN, Jalink K. Activation of
TRPM7 Channels by Phospholipase
C-coupled Receptor Agonists. J Biol Chem
2007; 282:232-239
Figure I.5: 3-step model for the involvements of
TRPM7 in mechano-regulated cell adhesion.
Ponsioen B, Van Zeijl L, Moolenaar WH,
A) Vesicles containing TRPM7 channels localize at
Jalink K. Direct measurement of cyclic AMP
or close to the membrane in resting cells.
diffusion and signaling through connexin43
B) Through cell adhesion structures, cells sense
gap junctional channels. Exp Cell Res
mechanical stress such as increased fluid flow.
2007; 313:415-423
As a consequence, TRPM7 vesicles fuse with the
plasma membrane and the channels become active.
Stroeken PJM, Alvarez B, Van Rheenen J,
C) Increased local Ca2+ influx through the TRPM7
Wijnands YM, Geerts D, Jalink K, Roos E.
channel pore causes association of the kinase domain
Integrin cytoplasmic domain-associated
with its substrates at the cell adhesion site. This leads
protein-1 (ICAP-1) interacts with the
to the formation of podosomes and the further
ROCK-I kinase at the plasma membrane.
recruitment of other proteins in the complex.
J Cell Physiol 2006; 208:620-628.
24
MOLECULAR CARCINOGENESIS
II D IV IS ION OF M OLECU LA R
CAR CIN OG ENES IS
FUNCTIONAL GENOMICS
Division head, Group leader RenГ© Bernards
RenГ© Bernards PhD Group leader
Katrien Berns PhD Post-doc
My group uses functional genomics technologies to identify novel cancer-relevant
genes. We use both high-throughput gain-of-function genetic screens and loss-offunction genetic screens to identify additional components of the major cancerrelevant pathways. We also use these functional genetic approaches to understand the
mechanisms of action of anti cancer drugs.
Luis Borlado PhD Post-doc
Annette Dirac PhD Post-doc
Pieter Eichhorn PhD Post-doc
Sidong Huang PhD Post-doc
Rianne Oosterkamp MD Clinical fellow
Benjamin Rowland PhD Post-doc
Miguel Rubio PhD Post-doc
Linda Smit PhD Post-doc
Mirjam Epping MSc Graduate Student
Ernst-Jan Geutjes MSc Graduate student
Roderik Kortlever MSc Graduate Student
Jasper Mullenders Msc Graduate Student
Functional identification of cancer-relevant genes One of the major remaining
deficits in our understanding of the human genome is that the function of only one
quarter of the approximately 30,000 genes is known to date. Many of these hitherto
anonymous genes are potential targets for the development of new anti-cancer drugs.
It is therefore important to functionally annotate the thousands of genes for which
this information is currently lacking. My laboratory has developed functional genetic
approaches to obtain information regarding gene function using high-throughput
screens in mammalian cells. We have developed both gain-of-function genetic
screens (using retroviral cDNA expression libraries) and loss-of-function genetic
screens (using RNA interference libraries) to carry out large-scale genetic screens in
mammalian cells.
Miranda Van Dongen Technical staff
Marielle Hijmans MSc Technical staff
Mandy Madiredjo Technical staff
Publications
Bernards R. Exploring the uses of RNAi Gene knockdown and the nobel prize.
N Engl J Med 2006; 355:2391-2393
Bernards R, Brummelkamp TR,
Beijersbergen RL. shRNA libraries and
their use in cancer genetics. Nat Meth
2006; 3:701-706
Brummelkamp TR, Fabius AWM,
Gain-of-function genetic screens These screens involve the infection of a cell
population with a high-complexity retroviral cDNA expression library, selection of
cells with altered phenotype, followed by identification of the cDNA responsible for
the phenotype. In the past years, we have used gain-of-function genetic screens to
identify genes that confer resistance the anti-proliferative effects of a group of
experimental anti cancer drugs: histone deacetylase inhibitors (HDACIs). The aim of
this study was the identification of biomarkers that predict resistance to HDACI
therapy. Using this approach, we have identified cDNAs that confer resistance to this
group of anti-cancer drugs. A remarkable common feature of all three cDNAs
identified to date is their ability to inhibit transactivation by retinoic acid receptor in
response to its natural ligand, retinoic acid. Conversely, suppression of these genes
by RNA interference increases sensitivity to the anti-proliferative effects of HDACI
in vitro and in vivo. Importantly, we found that RA and HDACI act synergistically in
the inhibition of tumor growth in a mouse xenograft model. Thus, this study
identified biomarkers of HDACI resistance and suggests strategies to increase the
efficacy of HDACIs in a clinical setting.
Mullenders J, Madiredjo M, Velds A,
Kerkhoven RM, Bernards R,
Beijersbergen RL. An shRNA barcode
screen provides insight into cancer cell
vulnerability to MDM2 inhibitors.
Nat Chem Biol 2006; 2:202-206
Creyghton MP, RoГ«l G, Eichhorn PJA,
Vredeveld LC, DestrГ©e O, Bernards R.
PR130 is a modulator of the Wnt-signaling
cascade that counters repression of the
antagonist Naked cuticle. Proc Natl Acad
Loss-of-function genetic screens Using a mammalian expression vector, which
directs the synthesis of short hairpin transcripts (shRNAs), we have inhibited all
members of a conserved family of Protein Phosphatase 2A (PP2A) regulatory
subunits. These regulatory subunits are thought to act as bridging factors between
the PP2A catalytic subunit and its substrate. Using this gene family knockdown
library, we identified PR55g as an inhibitor of c-Jun N terminal kinase (JNK)
activation by UV irradiation (Figure II.1). We found that PR55g binds c-SRC and
modulates the phosphorylation of serine 12 of c-SRC, a residue we found to be
required for JNK activation by c-SRC. We also found that the physical interaction
between PR55g and c-SRC is sensitive to UV irradiation, suggesting a novel pathway
of c-SRC activation in response to cellular stress.
Sci U S A 2006; 103:5397-5402
Kortlever RM, Higgins PJ, Bernards R.
Plasminogen activator inhibitor-1 is a
critical downstream target of p53 in the
induction of replicative senescence. Nat Cell
Biol 2006; 8:878-884
The tumor suppressor protein p53 limits the proliferative capacity of most primary
cells in culture through the induction of a state of growth arrest named replicative
senescence. However, little is known concerning the downstream target genes of
p53 in this growth-limiting response. In a second study, we asked which (if any) of
the transcriptional targets of p53 is responsible for the induction of replicative
senescence. We found that suppression of the p53 target gene encoding plasminogen
activator inhibitor-1 (PAI-1) by RNA interference leads to escape from replicative
senescence both in primary mouse embryo fibroblasts and primary human BJ
25
MOLECULAR CARCINOGENESIS
Publications (continued)
fibroblasts. Our data indicate that PAI-1 knockdown results in sustained activation of
the PI3K-PKB-GSK3b pathway and nuclear retention of cyclin D1, consistent with a
role for PAI-1 in regulating growth factor signaling. In agreement with this, we found
that the PI3K-PKB-GSK3b-cyclin D1 pathway is also causally involved in cellular
senescence. Conversely, we found that ectopic expression of PAI-1 in proliferating
p53-deficient murine or human fibroblasts induced a phenotype displaying all the
hallmarks of replicative senescence. These data indicate that PAI-1 is not merely a
marker of senescence, but both necessary and sufficient for the induction of
replicative senescence downstream of p53 (Figure II.2).
Unresponsiveness to therapy is a persistent problem in the treatment of cancer.
It is therefore important to identify the molecular pathways that contribute to
unresponsiveness to cancer therapeutics. We also use loss-of-function genetic screens
to identify genes that contribute to drug resistance, in particular to new classes
of targeted therapeutics. For these studies, 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 a new way to rapidly screen shRNA libraries: siRNA
bar code screening (in collaboration with R Beijersbergen, division of Molecular
Carcinogenesis, see below). In a first proof-of-concept study, we identified genes
required for the cytotoxicity of a promising new cancer drug named Nutlin-3. Nutlin-3
is a small molecule inhibitor of MDM2, which can activate the p53 pathway. We have
identified that a component of the DNA damage-induced signaling network, 53BP1, is
a critical mediator of Nutlin-3 induced cytotoxicity. This result suggests that Nutlin-3’s
cytotoxic effect results from its ability to turn a cancer cell specific property, activated
DNA damage signaling, into a weakness that can be exploited therapeutically. We are
currently using a similar approach to identify genes whose inactivation causes
resistance to new classes of anti cancer drugs, such as Herceptin, Iressa and Velcade.
We have also used our genome-wide shRNA library to identify genes whose
suppression can cause resistance to growth arrest induced by the retinoblastoma
tumor suppressor protein pRB. We generated human BJ fibroblasts that express a
mutant form of pRB in which 15 of the CDK phosphorylation sites are mutated. This
pRB-О”CDK is constitutively active as a growth suppressor. However, the growthinhibitory capacity of this protein can be overruled by a temperature sensitive mutant
of SV40 T antigen. This mutant T antigen protein binds to pRB at 32В°C, but not at
39В°C. As a result, cells co-expressing the pRB-О”CDK and the temperature sensitive
mutant of SV40 T antigen proliferate at 32В°C, but not at 39В°C, when the constitutively
active pRB is released from T antigen to inhibit proliferation. Using this cell system,
we searched for genes whose suppression by RNA interference causes a bypass of the
pRB-induced growth arrest. To date, we have identified one gene, RB1CC1, whose
suppression allows cells to proliferate, in the presence of active pRB. We are currently
studying the mechanism of action of RB1CC1 in the pRB pathway. Interestingly,
RB1CC1 is mutated in a significant fraction of human primary breast cancers,
suggesting that loss of this gene may also contribute to resistance to pRB growth
arrest in vivo.
Nijman SMB, Hijmans EM,
El Messaoudi S, Van Dongen MMW,
Sardet C, Bernards R. A functional genetic
screen identifies TFE3 as a gene that confers
resistance to the anti-proliferative effects of
the retinoblastoma protein and
transforming growth factor-b. J Biol Chem
2006; 281:21582-21587
Oosterkamp HM, Neering H,
Nijman SMB, Dirac AMG, Mooi WJ,
Bernards R, Brummelkamp TR. An
evaluation of the efficacy of topical
application of salicylic acid for the treatment
of familial cylindromatosis. Br J Dermatol
2006; 155:182-185
Rowland BD, Bernards R. Re-Evaluating
Cell-Cycle Regulation by E2Fs. Cell
2006; 127:871-874
Figure II.1: RNAi-based PP2A “B” subunit
genetic screen. (Top): Schematic of the
PP2A holoenzyme and outline of the b
regulatory subunit families. (Bottom):
U2-OS cells were co-transfected with a set
of PP2A shRNAs targeting a single PP2A
“B” subunit or a control vector. Levels of
phosphorylated JNK (a- pJNK) or JNK1
and JNK2 (a-JNK) are shown in cell lysates
for the samples 60 minutes after UV
An exciting new concept in the development of cancer drugs is the genotype-specific
drug target—the protein whose inactivation is toxic only to cells carrying a defined
(cancer-specific) genetic lesion. In theory, drugs targeting the products of such genes
would be more selective for cancer cells than the current generation of broadly acting
cytotoxic drugs, as they require the presence of a cancer-specific lesion to exert their
cytotoxic effects. RNAi technology is exquisitely suited for uncovering such genetic
interactions in mammalian cells. A new goal is to identify such interactions in
mammalian cells, as this may ultimately lead to the generation of “genotype-specific”
drugs, which act with a considerable degree of specificity on tumor cells.
Figure II.2: Proposed model of senescence in fibroblasts. p53 induces PAI-1 and p21CIP1 during ageing in
culture. PAI-1 antagonizes uPA/GF (growth factor) signaling to cyclin D1 via PI3K-PKB-GSK3b and
p21CIP1 blocks cyclin D1 activity directly. The PAI-1-cyclin D1 connection is dominant over p21CIP1 activity
and controls induction of the senescence response downstream of p53 and upstream of pRb.
treatment of the cells.
26
MOLECULAR CARCINOGENESIS
RNA INTERFERENCE BASED LOSS-OF-FUNCTION SCREENING
Group leader Roderick Beijersbergen
Roderick Beijersbergen PhD Group leader
David Egan PhD Post-doc
Armida Fabius MSc Graduate student
Johan Kuiken MSc Graduate student
Our research is focussed around the development and application of technologies
based on RNA interference to perform loss-of-function genetic screens. We have
completed our human and mouse collections of shRNA expression vectors for
cell based loss of function screens. In addition we have, within the RNAi global
consortium, acquired a full genome synthetic siRNA collection (21.500 genes) that
is used for high throughput arrayed format screens. This collection enables us to
develop screening systems based on more complex phenotypes using high content
imaging. We have continued to improve and apply our bar code screening technology
to identify novel components in disease-associated signaling pathways and to achieve
a more complete understanding of drug action, enabling more rational decisions on
drug application.
Jeroen Nijwening MSc Graduate student
Wouter Nijkamp Technical staff
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.
In this way the effect of gene inactivation on the phenotype of individual cells can
be followed. The design of the NKi shRNA library 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 (“bar code”) 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 bar code sequences. By using this technique, large complex populations of
shRNA expressing cells can be monitored for the abundance of each individual
shRNA expressing cell (Figure II.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 identification of genes
required for the cytotoxicity of a new cancer drug named Nutlin-3. Nutlin-3 is a small
molecule inhibitor of MDM2, which can activate the p53 pathway. We have identified
that a component of the DNA damage induced signaling network, 53BP1, is a critical
mediator of Nutlin-3 induced cytotoxicity. This result suggests that Nutlin-3’s
cytotoxic effect results from its ability to turn a cancer cell specific property, activated
DNA damage signaling, into a weakness that can be exploited therapeutically.
At this moment several cancer drugs are used to identify genes that either enhance
the drug’s effect or result in resistance to drug treatment.
Figure II:3: Large populations of shRNA
expressing cells are analyzed in parallel using
molecular barcode screens. Each shRNA vector is
tagged with a unique “bar code” sequence (the 19mer 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 bar code
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.
27
MOLECULAR CARCINOGENESIS
Publications
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; 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 multiple isogenic cell lines that contain single or
multiple defined genetic alterations in oncogene – or tumor-suppressor pathways.
These are used in high throughput single well assays in combination with genome
wide siRNA collections to identify siRNAs that result in enhanced lethality only in the
background of these tumor specific genetic alterations. The identification of the
proteins or pathways that are essential for the survival of tumor cells with specific
genetic alterations could result in a novel class of cancer therapeutics.
Bernards R, Brummelkamp TR,
Beijersbergen RL. shRNA libraries and
their use in cancer genetics. Nat Meth
2006; 3:701-706
Brummelkamp TR, Fabius AWM,
Mullenders J, Madiredjo M, Velds A,
Kerkhoven RM, Bernards R,
Beijersbergen RL. An shRNA barcode
screen provides insight into cancer cell
vulnerability to MDM2 inhibitors.
Nat Chem Biol 2006; 2:202-206
Kedde M, Le Sage C, Duursma A,
Zlotorynski E, Van Leeuwen B,
Nijkamp W, Beijersbergen R, Agami R.
Telomerase-independent Regulation of ATR
by Human Telomerase RNA. J Biol Chem
2006; 281:40503-40514
Figure II.4: Automated cell based assay system and automated confocal fluorescence microscope for high
content imaging of the NKI Robotics and Screening Center.
NKI Robotics and Screening Center The NRSC was started early 2005 with the
goal to develop an efficient technology platform to discover gene function, to unravel
molecular pathways and mechanisms, to discover novel drug targets and to support
the identification of small molecules both for biological tools and novel drug leads.
The NSRC is also a resource center that provides the technology for medium to high
throughput applications, provides support and expertise for automated cell and noncell based assays and is used for the development, production and maintenance of
large screening reagent collections (Figure II.4).
Recently, the NSRC has also acquired an automated fluorescence confocal
microscope to enable high content screening at single cell level with multiple cellular
markers (Figure II.5).
Figure II.5: Automated image analysis of a cell
based assay
Images obtained with different fluorescent
markers are used to identify the region of
interest (nucleus and cytoplasm) followed by
quantification of the fluorescent signal in both
compartments. Results are used to calculate the
cytoplasmic to nuclear ratio as measure for
pathway activation.
28
MOLECULAR CARCINOGENESIS
STRUCTURAL BIOLOGY
Group leader Titia Sixma
Titia Sixma PhD Group leader
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 proteins and the DNA involved. We use X-ray crystallography as a tool to provide
three-dimensional structures and we interpret the structural data using a variety of
biochemical and biophysical techniques. These studies provide more insight in the
molecular processes and they can also provide targets for drug design studies. In our
group we focus mainly on proteins involved in ubiquitin conjugation in chromatin
regulation, on DNA mismatch repair and nicotinic receptor signaling.
Joyce Lebbink PhD Senior Post-doc
Gretel Buchwald PhD Post-doc
Alex Fish PhD Post-doc
Valerie Notenboom PhD Post-doc
Chris Ulens PhD Post-doc
Rick Hibbert PhD Post-doc
Alex Faesen MSc Graduate student
Puck Knipscheer MSc Graduate student
Mark Vargas MSc Graduate student
Annet Reumer MSc Graduate student
Francesca Mattiroli MSc Graduate student
Pim Van Dijk Technical staff
Sari Van Rossum Technical staff
Herrie Winterwerp Technical staff
Publications
Aricescu AR, Assenberg R, Bill RM,
DNA mismatch repair DNA mismatch repair plays a crucial role in ensuring
genomic stability. Defects in the mismatch repair cascade in humans predispose
to hereditary non-polyposis colorectal cancer and are associated with a variety
of sporadic cancers. DNA mismatch repair is initiated by the protein MutS
(in Escherichia coli) or its MSH homologs. MutS recognizes and binds to mismatches
or unpaired bases that have escaped the proofreading capacity of the DNA replication
machinery or are present in the genome during recombinatorial events between nonfully complementary DNA strands. Mismatch binding triggers the uptake of ATP in
the MutS nucleotide binding domain and it is this mismatch-dependent 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.
One persistent problem in our structural studies of MutS complexes has been that we
could only crystallize it in a single crystal form. Recently, we have managed to
produce a new crystal form that shows that our previous conclusions were correct
that the DNA adapts to the protein, since we see the exact same protein-DNA
interaction. Nevertheless this structure was surprising as it gave us new insight in
the ATPase cycle of the MutS protein, which we are currently studying.
Busso D, Chang VT, Davis SJ,
Dubrovsky A, Gustafsson L, Hedfalk K,
Heinemann U, Jones IM, Ksiazek D,
Lang C, Maskos K, Messerschmidt A,
Macieira S, Peleg Y, Perrakis A,
Poterszman A, Schneider G, Sixma TK,
Sussman JL, Sutton G, Tarboureich N,
Zeev-Ben-Mordehai T, Jones EY.
Eukaryotic expression: Developments for
structural proteomics. Acta Crystallogr D
Biol Crystallogr 2006; 62:1114-1124
Ubiquitin and SUMO conjugation Ubiquitin conjugation processes have emerged
as a critical signaling system that is essential for cell stability, by controlling
degradation of short-lived proteins and signaling processes. Because of its
importance for regulating apoptosis, cell cycle, chromatin regulation and DNA
repair deregulation of ubiquitin-dependent processes often leads to cancer.
Structure analysis of the proteins involved in ubiquitin and SUMO conjugation could
be important in the development of novel drugs inhibiting critical pathways.
The process of conjugation by ubiquitin(-like) proteins involves covalent linking
of one or more 76-amino-acid ubiquitins to a target protein by an E1/E2/E3 cascade
of enzymes. Correct ubiquitination requires the complex spatial arrangement of
ubiquitin, E2, E3 proteins and the target simultaneously in a precise but flexible
manner. Although the overall mechanism has been defined, the atomic details have
been lacking and the specificity determining factors are unclear. We study several
E2/E3 complexes involved in conjugation of ubiquitin and the related modifier
SUMO as well as proteins involved in de-ubiquitination.
Polycomb complexes are transcriptional repressors involved in gene silencing and
maintenance of stem cell character. Together with the group of Maarten van
Lohuizen we studied the Ring-Ring complex of Bmi1-Ring1b (Figure II.6).
We showed in vitro E3 ubiquitin ligase activity for Ring1b against Histone2A in
nucleosomes and found that Bmi1 enhances this activity. We determined the
minimal Ring-Ring domain that is still active and solved its crystal structure. This
showed that the N-terminal arm of Ring1b embraces the Bmi1 Ring-domain, which
is essential for dimer formation and activity. We found that Ring1a has a similar
interaction with Bmi1. With point mutants we could show that activity in the
heterodimer is limited to Ring1b and not Bmi1. Currently we are expanding our
studies to the full length Ring1b/Bmi1 complex.
Figure II.6: The Ring1b-Bmi1 complex
showing the minimal Ring-Ring domain
that is active in ubiquitin conjugation of
Histone2A in nucleosomes
Nicotinic Acetylcholine receptor homolog AchBP In collaboration with Guus
Smit at the Free University in Amsterdam we study the Acetylcholine Binding
Proteins (AChBP) as tools for understanding the ligand binding in pentameric
29
MOLECULAR CARCINOGENESIS
Publications (continued)
ligand-gated ion-channels. AChBP has strong sequence similarity to the -subunits of
the nicotinic acetylcholine receptor ligand-binding domain and our AChBP crystal
structures are the established high-resolution model for the ligand-binding domains
in this class of ion channels. We have determined the 2.2-Г… crystal structure of a
homolog of the ligand-binding domain of the nAChR, Aplysia californica AChBP
(Ac-AChBP), in complex with a-conotoxin ImI (Figure II.7).
Banci L, Bertini I, Cusack S, De Jong RN,
Heinemann U, Jones EY, Maskos K,
Kozielski F, Owens R, Perrakis A,
Siebold C, Silman I, Messerschmidt A,
Poterszman A, Schneider G, Sixma T,
Sussman JL, Stewart-Jones G, Thierry JC,
Fig II.7: Crystal of the complex of the Aplysia
Moras D. First steps towards effective
californica Acetylcholine binding protein complexed
methods in exploiting high-throughput
to a-conotoxin ImI.
technologies for the determination of human
protein structures of high biomedical value.
Acta Crystallogr D Biol Crystallogr
2006; 62:1208-1217
Buchwald G, Van der Stoop P,
Weichenrieder O, Perrakis A,
This conotoxin is unique in its selectivity toward the neuronal a3b2 and a7 nicotinic
acetylcholine receptors, a feature that is reflected in its selective binding to Ac-AChBP
compared with other AChBP homologs. We observe a network of interactions
between the residues of the ligand-binding site and the toxin, in which ImI Arg-7 and
Trp-10 play a key role.
The toxin also forms interactions in the ligand-binding site that we did not see in the
complex of Ac-AChBP with PnIA(A10L D14K), a conotoxin variant that lacks binding
selectivity to AChBP homologs. In collaboration with the group of Daniel Bertrand
we could show on the basis of electrophysiological recordings obtained using the
wild-type a7 nAChR and L247T mutant, that conotoxin ImI inhibits ion conduction
by stabilizing the receptor in a desensitized conformation. Comparison of the AcAChBP–ImI crystal structure with existing AChBP structures gave new structural
insight into the extent of flexibility of the interface loops and how their movement
may couple ligand binding to channel gating in the context of a nAChR.
Van Lohuizen M, Sixma TK. Structure
and E3-ligase activity of the Ring-Ring
complex of Polycomb proteins Bmi1 and
Ring1b. EMBO J 2006; 25:2465-2474
Knipscheer P, Sixma TK. Divide and
conquer: The E2 active site. Nat Struct Mol
Biol 2006; 13:474-476
Lebbink JHG, Georgijevic D, Natrajan G,
Fish A, Winterwerp HHK, Sixma TK,
De Wind N. Dual role of MutS glutamate
38 in DNA mismatch discrimination and
in the authorization of repair. EMBO J
2006; 25:409-419
Natrajan G. MutS: Recognition of DNA
mismatches and initiation of repair.
Rotterdam: Erasmus Universiteit, 2006
Romier C, Ben Jelloul M, Albeck S,
Buchwald G, Busso D, Celie PHN,
Christodoulou E, De Marco V,
Van Gerwen S, Knipscheer P, Lebbink JH,
Notenboom V, Poterszman A, Rochel N,
Cohen SX, Unger T, Sussman JL,
Moras D, Sixma TK, Perrakis A.
Co-expression of protein complexes in
prokaryotic and eukaryotic hosts:
Experimental procedures, database tracking
and case studies. Acta Crystallogr D Biol
Crystallogr 2006; 62:1232-1242
Ulens C, Hogg RC, Celie PH, Bertrand D,
Tsetlin V, Smit AB, Sixma TK. Structural
determinants of selective a-conotoxin
binding to a nicotinic acetylcholine receptor
homolog AChBP. Proc Natl Acad Sci
USA 2006; 103:3615-3620
30
MOLECULAR CARCINOGENESIS
STRUCTURAL BIOLOGY
Group leader Anastassis Perrakis
Anastassis Perrakis PhD Group leader
Oliver Weichenrieder PhD Senior Post Doc
Patrick Celie PhD Post doc
Serge Cohen PhD Post Doc
Valeria De Marco PhD Post Doc
Mark Hilge Post Doc
Dene Littler Post Doc
Our main research themes continued to keep us occupied and are discussed below.
Dr. Oliver Weichenrieder who was leading the team studying function/structure
relationships in retrotransposition mechanisms - the first and longest lasting project
of our group - has accepted a group leader appointment at the Max Plank Institute in
Tubingen and he will continue his research on retrotransposons there. A couple of
new projects have been started and are ongoing in a collaborative effort; details for
these can be found in the reports of the groups of T Schumacher’s (Division of
Immunology) and M Fornerod (Division of Tumor Biology). While X-ray
crystallography remains the work horse for structure determination, new structural
techniques are being established in my laboratory: Small Angle X-ray Scattering
(SAXS) measurements and analyses are now done routinely; we are proceeding with
NMR structure determination of two protein domains; single particle reconstruction
Electron Microscopy (EM) pictures have also been obtained at high resolution for one
of our projects.
Krista Joosten Post Doc
Kostas Repanas MSc Graduate Student
Evangelos Christodoulou Technical Staff
Angelina Huseinovic Technical Staff
Suzan Van Gerwen Technical Staff
Mobien Kassiem Technical Staff
Marouane Ben Jelloul Software Engineer
Diederick De Vries Software Engineer
The role of Geminin in DNA replication licensing Geminin inhibits the action of
Cdt 1, a necessary factor for the formation of the pre-replication complex or
�replication license’, which is necessary to be assembled prior to DNA replication in
eukaryotic cells. We have crystallized various truncation constructs of the complex
between Cdt 1 and Geminin (Figure II.8) and determined the crystal structure of the
human truncated geminin/Cdt 1 complex at a resolution of 3.3 Г… from crystals grown
in conditions very close to physiological (pH 7.5, and about 50 mM NaCl). This
structure allowed us to establish important structural differences with previously
published data. To strengthen and validate our findings, the same molecules were
studied in solution by SAXS. Finally, the structure of the full-length geminin/
Cdt 1 complex has been characterized by SAXS and we have obtained high-resolution
EM micrographs. We clearly show that the �core complex’ of two geminin molecules
and one Cdt 1 (2Gem-1Cdt 1) forms a higher order dimer 2x(2Gem-1Cdt 1) under
physiological conditions in solution (Figure II.8). The dimerization interface of
geminin has been shown in literature to be crucial for function and our structural
data clearly explains its role and the biochemical data; the corresponding
dimerization region of Cdt 1 has been �uncharted’ and we are in the process of
characterizing its role in vivo.
Geminin also interacts with members of the Hox family of transcription regulators,
and with the Hox homologue Six3, a protein important for forebrain development.
This dual role of geminin has fundamental implications on how regulatory solutions
are �re-used’ in nature, besides the obvious questions about the mode of molecular
recognition of Hox proteins. We have cloned and purified Gemin and Six3 from
human, mouse and medaka fish and made complexes of Six3 with its target DNA.
We have obtained 2D NMR spectra for Six3 (Figure II.7) and are trying to improve the
spectra quality to determine the structure of Six3 alone and in complex with DNA.
In parallel we are trying to crystallise these complexes and perform affinity
measurements of the system components to elucidate how Six3 interacts with
geminin and how DNA affects that interaction.
Figure II.8: A model of the truncated
Geminin (grey cartoon) and Cdt 1
(darker-grey cartoon); Geminin forms a
parallel coiled coil, strongly attached to
one Cdt 1 molecule. Two such hetero-trimers
dimerize to form the biologically active
complex. The light-grey cloud is the low
resolution envelope of the full length
Geminin-Cdt 1 complex, as determined by
SAXS, depicting the additional domains.
In the background various crystal forms
of various truncation constructs of
the complex.
Structural studies of the plk1 Polo kinase Polo-like kinases play an important but
highly controversial role in cell division. If the molecular determinants for the
different roles of Plk- 1, 2, 3 and 4 are understood at the structural level we can
evaluate better the potential of Polo like kinases potential for the design of specific
drugs, targeting for example only Plk- 1 in tumor cells. We focus our efforts towards
two goals. Firstly, we want to characterize the full-length human Plk- 1; no crystals
have been obtained yet. Secondly, we have purified the polo domain of all four Plk
homologues. We have performed pull-down experiments that in combination with in
vivo results of our collaborators, and we demonstrate a partial functional overlap but
also distinct roles of the different PBDs: PBD2 appears to be more similar to PBD1
function, while PBD3 and PBD4 can only mimic PBD1 function in early mitotic
events.
31
MOLECULAR CARCINOGENESIS
Publications (continued)
Structural studies of Autotaxin Autotaxin (NPP 1) hydrolyzes circulating
lysophosphatidylcholine. The large-scale culture of human cells deficient in
glycosylation (HEK293 derivatives) and use of antibiotics have lead to production of
homogenous protein for crystallization experiments. We are still unable to obtain
crystals and provide structural information towards the collaborative screening effort
for drug discovery, which is currently ongoing in the NKI.
Albeck S, Ben Jelloul M, Perrakis A,
Henrick K. SPINE bioinformatics and
data-management aspects of highthroughput structural biology. Acta
Crystallogr D Biol Crystallogr 2006;
62:1184-1195
Structural studies of JBP 1 The JBP 1 protein binds to DNA that contains base J
(J-DNA). It was discovered by the P. Borst group, and has been shown to be essential
for survival in many major protozoan human pathogens such as T. brucei (sleeping
sickness), T. cruzi (Chagas’ disease) and Leishmania species (various types of
Leishmaniasis). We aim to elucidate the structure of JBP 1 and contribute to an
international drug discovery effort. We have worked out procedures to produce from
E. coli mg amounts of soluble purified Crithidia fasciculata and Leishmania tarentolae
JBP 1 that selectively bind J-DNA. Moreover, we have very recently identified a ~19 kD
domain of JBP 1 that binds J-DNA selectively (mini-JBP 1), and we can produce mg
amounts of it in purified soluble form. Surface plasmon resonance (SPR)
experiments have shown mini-JBP 1 to bind J-DNA with the same affinity as the fulllength protein and NMR experiments have shown it to be well folded and suitable for
structure solution by NMR. The shape of both mini-JBP 1 and JBP 1 has been
calculated by SAXS experiments. That allows us to establish that mini-JBP 1 is a
distinct small domain of the 100 kD JBP 1 protein, that is sufficient for binding JDNA in a specific manner.
Alzari PM, Christodoulou E,
Perrakis A, Owens RJ. Implementation of
semi-automated cloning and prokaryotic
expression screening: the impact of SPINE.
Acta Crystallogr D Biol Crystallogr
2006; 62:1103-1113
Aricescu AR, Perrakis A, Jones EY.
Eukaryotic expression: Developments for
structural proteomics. Acta Crystallogr
D Biol Crystallogr 2006; 62:1114-1124
Bahar M, Cohen SX, Perrakis A,
Wilson KS. SPINE workshop on automated
X-ray analysis: a progress report. Acta
Crystallogr D Biol Crystallogr 2006;
Structural characterisation of the human L1 retrotransposition machinery The
human L1 endonuclease (L 1EN) is encoded by the L 1 non-LTR retrotransposon that is
responsible for more than 1.5 million genomic re-integration events in the history of
the human genome. This has resulted in more than a quarter of our genomic DNA,
suggesting important roles for L1 elements for the “fluidity” and evolution of
genomes. Based on our structure of L1 endonuclease, we have designed a series of
L 1EN site and deletion mutants that were tested in a variety of in vitro DNA cleavage
and binding assays and in vivo assays by our collaborators. Several structure of such
mutants have been determined by X-ray crystallography. We have shown a 7-residue
loop to be important for target site recognition and we have managed to manipulate
DNA specificity by loop-exchange mutants. This brings us one step closer to
modulate the sequence specificity of L1 endonucleases and could help convert the
respective retrotransposon into a genetic tool for i.e. gene therapy.
62:1170-1183
Banci L, Maskos K, Perrakis A, Moras D.
First steps towards effective methods
in exploiting high-throughput technologies
for the determination of human protein
structures of high biomedical value. Acta
Crystallogr D Biol Crystallogr 2006;
62:1208-1217
Berry IM, Perrakis A, Messerschmidt A.
SPINE high-throughput crystallization,
crystal imaging and recognition techniques:
current state, performance analysis, new
Methods for high throughput Structural Biology The end of the EU SPINE
project and the start of SPINE-2 marked this year. Many of the methodological
contributions of this collaborative research project, in most of which our lab had an
active contribution, are summarized in a collection of publications that span diverse
but necessary fields for efficient structure determination: Protein (co)-expression in
eukaryotic and prokaryotic hosts, crystallization, information data management
and computational methods. SPINE (technology development for HTP structural
characterization) is dead! Long live SPINE-2 (macromolecular complexes of
biomedical interests)!
technologies and future aspects. Acta
Crystallogr D Biol Crystallogr 2006;
62:1137-1149
Haramis APG, Perrakis A. Selectivity and
promiscuity in Eph receptors. Structure
2006; 14:169-171
Mamely I, Van Vugt MA, Smits VA,
Semple JI, Lemmens B, Perrakis A,
Our lab keeps coordinating Crystallization research in the BIOXHIT EU program,
focusing on automation as well as new concepts. Our activity for devising laboratory
information management systems for structural biology is continuing within the
PIMS-LIMS European initiative.
Medema RH, Freire R. Polo-like Kinase-1
Methods for X-ray crystallography After the successful launch and establishment
of ARP/wARP version 6.1 early last year, time for new science to improve our
software was available again! Our latest endeavors include the implementation of
a Naive Bayess and a Multilayer Perceptron pattern classifier that allow better
assignment of sequence information to structures through prior knowledge from
the databases; algorithms to build more disordered regions in protein structures;
and an �intelligent’ decision system or driving better the ARP/wARP modules.
Romier C, Ben Jelloul M, Celie PHN,
Controls Proteasome-Dependent
Degradation of Claspin during Checkpoint
Recovery. Curr Biol 2006; 16:1950-1955
Christodoulou E, De Marco V,
Van Gerwen S, Cohen SX, Perrakis A.
Co-expression of protein complexes in
prokaryotic and eukaryotic hosts:
experimental procedures, database tracking
and case studies. Acta Crystallogr D Biol
Crystallogr 2006; 62:1232-1242
32
CELLULAR BIOCHEMISTRY
III D IV IS ION OF CELLU LA R
B IO CHEM IS TR Y
LIPID GROWTH FACTORS AND THEIR ROLE IN
DEVELOPMENT AND CANCER
Division head, Group leader Wouter Moolenaar
Wouter Moolenaar PhD Group leader
Nicolai Savaskan PhD Post-doc
Catelijne Stortelers PhD Post-doc
Anna Houben MSc Graduate student
Laurens Van Meeteren MSc Graduate student
Bas Ponsioen MSc Graduate student
Leonie Van Zeijl MSc Graduate student
Trudi Hengeveld Technical staff
Adrian Rzadkowski Technical staff
Our group has a longstanding interest in lipid growth factors, particularly
lysophosphatidic acid (LPA) and sphingosine 1-phosphate (S 1P), their signaling
properties and their role in health and disease. LPA and S 1P 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
ATX, the regulation of its activity as well as its role in embryonic development and
tumor progression. We found that ATX knockout mice die at midgestation,
apparently due to defective vascular development. In addition, we have developed
sensitive ATX activity assays, which have successfully been used for high-throughput
screening of small-molecule compound libraries. Since ATX is found overexpressed
in certain cancers and LPA promotes cancer progression, ATX qualifies as a potential
target for therapy. Our work should lead to novel ways of interfering with LPA
receptor signaling in tumor cells and with inappropriate LPA production in their
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: gene transcription To examine how LPA receptor
signaling influences global gene transcription, we subjected mouse embryonic
fibroblasts 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. We find that LPA induces many genes encoding secreted factors,
including growth factors, pro-angiogenic factors, pro-inflammatory cytokines and
metalloproteases, as confirmed by quantitative RT-PCR. We are currently analyzing
the upstream receptor-linked signaling pathways and to what extent the LPAassociated gene signature shows overlap with that induced by peptide growth factors.
These studies highlight the importance of LPA in generating fibroblast-derived
mediators of wound healing, angiogenesis and tumor progression.
Figure III.1: Abnormalities in ATX-deficient
embryos compared to heterozygous embryos.
Homozygous knockouts die at midgestation
due to severe vascular defects in yolk sac (A)
and embryo proper (B; arrows; note also
defective closure of the neural tube).
Knockout embryos show severe
malformation and dorsal effusions (C).
Autotaxin, a secreted lysophospholipase D implicated in vascular
development and tumor progression
Regulation of ATX and discovery of small-molecule inhibitors
ATX is highly expressed in brain as well as in malignant glioblastoma cells. In adult
rat brain, ATX was detected mainly in leptomeningeal cells and oligodendrocyte
precursor cells, but not in mature neurons. Following lesion-induced neurotrauma,
ATX is strongly upregulated in reactive astrocytes adjacent to the lesion, suggesting a
role for the ATX-LPA axis in brain injury. ATX is unique among the ecto-nucleotide
phosphodiesterases in that it primarily functions as a lysoPLD, using a single
catalytic site for the hydrolysis of both lipid and non-lipid phosphodiesters. We
recently discovered that ATX is potently inhibited by LPA and S 1P in a mixed-type
manner. Thus, by repressing ATX activity, LPA can regulate its own biosynthesis in
the extracellular environment. Furthermore, these results reveal a novel role for S 1P
as an inhibitor of ATX, in addition to its well-established role as a receptor ligand. To
identify non-lipid inhibitors of ATX activity, we have performed high-throughput
screening of chemical compound libraries (in collaboration with David Egan and
Huib Ovaa). Screening of a 20,000 small-molecule library yielded several positive
“hits” that inhibit ATX activity as well as ATX-mediated tumor cell migration by more
than 70% (see the report by Huib Ovaa). Among these hits are several new chemical
structures with IC(50) values in submicromolar range. Specificity, selectivity and
toxicity of these pharmacological inhibitors are now under investigation and will be
further evaluated in cell-based and animal studies.
33
CELLULAR BIOCHEMISTRY
Publications
ATX in vascular development and tumor progression
ATX is widely expressed, but its in vivo role is unknown. We find that ATX-deficient
mice die at midgestation (E9.5) with profound vascular defects in yolk sac and
embryo. Additional abnormalities in the ATX-deficient embryos include allantois
malformation, neural tube defects, dorsal effusions and asymmetric headfolds. ATX
heterozygous mice appear healthy but show half-normal ATX activity and plasma
LPA levels. Our results reveal a critical role for ATX in vascular development and
indicate that ATX is the major LPA-producing enzyme in vivo. The ATX-deficient
phenotype is reminiscent of the Ga13 knockout. Since LPA receptors couple strongly
to Ga13 the ATX-deficient phenotype might be explained by loss of LPA signaling
through Ga13. Ex vivo yolk sac studies are now being done in attempt to explain the
ATX-deficient phenotype in further mechanistic detail. 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 cancer-prone
mouse models. Furthermore, by deleting ATX in specific tissues we hope to learn
more about ATX function in adult life 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.
Clark K, Langeslag M, Van Leeuwen B,
Ran L, Ryazanov AG, Figdor CG,
Moolenaar WH, Jalink K,
Van Leeuwen FN. TRPM7, a novel
regulator of actomyosin contractility and cell
adhesion. EMBO J 2006; 25:290-301
Ferguson CG, Bigman CS,
Richardson RD, Van Meeteren LA,
Moolenaar WH, Prestwich GD.
Fluorogenic Phospholipid Substrate to
Detect Lysophospholipase
D/Autotaxin Activity.
Org Lett 2006; 8:2023-2026
Langeslag M, Clark K, Moolenaar WH,
Van Leeuwen FN, Jalink K. Activation of
TRPM7 Channels by Phospholipase
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-to-cell 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, such as endothelin, thrombin
and ATP. The mechanism underlying G protein-regulated Cx43 gating has remained
elusive to date. Our studies reveal that the closure and reopening of Cx43 channels is
tightly controlled by phosphatidylinositol 4,5-bisphosphate (PIP2). Phospholipase C
(PLC)-mediated depletion of PIP2 results in Cx43 channel closure, whereas
knockdown of PLC or overexpression of the PIP2-synthesizing enzyme,
phosphatidylinositol-4-phosphate 5-kinase, keeps Cx43 channels open. Similarly,
knockdown of the Cx43-associated scaffold protein ZO-1 renders gap junctional
communication refractory to receptor stimulation. Our results identify PIP2 as a key
regulator of Cx43 gap junctional communication, and suggest that ZO-1 serves to
assemble PLC and Cx43 into a signaling complex to allow exquisite regulation of
cell-cell coupling by localized changes in PIP2.
C-coupled Receptor Agonists. J Biol Chem
2007; 282:232-239
Ponsioen B, Van Zeijl L, Moolenaar WH,
Jalink K. Direct measurement of cyclic AMP
diffusion and signaling through connexin43
gap junctional channels. Exp Cell Res
2007; 313:415-423
Savaskan NE, Rocha L, Kotter MR, Bär A,
Lubec G, Van Meeteren LA, Kishi Y, Aoki J,
Moolenaar WH, Nitsch R, Bräuer AU.
Autotaxin (NPP-2) in the brain: cell
type-specific expression and regulation
during development and after neurotrauma.
Cell Mol Life Sci 2007;Online publication
Sayas CL, Ariaens A, Ponsioen B,
Moolenaar WH. GSK-3 Is Activated by
the Tyrosine Kinase Pyk2 during LPA1mediated Neurite Retraction. Mol Biol Cell
2006; 17:1834-1844
Van Meeteren LA, Ruurs P, Stortelers C,
Bouwman P, Van Rooijen MA,
PradГЁre JP, Pettit TR, Wakelam MJO,
Saulnier-Blache JS, Mummery CL,
Moolenaar WH, Jonkers J. Autotaxin,
a secreted lysophospholipase D, is essential
for blood vessel formation during
development. Mol Cell Biol
2006; 26:5015-5022
Van Rossum AGSH, Moolenaar WH,
Schuuring E. Cortactin affects cell
migration by regulating intercellular
adhesion and cell spreading. Exp Cell Res
2006; 312:1658-1670
34
CELLULAR BIOCHEMISTRY
LIPID METABOLISM IN SIGNAL TRANSDUCTION
Group leader Wim Van Blitterswijk
Wim Van Blitterswijk PhD Group leader
Marcel Verheij MD PhD Academic staff
Menno Van Lummel PhD Postdoc
Membrane lipids not only have a structural function but also can serve as signaling
molecules. Our current studies focus on the production, function and conversion
of three types of lipids. First, the sphingolipids that associate with cholesterol
in microdomains known as lipid rafts. Biosynthesis of the raft constituent
sphingomyelin (SM) plays a crucial role in the sensitivity of tumor cells to undergo
apoptosis by alkyl-lysophospholipids and other stimuli. Short-chain sphingolipids can
enhance the cellular uptake of the anti-cancer agent doxorubicin. Two other lipids,
diacylglycerol (DAG) and phosphatidic acid (PA), can function as second messengers
and are the respective substrate and product of diacylglycerol kinases. The latter
enzymes are the subject of our ongoing research.
Jurgen Van Baal PhD Postdoc
Alrik Los MSc Graduate student
John De Widt Technical staff
Shuraila Zerp Technical staff
Jeffrey Klarenbeek Technical staff
Sphingomyelin synthase 1 determines tumor cell sensitivity to alkyllysophospholipid and other apoptotic stimuli Synthetic alkyl-lysophospholipids
such as Edelfosine (ALP; 1-O-octadecyl-2-O-methyl-glycero-3-phosphocholine) and
Perifosine (a piperidine analog) induce apoptosis in tumor cells, such as S49
lymphoma; they are used as anti-cancer agents in the clinic. ALP rapidly inserts into
the plasma membrane, interferes with lipid signaling, accumulates in lipid rafts and
then undergoes endocytosis. Prolonged culturing of S49 cells in the presence of ALP
resulted in ALP-resistance (S49AR cells) and, intriguingly, cross-resistance to other
apoptotic stimuli, such as DNA damage (gв€’radiation, etoposide) and Fas/CD95
death-receptor activation. Resistant cells were devoid of the raft constituent
sphingomyelin (SM) due to downregulated expression of SM synthase (SMS 1).
SiRNA-induced suppression of SMS 1 in S49 cells blocked SM synthesis, causing
resistance to all apoptotic stimuli. The resistance to Fas ligation was overcome by
exogenous short-chain SM, which enhanced Fas surface expression, and by a
proteasome inhibitor that prevented Fas degradation. Resistance to ALP and DNA
damage was due to lack of SMS 1 activity, not to the mere lack of SM content. SMS 1
activity is important for raft formation and membrane vesicular trafficking.
Inhibition of Nieman-Pieck-C1 protein-dependent vesicular transport of cholesterol
from endosomes to the Golgi, blocked apoptosis and SM synthesis (Figure III.2). We
propose that ALP internalization via rafts represents the retrograde route of vesicular
recycling, and that the resistance to ALP and DNA damage relates to a defect in
recycling. SMS 1 deficiency was accompanied by downregulation of SHIP 1, a PIP3
phosphatase. This resulted in upregulation of the PI3-kinase/PKB survival pathway.
We are now examining how apoptotic cross-resistance relates to SMS 1- and SHIP 1deficiency.
For clinical applications of ALP-related lipids, see the report by M Verheij, Division
IX, Radiotherapy.
Short-chain glucosylceramide facilitates uptake of liposomal doxorubicin by
tumor cells in vitro and in vivo The anti-cancer agent doxorubicin (DOX) is often
administered as a liposomal formulation (CaelyxВ®), from which it is gradually
released into the interstitial space and then taken up by tumor cells. We discovered
that short-chain sphingolipids such as N-octanoyl-glucosylceramide (GC) accelerate
the cellular uptake of DOX. GC also enhanced DOX accumulation in A431 carcinoma
cells. 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 (Figure III.3). This new
liposomal formulation of DOX, which we have patented, undergoes further testing
and seems promising for clinical application.
Figure III.2: Biosynthesis of sphingomyelin
(SM) in the Golgi is blocked when vesicular
transport of cholesterol from endosomes to
the Golgi is abrogated by U18666A
(2 Вµg/ml). Radiolabeled lipids were
extracted and separated by thin-layer
chromatography. PC, phosphatidylcholine;
LPC, lysoPC.
Diacylglycerol kinases (collaboration with N Divecha). Diacylglycerol kinase (DGK)
phosphorylates the second messenger DAG, activator of protein kinase C (PKC), to
yield PA. We have identified DGKВЈ and reported on its translocation to the plasma
membrane in response to noradrenaline. We investigated how the localization of
DGKВЈ is controlled by G protein-coupled receptors. In A431 cells, hormone-induced
activation of PKCe and -h regulated the translocation of DGKВЈ to the plasma
membrane. DGKВЈ translocation required additional tyrosine kinase activity of the
EGF receptor. Furthermore, during this process there was physical interaction and
35
CELLULAR BIOCHEMISTRY
Publications
colocalization of DGKВЈ with the EGF receptor. Expression of DGKВЈ inhibited
bradykinin-induced, PKC-mediated Thr654 phosphorylation of the EGF receptor,
whereas knockdown of DGKВЈ increased phosphorylation. Our results suggest that G
protein-coupled receptor-mediated DGKВЈ translocation can modulate PKC-mediated
events.
Los AP, De Widt J, Topham MK,
Van Blitterswijk WJ, Divecha N. Protein
kinase C inhibits binding of diacylglycerol
kinase-[zeta] to the retinoblastoma protein.
Biochimica et Biophysica Acta (BBA) Molecular Cell Research 2007; In Press
Los AP, Vinke FP, De Widt J, Topham MK,
Van Blitterswijk WJ, Divecha N. The
Figure III.3: N-octanoyl-glucosylceramide (GC)
Retinoblastoma Family Proteins Bind to
incorporated in doxorubicin-containing liposomes
and Activate Diacylglycerol Kinase{zeta}.
(GC-DOX) results in more effective reduction of
J Biol Chem 2006; 281:858-866
tumor size in vivo compared to regular liposomes
(CaelyxВ®) (DOX). Nude mice bearing A431
Van der Luit AH, Budde M, Zerp S,
tumors received 6 mg liposomal DOX/kg
Caan W, Klarenbeek JB, Verheij M,
bodyweight intravenously, after which tumor size
Van Blitterswijk WJ. Resistance
was measured daily.
to alkyl-lysophospholipid-induced
apoptosis due to downregulated
The DGKz isotype localizes to the nucleus, where it binds the retinoblastoma protein
pRb. DGKz interacts with pRb in vitro and in vivo. PKC-mediated phosphorylations of
DGKz negatively regulated DGKz binding to pRB. DGKz did not affect the ability of
pRB to regulate transcription; however, pRB stimulated DGKz activity in vitro.
Overexpression of DGKz in pRB-null fibroblasts reconstituted a cell cycle arrest
induced by g-irradiation. pRB-negative cells show increased Ras activity, suggesting
that DGKz may stimulate cell cycle arrest by inhibiting Ras signaling.
sphingomyelin synthase 1 expression with
consequent sphingomyelin- and cholesteroldeficiency in lipid rafts. Biochem J
2007; 401:541-549
Vink SR, Lagerwerf S, Mesman E,
Schellens JHM, Begg AC,
Van Blitterswijk WJ, Verheij M.
pRB is known to regulate muscle differentiation. In C2C12 myoblasts, DGKz
enhanced the expression of muscle-specific genes. Microarray analysis revealed that
DGKz increased expression of diverse muscle genes, especially targets of the
myogenic transcription factor MyoD. Indeed, DGKz stimulated promoter activity of
the MyoD target genes. DGKz knock-out cells were unable to differentiate or to
express muscle-specific genes.
Radiosensitization of squamous cell
carcinoma by the alkylphospholipid
perifosine in cell culture and xenografts.
Clin Cancer Res 2006; 12:1615-1622
Vink SR, Van Blitterswijk WJ,
Schellens JHM, Verheij M. Rationale and
clinical application of alkylphospholipid
analogues in combination with
radiotherapy. Cancer Treatment Rev 2007;
in press.
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: Antiangiogenic properties of a new class of antitumor agents. Anti-Cancer Drugs 1997;
in press.
36
CELLULAR BIOCHEMISTRY
SIGNALING THROUGH INOSITOL PHOSPHOLIPIDS
INTRODUCTION
Group leader Nullin Divecha
Nullin Divecha PhD Group leader
Jonathon Halstead PhD Post-doc
David Jones PhD Post-doc
Jurgen Van Baal PhD Post-doc
Alrik Los MSc Graduate student
Cells constantly experience various forms of cellular stress such as hypoxia and
oxidative imbalance, which can lead to genetic changes and eventually to the onset of
tumorigenesis. Cells therefore have a myriad of mechanisms which sense cell stress
and induce processes to counteract them. In extreme cases elimination (apoptosis) of
the cell benefits the whole organism. Furthermore the process of tumorigenesis
inevitably leads to the selection of cells with upregulated signalling pathways that can
confer increased resistance to cell stress, increased proliferation in the absence of
extracellular hormones and an increase in the ability of cells to migrate. We are
interested in the role of phosphoinositides, a family of phospholipid second
messengers, in the regulation of cellular stress pathways and the process of
tumorigenesis.
Willem-Jan Keune MSc Graduate student
Dalila Elouarrat MSc Graduate student
Yvette Bultsma Ing Technician
Mireille Snel Ing Technician
Figure III.4: The figure shows that
PIP4KII-beta is phosphorylated at five
different sites (P) and is acetylated at nine
different lysine residues (Ac). We generated
antibodies specific for phosphorylated S326
and showed that S326 becomes
phosphorylated in a stress (UV) dependent
manner (Inset top panel). RNAi-mediated
suppression of p38 map kinase expression
(pRetro-RNAi p38) showed that s326
phosphorylation in response to UV
irradiation was dependent on p38 activity
(inset). Antibodies to other phosphorylation
and acetylation sites have been generated
and are being to used to study their role in
vivo.
Phosphoinositides and Stress induced signalling PIP4KII-beta is a nuclear lipid
kinase that is able to phosphorylate the phosphoinositide PtdIns5P. PIP4KII-beta is
upregulated in breast tumours and is a component of a stress pathway that can
ultimately impinge on the function of the human tumour suppressor protein p53.
Upon exposure to various cellular stressors PIP4KII-beta regulates the levels of
nuclear PtdIns5P. How stress dependent changes in nuclear PtdIns5P are translated
into cellular phenotypes is beginning to be unravelled. Plant Homology Domains
(PHD) are zinc fingers that are able to interact with phosphoinositides and are
present in many proteins that are involved in regulating gene transcription, DNA
methylation and DNA repair. The PHD containing protein, Inhibitor of growth-2
(ING2), interacts with PtdIns5P and can regulate numerous nuclear functions
through its interaction with chromatin remodelling complexes and histone acetylases
and deacetylases. We now show that the intra-nuclear localisation of ING2 is subject
to control by cellular stress dependent changes in nuclear PtdIns5P and is an in vivo
sensor for changes in nuclear phosphoinositides.
To understand how the level of nuclear PtdIns5P is controlled we have identified
numerous stress dependent changes in post-translational modifications of PIP4KIIbeta using mass spectrometry (in collaboration with Prof. AJR Heck, Department of
Biomolecular Mass Spectrometry Utrecht and Dr C D’Santos, Probe Bergen Norway).
PIP4KII-beta is phosphorylated at five different positions and acetylated at
nine different lysine residues. We have generated antibodies specific to two of
these phosphorylation sites and show that one of them, serine 326 (S326), is
phosphorylated in response to cellular stress by the stress dependent protein kinase
p38 (Figure III.4). Furthermore phosphorylation negatively regulates PIP4KII-beta
activity and modulates its interaction with other proteins. How these various posttranslational modifications are coordinated into regulation of PIP4KII-beta activity
and localisation is under investigation.
PtdIns(4,5)P 2 : a role in regulating stress induced apoptosis How apoptotic
pathways are engaged in response to cell stress is not well characterized. We have
found that different types of cell stress lead to a rapid and irreversible decrease in the
cellular level of PtdIns(4,5)P2. PtdIns(4,5)P2 is a key intracellular signaling lipid and
is required for the regulation of many important cellular pathways including cell
survival, vesicle trafficking and growth factor signaling. Stress induced decrease in
PtdIns(4,5)P2 occurs in part via the delocalisation from the plasma membrane and
catalytic inactivation of PIP5K, an enzyme that synthesizes PtdIns(4,5)P2. Importantly
overexpression of PIP5K prevents the decrease in PtdIns(4,5)P2 and attenuates the
induction of apoptosis in response to various stressors. We postulate that cells
constantly monitor their PtdIns(4,5)P2 levels and that an irreversible decrease in
PtdIns(4,5)P2, induced by cellular stress, may act as a trigger to initiate apoptosis.
Interestingly, tumors often show enhanced PIP5K activity, which may be a
mechanism to avoid stress induced apoptosis. How increased PtdIns(4,5)P2
synthesis attenuates stress induced apoptosis is not clear, however we have found
that overexpression of PIP5K stimulates and maintains activation of the
p42/44 mapkinase signaling pathway after cellular stress induction. The p42/44
37
CELLULAR BIOCHEMISTRY
Publications
mapkinase is intimately involved in stimulating both cell survival and proliferation.
Further studies will define how PtdIns(4,5)P2 can regulate Map kinase signaling.
Bins A, Van Rheenen J, Jalink K,
Halstead J, Divecha N, Spencer D,
PIP5Ks and cytoskeletal dynamics The ability to migrate from their natural
environment and to invade other tissues is a hallmark of metastatic cancer cells and
its onset often defines a poor prognosis for the patient. Understanding signal
transduction pathways involved in migration is of the utmost importance in defining
novel anti cancer therapeutics. A key player in migration is the small molecular
weight G protein Rac, which regulates cytoskeletal dynamics. Rac can interact with
PIP5K although the consequence of this interaction is not clear. We have defined
specific regions of PIP5K-alpha that interact with Rac and have established a mutant
of PIP5K that no longer interacts with Rac (PIP5K-RacM). Interestingly this mutant
no longer localizes to the membrane and is also compromised in its ability to
synthesize phosphoinositides in vivo, but not in vitro. We have previously
demonstrated that PIP5K is required for inducing changes in neuronal morphology
in response to numerous signaling receptors such as lysophosphatidic acid (LPA).
We have used this cell system to demonstrate that both PtdIns(4,5)P2 synthesis and
interaction with Rac are required for remodeling of focal adhesions complexes by
PIP5K (Figure III.5).
Haanen J, Schumacher T. Intravital
imaging of fluorescent markers and
FRET probes by DNA tattooing. BMC
Biotechnology 2007; 7:2
Guillou H, Lecureuil C, Anderson KE,
Suire S, Ferguson GJ, Ellson CD, Gray A,
Divecha N, Hawkins PT, Stephens LR.
Use of the GRP1 PH domain as a tool
to measure the relative levels of
PtdIns(3,4,5)P3 through a protein-lipid
overlay approach. J Lipid Res
2006;D600038-DJLR200
Halstead JR, Van Rheenen J, Snel M,
Meeuws S, Mohammed S, D’Santos CS,
Heck A, Jalink K, Divecha N. A Role for
PtdIns(4,5)P2 and PIP5Ka in Regulating
Stress-Induced Apoptosis. Curr Biol
2006; 16:1850-1856
Jones DR, Bultsma Y, Keune WJ,
Halstead JR, Elouarrat D, Mohammed S,
Heck AJ, D’Santos CS, Divecha N.
Nuclear PtdIns5P as a Transducer of Stress
Signaling: An In Vivo Role for PIP4Kbeta.
Mol Cell 2006; 23:685-695
Figure III.5: Neuronal cells (N1E-115) were transfected with either a control vector or a mutant of PIP5K
unable to interact with Rac (PIP5K-RacM) or a Kinase inactive PIP5K (PIP5K-KD) which is unable to
Los AP, De Widt J, Topham MK,
generate PtdIns(4,5)P2 and acts to inhibit morphological changes in cells in response to LPA. Cells were
Van Blitterswijk WJ, Divecha N. Protein
maintained as controls (control) or stimulated with LPA. The cells were then fixed after 15 minutes and
kinase C inhibits binding of diacylglycerol
stained for the focal adhesion protein vinculin (shown as white areas). Stmulation with LPA leads to a
kinase-[zeta] to the retinoblastoma protein.
rapid dissolution of vinculin from focal adhesion complexes, which is blocked by the expression of both
BBA Mol Cell Res 2006; In Press
PIP5K mutants. This suggests that both PtdIns(4,5)P2 synthesis and the interaction of PIP5K with Rac is
required for regulating focal adhesion disassembly.
Vermeer JEM, Van Leeuwen W,
Tobena-Santamaria R, Laxalt AM,
Our demonstration of the role of PIP5K in regulating apoptosis and cytoskeletal
dynamics has led to the development of a targeted screen for small molecule
inhibitors of PIP5Ks. We hope that in the future these inhibitors may be useful in
cancer therapy.
Jones DR, Divecha N, Gadella TW, Jr.,
Munnik T. Visualization of PtdIns3P
dynamics in living plant cells. Plant J
2006; 47:687-700
Weinkove D, Halstead JR, Gems D,
Divecha N. Long-term starvation and
ageing induce AGE-1/PI 3-kinase-dependent
translocation of DAF-16/FOXO to the
cytoplasm. BMC Biology 2006; 4:1
38
CELLULAR BIOCHEMISTRY
ORGANIC SYNTHESIS AND CHEMICAL BIOLOGY
Group leader Huib Ovaa
Huib Ovaa PhD Group leader
Danny Burg PhD Post-doc
The organic synthesis and chemical biology laboratory at the Netherlands Cancer
Institute specializes in the design, synthesis and development of diagnostic-,
imaging- and proteomics tools. The group has extensive experience in organic
synthesis, intein chemistry, expressed protein ligation, mass spectrometry, protein
purification, fluorescence-based techniques, FACS analysis, confocal microscopy and
various standard biochemical techniques.
Research is centered around one central theme: chemical biology of protein
degradation and antigen presentation, and divided into three different topics:
Silvia Cavalli PhD Post-doc
Boris Rodenko PhD Post-doc
Fijs Van Leeuwen PhD Post-doc
Erica Van Tilburg PhD Post-doc
Topic 1: Ubiquitin proteomics
Topic 2: Proteasome activity
Topic 3: Antigen presentation
Celia Berkers MSc Graduate student
Rieuwert Hoppes MSc Graduate student
Henk Hilkmann Technical staff
Publications
Crawford LJA, Walker B, Ovaa H,
Chauhan D, Anderson KC, Morris TCM,
Irvine AE. Comparative selectivity and
specificity of the proteasome inhibitors
BzLLLCOCHO, PS-341, and MG-132.
Cancer Res 2006; 66:6379-6386
Groll M, Berkers CR, Ploegh HL, Ovaa H.
Crystal Structure of the Boronic Acid-Based
Proteasome Inhibitor Bortezomib in
Complex with the Yeast 20S Proteasome.
Structure 2006; 14:451-456
Hol EM, Fischer DF, Ovaa H, Scheper W.
Ubiquitin proteasome system as a
The group develops techniques to profile and interfere with enzymatic activities
associated primarily with ubiquitin-mediated proteasomal degradation and antigen
presentation and uses an organic synthesis and mass spectrometry driven approach
in order to gain further understanding of the biochemical processes under
investigation.
Antigen production and antigen presentation by MHC complexes leading to
T cell activation T lymphocytes serve to recognize and eliminate cells that express
foreign (e.g. virus-derived or tumor-specific) proteins. The recognition of antigen
presenting cells is based on the binding of the T cell receptor to MHC complexes
that have bound a peptide derived from a foreign protein (Figure III.6). Major
Histocompatibility Complexes (MHCs) can be divided into MHC class I and class II
complexes that are specialized to present antigens on the cell surface originating
from different sources. Whereas the class II pathway is responsible for the
presentation of extracellular proteins, the class I pathway presents endogenously
synthesized antigens, e.g. viral protein fragments, sequestered within the infected
cell, although some crosstalk between these pathways exists. Recognition of displayed
pMHC class I complexes by cytotoxic T cells leads to death of the antigen-expressing
cell whereas pMHC class II complexes are recognized by helper T cells, resulting in
immune activation. We currently study the loading of MHC class I molecules and
MHC class I binding to T cells.
pharmacological target in neurodegeneration.
Expert Rev Neurother 2006; 6:1337-1347
Kraus M, Ruckrich T, Reich M, Gogel J,
Beck A, Kammer W, Berkers CR, Burg D,
Overkleeft H, Ovaa H, Driessen C. Activity
patterns of proteasome subunits reflect
bortezomib sensitivity of hematologic
malignancies and are variable in primary
human leukemia cells. Leukemia 2007; 21:84-92
Figure III.6: MHC class I-mediated immunity: antigen processing, presentation and T cell activation. The
Risseeuw MDP, Berkers CR, Ploegh HL,
proteasome is responsible for protein degradation, assisted by other (endo)-proteolytic activities, affording
Ovaa H. Synthesis of tritium labeled KRN7000
epitopes that can be loaded into MHC class I complexes. Recognition of the presented pMHC complex by
Tetrahedron Lett 2006; 47:3677-3679
the TCR results in a T cell-mediated kill of the antigen presenting cell.
Rodenko B, Toebes M, Hadrup SR,
Ubiquitin-mediated protein degradation by the proteasome Ubiquitin
modification is a post-translational modification at the core of proteasome-mediated
protein degradation. The C-terminal residue of Ub is conjugated most often to the
e-amino group of a lysine residue of another protein forming a ubiquitin polymer
linked through isopeptide bonds. These polymers regulate proteasome-mediated
destruction and hence stability of most proteins. The action of Ub specific ligases is
counterbalanced by the action of Ub specific proteases (USPs) similar to the way
phosphorylation, mediated by the action of kinases, is counterbalanced by the action
of phosphatases. Malfunction of individual members of these enzyme classes are
Van Esch WJE, Molenaar AM, Schumacher TNM,
Ovaa H. Generation
of peptide-MHC class I complexes through UVmediated ligand exchange.
Nat Protocols 2006; 1:1120-1132
39
CELLULAR BIOCHEMISTRY
Publications (continued)
known to cause disease, which underscores the importance of assessing actual
catalytic activity profiles of USPs in different cell types and in cells corresponding to
discrete stages of differentiation, activation, and malignant transformation. A variety
of USPs act either as oncoprotein or as tumor suppressor protein and USPs also
play important regulatory roles by determining the conjugation status of substrate
proteins. The combined action of ligases and proteases is responsible for remodeling
the shape and size of “trees” such as seen in polyubiquitin chains, in which Ub
and probably ubiquitin-like molecules are conjugated onto other Ub molecules via
isopeptide bonds to form a polymer. Size, shape and composition of such trees
likely determine the half-life of a protein. We develop tools that can provide
information on the specificity, activity and identity of factors involved in ubiquitinmediated protein degradation. Studies aimed at a functional characterization of
proteolytic activity profiles under different physiological and pathological conditions
have been boosted by the development of such probes.
RolГ©n U, Kobzeva V, Gasparjan N,
Ovaa H, Winberg G, Kisseljov F,
Masucci MG. Activity profiling of
deubiquitinating enzymes in cervical
carcinoma biopsies and cell lines.
Mol Carcinog 2006; 45:260-269
Toebes M, Coccoris M, Bins A,
Rodenko B, Gomez R, Nieuwkoop NJ,
Van de Kasteele W, Rimmelzwaan GF,
Haanen JBAG, Ovaa H,
Schumacher TNM. Design and use of
conditional MHC class I ligands.
Nat Med 2006; 12:246-251
Conditional MHC class I ligands In collaboration with the Schumacher lab
(Division of Immunology) we recently developed a chemical strategy to load MHC
class I molecules on command. This strategy takes advantage of epitopes that can be
triggered to fragment, by UV irradiation (Figure III.7). Resulting in in situ-generated
peptide-receptive protein complexes that can be readily loaded with appropriate
ligands, enabling broad epitope scans. This strategy will be used in the rapid
identification of both MHC ligands and cytotoxic T cell responses and the value of
this strategy was demonstrated by the definition of an avian flu epitope.
Figure III.7: Principle of MHC class I exchange: A UV cleavable MHC ligand is triggered to fragment, in
situ generating an unstable, peptide receptive MHC molecule. In the presence of a rescue peptide, an
efficient ligand exchange takes place allowing the generation of large collections of MHC complexes.
High throughput small molecule screening In collaboration with the Dr David
Egan (Division of Molecular Carcinogenesis) and the group of Wouter Moolenaar we
recently completed the first high throughput small molecule screen carried out so far
at an academic institution in the Netherlands. This screen was carried out to find
inhibitors of autotaxin (ATX), a widely expressed phosphodiesterase that promotes
tumor cell motility, experimental metastasis and angiogenesis, leading to the
discovery of a series of low nanomolar ATX inhibitors. Several other small molecule
screens are planned for next year.
Verdoes M, Berkers CR, Florea BI,
Van Swieten PF, Overkleeft HS, Ovaa H.
Chemical Proteomics Profiling of
Proteasome Activity. In: Nedelkov D, Nelson
RW, editors. New and Emerging Proteomic
Techniques. Totowa, NJ: Humana Press,
2007: 51-70
40
CELLULAR BIOCHEMISTRY
EPIGENETIC REGULATION OF GENE EXPRESSION
Group leader Fred van Leeuwen
Fred Van Leeuwen PhD Group leader
Alex Faber PhD Post-doc
Floor Frederiks MSc Graduate student
In eukaryotic cells the DNA is packaged into chromatin by histone proteins. Posttranslational modifications of the histone proteins and methylation of DNA can
result in heritable changes in gene expression without changes in the actual genetic
code. These epigenetic changes have been shown to contribute to tumorigenesis.
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 and
gene expression.
Kitty Verzijlbergen MSc Graduate student
Tibor Van Welsem Technical staff
Figure III.8: Telomeric silencing in budding
yeast; red (dark) and white (light) sectors in
a yeast colony indicate epigenetic
inheritance of active and silent chromatin.
Figure III.9: Interdependence between
active and silent chromatin. Histone H3
acetylation (ac) and methylation (me) in
active chromatin introduced by
methyltransferases (MT) and histone
acetyltransferases (HAT) affect assembly of
the silencing complex (Sir2,3,4) at silent
chromatin.
Interdepence between active and silent chromatin We are studying how histones
and their post-translational modifications help set up and propagate domains of
active and silent chromatin. In yeast we previously identified a novel histone
methyltransferase Dot1, which methylates lysine 79 on histone H3 (H3K79) on the
top and bottom of the nucleosome core. This unusual enzyme is conserved from
yeast to humans and has been shown to be involved in certain human leukemias. In
yeast Dot1 is critical for gene silencing but is mainly present in active regions of the
genome. Our findings suggest that histone H3K79 methylation in active chromatin
prevents promiscuous binding of silencing proteins, and thereby enhances targeting
of silencing proteins to silent chromatin. Recent results showed that, in addition to
histone H3K79, several other histone modifications associated with active regions of
the genome positively affect silencing and that they do so by pathways independent
of Dot1. This proposes an important general interdependence between the active and
silent chromatin domains in the nucleus.
The role of histone H3 lysine 79 methylation in gene silencing Dot1 methylates
~90% of histone H3 in yeast and can add one, two, or three methyl groups to its
target lysine. In general, different forms of lysine methylation tend to have specific
localizations or functions in chromatin. We recently isolated a mutant of Dot1 that
lacks the ability to trimethylate H3K79 in vivo or in vitro. Characterization of this
mutant showed that trimethylation of H3K79, which is present on as much as 50% of
all histone H3, is not critical for silencing. Our data suggest that the overall degree of
H3K79 methylation rather than the amount of one specific methyl form determines
the degree of gene silencing. We are currently running genome-wide genetic screens
to determine the pathways and molecular mechanisms by which H3K79me affects
chromatin organization.
An assay to study the role of histones and their modifications in cellular
memory One of the main goals of our group is to understand how chromatin
modifications can have long-term effects on gene expression. 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 very dynamic. We have developed a novel
assay in yeast to determine protein turnover and protein-complex dynamics in vivo.
The assay involves differential labeling of existing and newly synthesized proteins.
We are now applying this assay to determine how parental histones and newly
synthesized histones and their post-translational modifications are assembled into
chromatin.
41
IMMUNOLOGY
IV DI VI S I ON OF IMMU NO LOG Y
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. Sustaining survival of activated
lymphocytes is expected to improve anti-tumor immunity by enlarging the tumorspecific lymphocyte pool and by enhancing long-term maintenance of tumor-specific
lymphocytes (immunological memory). The second goal of our work is to contribute
to the design of novel therapies aimed at killing cancer cells by activating apoptotic
pathways.
Division head, Group leader Jannie Borst
Jannie Borst PhD Group leader
Sabine Middendorp PhD Post-doc
Esther Wissink PhD Post-doc
Anna Keller MSc Graduate student
Chiel Maas MSc Graduate student
Victor Peperzak MSc Graduate student
Elise Veraar MSc Graduate student
Inge Verbrugge MSc Graduate student
Evert De Vries Technical staff
TNF receptor family members and control of the immune response From our
work, TNF receptor family member CD27 and its ligand CD70 have emerged as
interesting targets to improve anti-tumor immunity. This costimulatory system
promotes generation and maintenance of effector and memory T cells, which is
accomplished at least in part by rescue of activated T cells from apoptosis. To
determine by which molecular mechanisms CD27 directs the T cell response, we use
as a prime approach micro-array analysis of CD27-induced gene expression. We
employ an in vitro system of T cell stimulation, but are also able to analyze expression
profiles of T cells taken ex vivo from mice that are undergoing an immune response.
Findings are corroborated at the protein level and by functional assays. In this way,
we have determined that one of the key effects of CD27 is directing the synthesis of
and responsiveness to certain pro-survival cytokines. Its anti-apoptotic effect seems to
be primarily indirect, via these cytokines. We have also obtained the novel insight that
CD27 directs the synthesis of certain chemokines that have been implicated in the
recruitment of effector T cells. This ties in with the potential of CD27 to promote
accumulation of T cells at tissue sites.
We have demonstrated that CD27 improves the potential of CD8+ memory T cells to
expand upon secondary stimulation. Imprinting of memory characteristics into
CD8+ T cells has been reported to require the help of CD4+ T-cells during the
primary response. We have established that CD4+ T cells need CD27 to deliver such
help to the memory CD8+ T cell response. By protein and gene expression analysis of
CD4+ T cells undergoing an immune response in vivo, we have established that
CD27 promotes the capacity of CD4+ T cells to synthesize certain cytokines that have
been implicated in CD4+ T cell help.
The ligand for CD27, CD70, is expressed on activated lymphocytes and dendritic cells
(DC). Recent findings in the field suggest that the functional capabilities of T cells
are to a large extent imprinted in the priming phase, during T cell-DC contact.
To determine the exact impact on T cell function of CD27/CD70 interactions that
take place during this contact, we have made novel genetic tools. In the first place,
we have generated transgenic mice constitutively expressing CD70 under control
of the DC-selective CD11c promoter. While CD70 is normally acquired during DC
maturation, these mice express CD70 on immature DC (Figure IV.1). It is known that
the maturation stage of DC is decisive for the T cell response. Whereas immature DC
maintain T cell tolerance and give abortive responses to foreign antigen, mature DC
that express the full array of costimulatory ligands, induce T cell responsiveness.
Interestingly, we have found that the mere expression of CD70 on immature DC
suffices to induce T cell expansion to foreign peptide antigen. The activated T cells in
addition acquire full functional activity, sufficient to eliminate aggressive tumors.
This finding emphasizes the potential of exploiting CD27/CD70 interactions to
promote anti-tumor immunity. We have recently generated CD70-deficient mice,
which will be used as recipients to further address the potential of CD70 transgenic DC.
Gerda Van der Horst Technical staff
Figure IV.1: Characterization of CD70
transgenic mice. Flow cytometric analysis
indicated constitutive expression of CD70
on immature dendritic cells (CD11c+)
of CD70 transgenic (tg), but not wild-type
(WT) mice (A Keller, unpublished results).
42
IMMUNOLOGY
Publications
Aulwurm S, Wischhusen J, Friese M,
Borst J, Weller M. Immune stimulatory
effects of CD70 override CD70-mediated
immune cell apoptosis in rodent glioma
models and confer long-lasting antiglioma
immunity in vivo. Int J Cancer 2006;
118:1728-1735
Keller AM, Borst J. Control of Peripheral
T Cell Survival: A Delicate Division of
Labor between Cytokines and Costimulatory
Molecules. Hum Immunol 2006;
67:469-477
Ortiz F, Tait SW, Robledo G, De Vries E,
Borst J, LГіpez-Rivas A. The mitogenactivated protein kinase pathway can
inhibit TRAIL-induced apoptosis by
prohibiting association of truncated Bid
with mitochondria. Cell Death Differ 2006;
13:1857-1865
Wieland CW, Kerver ME, Florquin S,
Nolte MA, Borst J, Van Lier R,
Van Oers MHJ, Van der Poll T. CD27
contributes to the early systemic immune
response to Mycobacterium tuberculosis
infection but does not affect outcome.
Int Immunol 2006; 18:1531-1539
Wissink EHJ, Verbrugge I, Vink SR,
Schader MB, Schaefer U, Walczak H,
Borst J, Verheij M. TRAIL enhances
efficacy of radiotherapy in a p53 mutant,
Bcl-2 overexpressing lymphoid malignancy.
Radiother Oncol 2006; 80:214-222
Figure IV.2: Proof of principle of RNA
interference library screen. Barcode analysis
identified siRNA constructs enriched in
MCF-7 cells selected for resistance to
TRAIL-induced apoptosis. Among these was
siRNA construct for Caspase-8, a known
component of the pathway (C Maas,
unpublished results).
The cell biology of TNF family members CD70 is a type II transmembrane
molecule, like most other ligands of the TNF receptor family. Cell surface expression
of CD70 is transient and fully dependent on conditions of immune activation. We
have revealed that in cells that contain the machinery for MHC class II-restricted
antigen presentation, such as DC, CD70 is sorted exactly like MHC class II to the
inner vesicles of late endocytic compartments. In cells that lack MHC class II
presentation function, CD70 travels by default to the cell surface. We propose that the
specific intracellular trafficking of CD70 provides professional antigen-presenting
cells, such as DC, with a mechanism to coordinate CD70 cell surface expression both
temporally and spatially with antigen presentation. Indeed, when maturing DC were
exposed to specific T cells in the presence of the appropriate antigen, all newly
synthesized CD70 protein was routed via MHC class II compartments to the
immunological synapse formed between the T cell and DC. This work is carried out
in close collaboration with the group of J Neefjes (Division of Tumor Biology).
Apoptosis signaling and cancer therapy Death receptors, such as the TRAIL
receptors and 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.
We have performed a functional genetic screen for mediators of TRAIL-induced
apoptosis in MCF-7 breast cancer cells. The complete NKI RNA interference library
was transduced into these cells, which were subsequently selected for resistance to
TRAIL-induced apoptosis. SiRNA inserts were recovered and their identity was
determined by bar code array (collaboration with the Division of Molecular
Carcinogenesis). The system has worked reliably and revealed proof of principle,
since siRNA for Caspase-8 and Bid were selected (Figure IV.2). A dozen of novel
candidate regulators for TRAIL-induced apoptosis have been identified in this way,
which will be subject of further functional analysis.
After death receptor stimulation, Bid is cleaved by Caspase-8 and its carboxy-terminal
fragment subsequently translocates to mitochondria where it induces permeability of
the outer membrane and release of pro-apoptotic mediators. After DNA damage, Bid
can also be activated, according to our data, but by a different mechanism that is
currently under investigation in mouse embryo fibroblasts. We have found that the
amino-terminal fragment of Bid, upon its generation by Caspase-8 is ubiquitinated in
an unconventional way and subsequently degraded by the proteasome (work of postdoc S Tait, currently in Memphis TN). Extensive combined genetic and biochemical
analysis has allowed us to define the region in Bid that is subject to ubiquitination
and to reveal the likely target residues. Functional experiments have proven that
ubiquitination and consequent proteasomal degradation of the amino-terminal
fragment of Bid liberates its carboxy-terminal fragment for apoptosis-induction.
In collaboration with M Verheij of the Division of Radiotherapy we carry out a project
aimed to exploit death ligands to enhance the efficacy of radiotherapy. As proof of
principle, we have used human lymphoid tumor cells. In these cells, radiation
(and DNA damaging drugs) can sensitize for TRAIL receptor- and CD95 induced
apoptosis when the mitochondrial pathway is blocked and p53 is lacking.
Sensitization is not due to death receptor upregulation at the cell surface but appears
to entail increased caspase activity, resulting from a higher capacity of engaged
receptors to generate pro-apoptotic signals. The efficacy of single and combined
treatment was determined in a subcutaneous xenotransplant setting of the lymphoid
tumor. Tumor growth and regression was determined by bioluminescence as well as
by caliper measurement. For results of this study, the reader is referred to the report
of M Verheij in this issue. Our ultimate goal is to test efficacy of combined treatment
in solid tumor models, particularly those generated by A Berns and coworkers. These
models will be used to explore both TRAIL and a novel formulation of recombinant
CD95 ligand in combined modality treatment.
43
IMMUNOLOGY
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.
Group leader Ton Schumacher
In vivo visualization of T cell immunity (collaboration with Haanen lab) Aim
of this line of research is to define the rules that control the efficiency of vaccineinduced T cell responses and of effector T cell function, both through in vivo
imaging. In the past year, focus of this work has mostly been on the evaluation of the
DNA tattoo approach developed in the Haanen lab by confocal laser scanning
microscopy and the development of model systems in which in vivo T cell responses
can be traced by CLSM. These studies have demonstrated that a combination of DNA
tattoo and CLSM can be utilized to monitor cellular processes such as apoptosis in
skin. Furthermore, ongoing experiments suggest that it will be feasible to trace
anti-viral T cell responses by imaging, using a Herpes Simplex virus skin infection
model. In the coming year we will set out to utilize this latter model to visualize how
anti-viral T cells combat a viral infection in a physiological setting.
Ton Schumacher PhD Group leader
Ramon Arens PhD Post-Doc
Anna Calogero PhD Post-doc
Arne Bakker MSc Graduate student
Miriam Coccoris MSc Graduate student
Carmen Gerlach MSc Graduate student
Jeroen Van Heijst MSc Graduate student
Koen Schepers MSc Graduate student
Moniek De Witte MD Graduate student
Marly Van den Boom Technical staff
Jeanine Joling Technical staff
Erwin Swart Technical staff
MHC exchange technology (collaboration with Ovaa and Perrakis labs) MHC
tetramers and other types of MHC multimers have become an essential tool for the
analysis of T cell immunity both in animal model systems and in the clinic. However,
a major obstacle for the effective application of MHC tetramers is the inefficiency of
the classical strategy for MHC generation, and this precludes the generation of large
libraries of MHC multimers, or of a collection of clinical grade MHC multimer
reagents for cellular therapy.
In collaboration with H Ovaa (Division of Cellular Biochemistry) we have set out to
address this issue, by designing MHC class I molecules occupied with a general
producer peptide that can be exchanged with an antigenic peptide of interest at will.
We have now shown by X-ray crystallography that MHC molecules form the intended
complex with these designed conditional ligands (collaboration with T Perrakis and
P Celie, Division of Molecular Carcinogenesis). Furthermore, we have demonstrated
that 1) Recombinant MHC molecules can be produced with designed peptide variants
that are sensitive to UV exposure for a variety of MHC alleles 2) MHC complexes
created in this manner can then be utilized to screen collection of thousands of
potential MHC ligands by fluorescence anisotropy or ELISA. In addition we have
begun the development of MHC micro-arrays and technology for clinical grade
production of conditional MHC complexes for subsequent use in cellular therapy.
One specific area in which we consider the prospects of this type of technology
significant is that of allogeneic hematopoietic stem cell transplantation for
hematological malignancies.
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. MHC tetramer technology makes it
possible to follow the development of immunity at the T cell population level.
However, it doesn’t allow the analysis of cell fate and cellular differentiation pathways.
To address this issue, 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 microarray. Infection of cell
populations by these libraries of retroviral vectors and subsequent micro-array
analysis can then be used to trace the progeny of individual cells. In a set of proof-ofprinciple experiments we have first demonstrated that the technology that we have
developed can reveal kinship (or lack thereof) between T cell populations that are
formed in vivo. We have now used this approach to determine whether T cell
populations that migrate to different effector sites are derived from a single or from
distinct naГЇve T cells, the latter being consistent with the increasingly popular notion
Mireille Toebes Technical staff
Jos Urbanus Technical staff
Figure IV.3: Retroviral barcoding supports a
diaspora model for T cell migration.
Barcode analysis of T cells in lung and
tumor tissue and the same analysis of the
draining lymph nodes of the two sites earlier
during antigen encounter. Note that while
distinct barcodes are recovered within the
draining lymph nodes (left), the T cells
ending up at the effector site share the same
set of barcodes (right).
44
IMMUNOLOGY
Publications
Crawford A, MacLeod M, Schumacher T,
Corlett L, Gray D. Primary T cell expansion
and differentiation in vivo requires antigen
presentation by B cells. J Immunol 2006;
176:3498-3506
De Witte MA, Coccoris M, Wolkers MC,
Van Den Boom MD, Mesman EM,
Song JY, Van Der Valk M, Haanen JBAG,
of imprinting of T cell function by DC. Remarkably, although T cells that are
activated within the draining lymph nodes contain a distinct set of barcodes, the
T cells that are found at the site of infection are fully kin (Figure IV.3). These data
strongly support a “diaspora model’ for T cell migration, in which T cells randomly
migrate to different sites of inflammation, irrespective of the site of original T cell
priming. In addition to these experiments that are aimed at analyzing T cell
migration, we are currently setting up model systems to analyze naГЇve T cell
differentiation, hematopoietic stem cell and DC differentiation and a host of other
issues related to blood cell differentiation. A long-term aim in this line of research is
the development of technologies that will allow cellular barcoding in vivo.
Schumacher TNM. Targeting self-antigens
through allogeneic TCR gene transfer. Blood
2006; 108:870-877
Kessels HWHG, Schepers K,
Van Den Boom MD, Topham DJ,
Schumacher TNM. Generation of T cell
help through a MHC class I-restricted TCR.
J Immunol 2006; 177:976-982
Lukens MV, Claassen EAW,
De Graaff PMA, Van Dijk MEA,
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 virus- and tumor-specific 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. In the past
year, we have examined the value of TCR gene therapy in a mouse spontaneous
tumor model where the endogenous tumor-specific T cell repertoire is lacking, as is
the case for the majority of human tumor-associated self antigens. Importantly,
data obtained in these studies indicate that TCR gene transfer can successfully be
used to suppress tumor formation in a clinically relevant model (Figure IV.4).
Hoogerhout P, Toebes M,
Schumacher TN, Van der Most RG,
Kimpen JLL, Van Bleek GM.
Characterization of the CD8+ T cell
responses directed against respiratory
Figure IV.4: Suppression of tumor growth in a
syncytial virus during primary and
spontaneous prostate tumor model by TCR gene
secondary infection in C57BL/6 mice.
therapy. Mice were either left untreated, treated
Virology 2006; 352:157-168
by vaccination, or by vaccination plus TCR gene
transfer. Tumor progression was analyzed by
MacLeod M, Kwakkenbos MJ, Crawford A,
histopathological analysis at 28 wks and
Brown S, Stockinger B, Schepers K,
compared to mice at the age treatment was
Schumacher T, Gray D. CD4 memory T
started. Note the marked reduction in tumor
cells survive and proliferate but fail to
progression upon TCR gene therapy.
differentiate in the absence of CD40. J Exp
Med 2006; 203:897-906
Rodenko B, Toebes M, Hadrup SR,
Van Esch WJE, Molenaar AM,
Schumacher TNM, Ovaa H. Generation of
peptide-MHC class I complexes through
UV-mediated ligand exchange.
Nat Protocols 2006; 1:1120-1132
Schepers K. Dissection and manipulation
of antigen-specific t cell responses. Leiden:
Universiteit Leiden, 2006
Toebes M, Coccoris M, Bins A,
Rodenko B, Gomez R, Nieuwkoop NJ,
Van De Kasteele W, Rimmelzwaan GF,
Haanen JBAG, Ovaa H,
Schumacher TNM. Design and use of
conditional MHC class I ligands.
Nat Med 2006; 12:246-251
In addition, we have tested the value of a series of treatments, such as high dose IL-2,
TGF-b inhibitors and lymphodepletion, as strategies to enhance the persistence and
anti-tumor effect of TCR gene modified T cells. These studies have been highly
informative with regard to future clinical trial design. With respect to such trials,
from a collection of four gene-optimized high affinity T cell receptors specific for
human melanoma antigens, we have selected the most potent TCR. This TCR will
be used in a planned phase I clinical trial of TCR gene therapy in patients with
metastatic melanoma.
45
IMMUNOLOGY
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 (in close
collaboration with T Schumacher). 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 to evaluate specific and cytokine-based
immunotherapies, using advanced technologies for characterization of immune
responses in peripheral blood and at the tumor site.
Group leader John Haanen
John Haanen MD PhD Group leader
DNA vaccination for the treatment of cancer Naked plasmid DNA when injected
into the skin or muscle is taken up and translated into protein. Although the process
of DNA uptake does not seem to be an efficient process, immune response against
antigens encoded in the naked DNA plasmids can be found, mostly upon in vitro
antigen-specific restimulation. Since DNA vaccines are relatively easy to produce,
cheap and appear to be safe, this mode of vaccination is explored for both protection
against infectious diseases and treatment of high risk cancer 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 have 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. Moreover, this vaccination
strategy confers protecting humoral immunity against influenza A infection within 2
weeks after the start of vaccination. The strength and speed of this newly developed
strategy will be beneficial in situations where immunity is required in the shortest
possible time. These results were confirmed in a non-human primate model (in
collaboration with BPRC, Rijswijk). We were able to show that DNA tattooing results
in 10-100-fold increase in vaccine-specific T cells compared to intramuscular
vaccination.
These positive results have convinced us that this strategy should be tested in early
phase clinical trials in humans. For this purpose a clinical grade DNA vaccine has
been produced in the recently built GMP DNA plasmid production unit at the NKI
pharmacy department for a phase I clinical study in HLA-A*0201-positive advancedstage melanoma patients.
Florry Vyth-Dreese PhD Senior staff scientist
Carina Lotz PhD Post-doc
Esther Tjin PhD Post-doc
Adriaan Bins MD Graduate student
Annelies Jorritsma MSc Graduate student
Yeung-Hyen Kim MSc Graduate student
Koen Oosterhuis Msc Graduate student
Trees Dellemijn Technical staff
Raquel Gomez MSc Technical staff
Willeke Van de Kasteele Technical staff
Johan Sein Technical staff
Tineke Vendrig Technical staff
DNA vaccination for the treatment of human papilloma virus associated
cancers Recently, ZonMW funding was obtained for a phase I/II clinical trial with
HPV 16+ penile and cervical cancer patients. Human papilloma virus infection
(serotypes 16 and 18) is strongly associated with the development of squamous cell
cancer of the penis and cervix. Because the persistence of oncogenic HPV proteins
E6 and E7 is required for carcinogenesis, these viral antigens are exquisite targets for
immunotherapeutic interventions. Indeed, therapeutic vaccinations targeting these
viral antigens have shown some promise in women 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. In preclinical studies, this strategy was shown to be much
more potent in the induction of E6/E7-specific T cell immunity than existing nonviral vaccination strategies, providing a strong rationale for its clinical evaluation.
Gene therapy with melanoma-specific human T cell receptor genes In close
collaboration with the group of T Schumacher and several foreign groups, we have
collected a number of T cell receptor (TCR) a and b genes coding for melanomaspecific TCRs, amongst others the MART-126-35 and gp100209-217-specific receptors.
These genes have been cloned into Moloney-based retroviral vectors. These retroviral
vector systems enabled us to transduce human peripheral blood T-lymphocytes (see
Fig. IV.5). Presently, we have chosen a novel MART-1-specific TCR displaying high
avidity towards melanoma tumor cells upon recognition of cognate antigen in both
cytolytic activity assay and IFN-g production assay, no detectable alloreactivity against
a large panel of cell lines expressing different HLA antigens and superb transduction
efficiency of both CD8+ and CD4+ peripheral blood T cells.
Figure IV.5. Virus titration of a panel of
optimized, melanoma-specific TCRs. The
Mart-1-specific 1D3 TCR is codon
optimized. Four days post transduction,
TCR expression was determined by staining
with PE anti-CD8 and APC-MART-1
(26-35. 27 A>L) tetramer.
46
IMMUNOLOGY
Publications
De Witte MA, Coccoris M, Wolkers MC,
A large translational gene therapy grant will allow us to perform a phase I clinical
study in melanoma patients in the next years.
Van Den Boom MD, Mesman EM,
Song JY, Van Der Valk M, Haanen JBAG,
Schumacher TNM. Targeting self-antigens
IMMUNOMONITORING
through allogeneic TCR gene transfer. Blood
This work is performed under supervision of F Vyth-Dreese, staff scientist in the
group. The primary aim of this work is to evaluate immunotherapy clinical trials,
using advanced technologies for characterization of immune responses in peripheral
blood and at the tumor site. Work is performed in close collaboration with G de Gast
(section X), A Bex (section XI) and W Nooijen (section XIII).
2006; 108:870-877
Haanen JBAG, Baars A, Gomez R,
Weder P, Smits M, De Gruijl TD,
Von Blomberg BME, Bloemena E,
Scheper RJ, Van Ham SM, Pinedo HM,
Van Den Eertwegh AJM. Melanomaspecific tumor-infiltrating lymphocytes but
not circulating melanoma-specific T cells
may predict survival in resected advancedstage melanoma patients. Cancer Immunol
Immunother 2006; 55:451-458
Overwijk WW, De Visser KE, Tirion FH,
De Jong LA, Pols TWH,
Van Der Velden YU, Van Den Boorn JG,
Keller AM, Buurman WA, Theoret MR,
Blom B, Restifo NP, Kruisbeek AM,
Kastelein RA, Haanen JBAG.
Immunological and antitumor effects of
IL-23 as a cancer vaccine adjuvant.
J Immunol 2006; 176:5213-5222
Immunotherapy of ongoing and future clinical trials in advanced-renal cell
carcinoma and melanoma Tools have been developed that allow us to monitor
immunotherapy-based studies in a state-of-the-art fashion. Recent studies of IL-2 and
IFN-a based therapies have enhanced insight into the mechanisms and potential of
immune responses in renal cell carcinoma patients.
Routinely, peripheral blood samples are stained for the presence of subtypes of
T cells, using CD4, CD8, Foxp3 and additional markers to distinguish helper,
cytotoxic and regulatory T cells, but also naГЇve, memory and effector T cells, NK cells
and NKT cells. Tumor-specificity is measured by ELIspot (IFN-g), intracellular
cytokine staining (ICS), conventional 51Cr release assays and LAMP-assays. Tumor
biopsies and biopsies from vaccination sites are analyzed by culturing infiltrating
T cells and their analysis using 6-color flow cytometry. In addition, tissue biopsies are
studied using immunohistochemistry and multicolor confocal laser scanning
microscopy for the presence and localization of T cells and dendritic cells in relation
to tumor cells and the expression of immunomodulating molecules on tumor cells,
including B7 family members (Figure IV.6). Tumor cell lines are generated from
clinical material for stimulation of T cells. This allows for detection of tumor-specific
T cells in an autologous fashion.
Toebes M, Coccoris M, Bins A,
Rodenko B, Gomez R, Nieuwkoop NJ,
Van de Kasteele W, Rimmelzwaan GF,
Haanen JBAG, Ovaa H,
Schumacher TNM. Design and use of
conditional MHC class I ligands.
Nat Med 2006; 12:246-251
Vuylsteke RJCL, Molenkamp BG,
Figure IV.6: Foxp3 positive regulatory
Van Leeuwen PAM, Meijer S,
T cells in renal cancer tissue.
Wijnands PGJT, Haanen JBAG,
Scheper RJ, De Gruijl TD. Tumor-specific
CD8+ T cell reactivity in the sentinel lymph
node of GM-CSF - Treated stage I
melanoma patients is associated with high
myeloid dendritic cell content. Clin Cancer
Res 2006; 12:2826-2833
Vyth-Dreese FA, Kim YH, Dellemijn TAM,
Schrama E, Haanen JBAG, Spierings E,
Goulmy E. In situ visualization of
antigen-specific T cells in cryopreserved
Detection of antigen-specific T lymphocytes in experimental and human
tissues Immunohistological techniques have been developed to visualize tetrameric
peptide/MHC class I and class II complexes binding tumor- and virus-specific
T lymphocytes in situ.
Recently, the in situ tetramer technique has been applied successfully 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, LUMC). We have developed a method to detect minor antigen
specific T cells, not only in fresh, but also in cryopreserved tissues. This enabled in
situ visualization of their functional status as cytotoxic and cytokine producing
effector lymphocytes in the skin microenvironment.
human tissues. J Immunol Methods 2006;
310:78-85
Development of a human ex vivo in situ skin model for vitiligo and melanoma
In collaboration with R Luiten (SNIP and Dermatology, AMC, Amsterdam) and
C Melief (LUMC) a human in situ skin model is being developed to study immune
and environmental factors involved in the development and potential therapy of
vitiligo and melanoma skin cancer. Using melanocyte specific T cell clones cocultured 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 are aimed to set up a similar
model in which melanoma explants can be visualized ex vivo in situ.
47
IMMUNOLOGY
PROGRAMMED MUTAGENESIS IN B CELLS
Antigen receptor diversification, genetic instability and lymphoma
development Lymphoma is the most common blood cancer and the third most
common cancer in childhood. Principally lymphoma is - like most other cancer types
- a genetic disease that causally relates to the accumulation of somatic mutations in
dominant and recessive cancer genes. Aberrant targeting of the Immunoglobulin (Ig)
remodeling machinery in B cells of the germinal center, seems to boost the
accumulation of oncogenic mutations in B cells. In fact, germinal center derived
lymphomas like Follicular Lymphoma and most Diffuse large B-cell lymphomas
account for more than 50 percent of all lymphomas. Given the mutagenic potential of
secondary Ig gene diversification, we focus on its molecular mechanism, regulation
and impact on genome stability. Our approaches involve basic and advanced
molecular genetics, biochemistry, immunology and recombinant mouse genetics.
Germinal center B cells - the physiological equivalent of the above mentioned
lymphomas – diversify the antigen specific Ig gene repertoire by somatic
hypermutation (SHM) and class switch recombination (CSR). While SHM efficiently
introduces point mutations into the Ig variable region, CSR is a process enabling
isotype switching within the Ig heavy chain constant region. Both SHM and CSR are
transcription-controlled and require the Activation Induced Deaminase (AID) to
deaminate cytosine in the variable- and switch-regions of Ig genes, respectively.
The resulting uracil can be removed by an uracil glycosylase, generating an abasic
site. Processing by the MRN complex (MRE11, Rad50 and NBS1) or mismatch repair
is thought to prohibit error free base excision repair, favoring mutagenic translesion
DNA synthesis as well as the generation of single and double strand DNA breaks.
Based on the translocation break points and somatic mutations in dominant and
recessive cancer genes, SHM and CSR have been implicated in the molecular
pathogenesis of germinal centre-derived lymphoma.
A genome wide binding profile of AID Given the mutagenic potential of AIDmediated DNA deamination, we address whether aspecific targeting of AID and the
Ig gene remodeling machinery explains why particularly B cells of the germinal
center are prone to malignant transformation. As oncogenic translocations provide
a selective growth advantage, do they just resemble the tip of the iceberg? Are
translocation hotspots targets of AID? To address these and related questions we have
set out to derive a genome wide chromatin-binding profile of AID. These profiles are
established under physiological (germinal center B cells) and patho-physiological
conditions (lymphomas with a germinal center signature). Putative binding sites are
verified by sequencing and the binding profile is related to other parameters known
to favor mutagenesis. This map will provide information on the mutability of non-Ig
genes and their contribution to malignant transformation of germinal center derived
lymphomas.
Error free versus error prone repair: Posttranslational modification of PCNA
Numerous DNA lesions are generated continuously by environmental factors and by
endogenous damage, including deamination of cytosine by AID. During replication
these 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,
two alternative pathways of DNA damage tolerance can be triggered: polymerase
switching or template switching. Yeast genetics has led to the identification of
the Rad6 pathway, a ubiquitin (Ub) conjugating-system that catalyses
monoubiquitination of a single ultra-conserved lysine, K164 of the proliferating
nuclear antigen (PCNAK164) to stimulate polymerase switching and
polyubiquitination of PCNAK164 to stimulate template switching. PCNA is a ring
shaped homo-trimer, that encircles the DNA and serves as a DNA sliding clamp and
molecular interaction platform enabling cross talk between proteins involved in DNA
replication, DNA repair, DNA modification, chromatin modeling, cell cycle control
and apoptosis.
Mono-ubiquitination at PCNAK164 is catalyzed by the ubiquitin conjugating/ligating
complex Rad6/Rad18. Monoubiquitinated PCNA triggers switching from a damage
Group leader Heinz Jacobs
Heinz Jacobs PhD Group leader
Silke Schnell MSc Graduate student
Marinus Heideman MSc Graduate student
Peter Krijger MSc Graduate student
Petra Langerak MSc Graduate student
Paul Van den Berk Technical Staff
48
IMMUNOLOGY
Publications
Jansen JG, Langerak P, Tsaalbi-Shtylik A,
Van den Berk P, Jacobs H, De Wind N.
Strand-biased defect in C/G transversions in
hypermutating immunoglobulin genes in
Rev1-deficient mice. J Exp Med 2006;
203:319-323
Langerak P, Nygren AOH, Schouten JP,
Jacobs H. Rapid and quantitative detection
of homologous and non-homologous
recombination events using three
oligonucleotide MLPA. Nucleic Acids Res
sensitive, high fidelity DNA polymerase (d or e) to one of the damage tolerant, low
fidelity translesion DNA synthesis (TLS) polymerases. TLS polymerases can continue
replication across non-Watson Crick base pairs. 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 suggest that this process may also determine the
choice between polymerase switching and template switching in mammals. To
establish this, we have generated ES cell clones carrying a conditional PCNA allele as
well as a mutant K164R allele. Recombinant mouse strains and cell lines thereof will
establish the role of posttranslational K164-linked PCNA modification in controlling
genetic stability. As TLS lies at the heart of mutagenesis, our investigations will
provide not only insights in to the control of SHM and lymphoma development but
cancer in general.
2005; 33:e188
Schnell S, DГ©molliГЁre C, Van den Berk P,
Kirberg J, Jacobs H. Constitutive expression
of the pre-TCR enables development of
mature T cells. Int Immunol 2006;
18:911-920
Schnell S, DГ©molliГЁre C, Van den Berk P,
Jacobs H. Gimap4 accelerates T-cell death.
Blood 2006; 108:591-599
Schnell SAC. Life and death in the T cell
lineage. Amsterdam: Universiteit van
Amsterdam, 2006
Somatic hypermutation in B cells lacking highly conserved K164
modifications of PCNA Somatic hypermutation of Ig genes enables B cells of the
germinal center to generate high affinity Ig variants. B cells make use of damage
tolerant, error prone TLS to generate somatic mutations at and around genetic
lesions caused by AID. To determine the role of PCNAK164 modifications in mouse
development and somatic hypermutation, PCNAK164R knock in mice have been
generated. PCNAK164R mutant mice are viable, somatic cells develop normally but are
highly sensitive to genotoxic stress. Moreover, PCNAK164R fails to recruit TLS
polymerase h, explaining the defective mutagenesis at template A/T in PCNAK164R
mutant B cells.
Establishing the role of TLS polymerases in SHM To address the role of the TLS
polymerase Rev1 in SHM, two recombinant Rev1 mutant mouse strains either
lacking the regulatory BRCT domain or the catalytic SCDE domain, respectively have
been analyzed. While the mutation frequencies in both mouse strains appear to be
normal, the base exchanges are significantly altered in the SCDE mutant Rev1 mice.
While mice lacking the BRCT domain show a normal base exchange pattern, mice
lacking the SCDE domain however, show an increase in A -> T and T -> C mutations
and a decrease in G -> C and C -> G mutations. The decrease in G -> C and C -> G
mutations can be explained by the proposed CMP-transferase activity of Rev1, the
increase in A -> T and T -> C mutations likely relates to a compensatory activity of the
A/T mutator polymerase h.
49
MOLECULAR BIOLOGY
V DI VI S I ON OF MOL ECU L A R BIOLOG Y
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
in mutation avoidance, (2) the role of cell cycle checkpoints in tumor suppression
and (3) control of mitosis.
Division head, Group leader Hein Te Riele
Hein Te Riele PhD Group leader
DNA MISMATCH REPAIR
Camiel Wielders PhD Post-doc
Genetic defects in DNA mismatch repair (MMR) underlie the cancer syndrome
hereditary non-polyposis colorectal cancer (HNPCC). The primary function of MMR
is correction of DNA replication errors. This is initiated by either MSH2/MSH6 or
MSH2/MSH3 protein complexes, which recognize base.base mismatches and small
loops of unpaired bases, respectively.
In addition, two other MMR functions have been identified. The first is suppression
of recombination between homologous but not perfectly identical DNA sequences.
We have recently found that this activity of MMR also imposes a strong impediment
to subtle gene modification by short single-stranded DNA oligonucleotides.
The second is suppression of DNA damage-induced mutagenesis by elimination of
damaged cells.
Rob Wolthuis PhD Post-doc
Marieke Aarts MSc Graduate student
Sietske Bakker MSc Graduate student
Elena Bardina MSc Graduate student
Floris Foijer MSc Graduate student
Wouter Van Zon Graduate student
Tinke Vormer MSc Graduate student
Eva Wielders Msc Graduate student
Marleen Dekker Technical staff
Elly Delzenne-Goette Technical staff
Sandra De Vries MSc Technical staff
Anja Van der Wal Technical staff
Oligonucleotide-directed gene modification Oligonucleotide-directed gene
modification (�oligo targeting’) is emerging as a powerful novel tool to subtly modify
the genome of mouse embryonic stem (ES) cells and the generation of mutant
mouse strains. We found that sequence-specific single-stranded oligonucleotides
consisting of ~38 deoxyribonucleotides can restore the activity of a disabled neo
reporter gene by deletion, insertion or substitution of one or a few base pairs.
However, the success of oligo targeting is severely hampered by DNA mismatch
repair activity (Dekker et al., NAR 2003;31:e27). Dependent on the type of alteration,
oligo targeting was suppressed by MSH2/6 or MSH2/3 activity, or both. Based on
this knowledge we have developed procedures for oligonucleotide-directed gene
inactivation and modification and shown that mutant alleles can be introduced into
the mouse germ line.
Publications
Aarts M, Dekker M, De Vries S,
Van der Wal A, Te Riele H. Generation of a
mouse mutant by oligonucleotide-mediated
gene modification in ES cells. Nucleic Acids
Res 2006; 34:e147
Oligo-directed insertion of three or four nucleotides can readily be achieved in
Msh3-deficient ES cells. As these cells do not show an overt mutator phenotype and
Msh3-deficient mice are not cancer prone, we are using oligonucleotide-directed
frameshift mutagenesis as an alternative to current knockout procedures. We have
thus far generated disruptions in Fancf, Fen1 and Esco2.
Oligo-directed base substitution is largely suppressed by MSH2/6 and therefore
relies on the absence of MSH2. When we transiently suppress MSH2 activity by RNA
interference, cells become permissive for oligonucleotide-mediated substitution of
three or four nucleotides, allowing modification of virtually any codon in the genome.
Under this condition the level of spontaneous mutagenesis remains low. We are
exploiting this procedure to study the pathogenicity of allelic variants found in the
human population, focusing on RB, p53 and MSH2/6.
Uncoupling of DNA MMR functions A tenfold reduced level of MSH2 protein
leads to uncoupling of MMR functions: replication errors are still largely corrected,
but DNA-damage-induced mutagenesis is no longer suppressed. In mice, ten percent
MSH2 level was sufficient to suppress spontaneous tumorigenesis in the thymus and
small intestine. However, in a sensitized background, reduced MSH2 level did
accelerate tumorigenesis. We are currently investigating whether this is the result of
the slightly reduced efficacy of mismatch correction or the full loss of appropriate
DNA damage responses. Furthermore, we are generating, by oligo targeting, specific
codon substitutions in Msh2 to uncouple MMR functions and to study the role of
each in tumor suppression.
Figure V.1: Extending the restriction point.
A Cell cycle progression is dependent on
Cyclin-CDK activity. B Mitogen starvation
results in cell cycle arrest in G1 through
degradation or suppression of Cyclin D and
CKI-mediated inhibition of Cyclin E-CDK2.
C Passage through the G1 restriction point in
the absence of mitogens (e.g., through RB
loss) results in cell cycle arrest in G2.
50
MOLECULAR BIOLOGY
Publications (continued)
CELL CYCLE CHECKPOINTS
Dannenberg J-H, Te Riele HPJ.
The Retinoblastoma Gene Family in Cell
Cycle Regulation and Suppression of
Tumorigenesis. In: Kaldis P, editor. Cell
Cycle Regulation. Berlin; Heidelberg; New
York: Springer, 2006: 183-225
Dekker M, Brouwers C, Aarts M,
Van der Torre J, De Vries S,
Van de Vrugt H, Te Riele H. Effective
oligonucleotide-mediated gene disruption in
The retinoblastoma suppressor protein pRB and its homologs p107 and p130
(collectively called “pocket proteins”) impede the G1-S transition of the cell cycle by
binding to and inhibiting E2F transcription factors. G1 arrest is relieved by
phosphorylation of pocket proteins by Cyclin D- and E-dependent kinases, whose
activity is dependent on mitogens. However, mitogenic signaling is only required
before the G1 restriction point after which cells can progress into S-phase in the
absence of mitogens. The G1 restriction point mechanistically coincides with the
requirement for pocket protein phosphorylation for S-phase entry. Thus, mouse
embryonic fibroblasts (MEFs) with defects in the retinoblastoma gene family
(TKO MEFs, lacking expression of Rb, p107 and p130) can pass through G1 and enter
S phase with similar kinetics in the presence or absence of mitogens.
ES cells lacking the mismatch repair protein
MSH3. Gene Ther 2006; 13:686-694
Foijer F, Te Riele H. Check, double check:
the G2 barrier to cancer. Cell Cycle 2006;
5:831-836
Foijer F. Braking news: The G2 restriction
point. Amsterdam: Vrije Universiteit, 2006
Foijer F, Te Riele H. Restriction beyond the
Growth-factor independence We found that after passage through S-phase,
proliferation of TKO MEFs was still restricted by: (1) massive apoptosis and (2) a G2
arrest that became apparent when apoptosis was suppressed by ectopic expression of
Bcl2. The G2 arrest was mediated by upregulation of the cyclin-dependent-kinase
inhibitors p21CIP1 and p27KIP1 that inhibited the G2 cyclins Cyclin A and Cyclin B1
through direct interaction. The arrest was reversible: re-addition of serum for 3-6 h led
to mitotic entry about 15 h later, thus defining a second restriction point operating in
G2 (Fig. V.1). Strikingly, inactivation of p53, overexpression of c-MYC or mutational
activation of RAS alleviated the G2 arrest response, providing a rationale for the
synergism between loss of pocket proteins and these oncogenic events in tumorigenesis.
restriction point: mitogen requirement for
G2 passage. Cell Division 2006; 1:8
Foiry L, Dong L, Savouret C, Hubert L,
Te Riele H, Junien C, Gourdon G. Msh3 is
a limiting factor in the formation of
intergenerational CTG expansions in
DM1 transgenic mice. Hum Genet 2006;
119:520-526
Zienolddiny S, Ryberg D, Svendsrud DH,
Eilertsen E, Skaug V, Hewer A,
Anchorage independence We previously showed that ablation of Rb and p107 or Rb
and p130 was sufficient to cause immortality and to reverse the growth inhibitory
effect of RASV12 into a proliferative stimulus. However, Rb-/-p107-/- or Rb-/-p130-/MEFs were not oncogenically transformed by RASV12: apparently, a cell cycle
mechanism still operates to restrict proliferation of these cells in soft agar. We
therefore performed screens for genetic events that permit anchorage-independent
growth of RASV12-expressing Rb-/-p130-/- MEFs. In one screen we identified the
immortalizing oncogene Tbx2 that has previously been shown to interfere with the
p53 pathway. As over-expression of Tbx2 and RASV12 in primary wild-type MEFs did
not result in anchorage-independent growth, this result indicates that concomitant
ablation of the p53 and pRB pathway facilitates oncogenic transformation.
Phillips DH, Te Riele H, Haugen A.
Msh2 deficiency increases susceptibility to
REGULATION OF MITOSIS
benzo[a]pyrene-induced lymphomagenesis.
In 2005, Rob Wolthuis joined the group of Hein Te Riele to continue his studies of
mitosis in normal and cancer cells. During mitosis, the duplicated genome is
irreversibly distributed over two newly formed cells. This process requires timely
inactivation of mitotic regulators by proteolysis. A multi-subunit assembly, the
Anaphase-Promoting Complex (APC/C), ubiquitinates key mitotic proteins such as
Cyclin B1 and Securin at highly defined mitotic time points, which targets them for
destruction by the 26S proteasome. By precisely regulating mitotic proteolysis, the
APC/C coordinates cell division with segregation of sister chromatids, which is
essential to guarantee cellular 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 established an innovative,
combinatorial approach of gene silencing, biochemistry and live-cell imaging (Figure
V.2). Hereby, we identified a novel role for Cdk2 in controlling Cyclin A destruction.
Furthermore, we discovered that Cyclin B1 and Securin are differentially recruited by
the APC/C in metaphase, a process that involves phosphorylation of the subunit
APC3. Furthermore, we identified a novel de-Ubiquitinating enzyme, which helps to
check APC/C-dependent destruction.
Int J Cancer 2006; 118:2899-2902
Figure V.2: Measuring APC/C activity by
time-lapse fluorescence microscopy. A
plasmid encoding fluorescent Cyclin B1 was
injected into G2-phase osteosarcoma cells, a
few hours before they started mitosis. The
top panel shows a cell undergoing mitotic
division, followed over time (min) 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 APC/C-dependent protein
destruction in live cells while they divide.
51
MOLECULAR BIOLOGY
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 all 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 (JBP 1) that binds with high specificity to
J-containing duplex DNA (Cross et al., EMBO J 1999; 18:6573-81). A JBP 1 gene KO
in T. brucei has the remarkable effect of reducing the J content of trypanosome DNA
to 5% of wild type, without any detectable physiological consequences. In the related
kinetoplastid Leishmania, a JBP 1 KO is lethal. Our recent work indicates that JBP 1 is
a hydroxylase that catalyses the first step of J biosynthesis, the conversion of T in
DNA into hydroxymethyluracil. JBP 1 appears to belong to the family of Fe2+ and
2-oxoglutarate-requiring dioxygenases, as does a second putative hydroxylase, JBP2.
We have shown that JBP2 is dispensable in Leishmania under the growth conditions
tested thusfar. To determine the function of J in Leishmania we are constructing a
conditional KO of the JBP 1 gene. We are also continuing attempts to set up assays for
hydroxylase activity in vitro 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 JBP 1-J-DNA complexes by crystallography; with Charles
Weissmann (Scripps Florida), we are trying to find inhibitors of the binding of JBP 1
to DNA and of its hydroxylase function.
Group leader Piet Borst
Piet Borst MD PhD Group leader
Henri Van Luenen PhD Academic staff
Jan Wijnholds PhD Academic staff, honorary
Sjofn Gunnarsdottir PhD Post-doc
Marina Pajic PhD Post-doc
Sven Rottenberg DVM PhD Dipl. ECVP Post-doc
Saara Vainio PhD Post-doc
Koen Van de Wetering PhD Post-doc
Cocky De Wolf PhD Post-doc
Paul-AndrГ© Genest MSc Graduate student
Marcel De Haas Technical staff
Liesbeth Van Deemter Technical staff
Wouter Feddema Technical staff
Ariena Kersbergen Technical staff
Bas Ter Riet Technical staff
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 (ABCB family); most recently we have studied the Multidrug
Resistance Protein (ABCC) family members MRP2, 3, 4, 5 and 9.
Publications
Borst P, Zelcer N, Van de Wetering K.
MRP2 and 3 in health and disease. Cancer
Lett 2006; 234:51-61
Borst P, De Wolf CJF, Van de Wetering K.
Multidrug resistance-associated proteins 3,
4, and 5. Pflugers Arch 2006;Published
online
Borst P, Zelcer N, Van de Wetering K,
Poolman B. On the putative co-transport of
drugs by multidrug resistance proteins.
FEBS Lett 2006; 580:1085-1093
MRP2 (ABCC2) and MRP3 (ABCC3) are organic anion transporters contributing to
the cellular export of endogenous or exogenous (toxic) compounds, conjugated to
glutathione, sulphate or glucuronate. Using KO mice, we have previously shown that
murine MRP3 is the major transporter mediating export of morphine-3-glucuronide
(M3G) from the liver into the blood (Zelcer et al., Proc. Natl. Acad. Sci. USA
2005;102:7274-9). In collaboration with the Schinkel group (NKI-AVL) we have now
shown that MRP2 is responsible for extrusion of M3G into bile. As long as either
MRP2 or MRP3 is present, the liver can efficiently dispose of M3G. In the
Mrp2-/- / Mrp3-/- double KO, M3G accumulates to high levels in the liver, but can still
be slowly exported by a transporter that remains to be identified.
MRP3 is also important for basolateral export of glucuronated compounds from the
gut epithelium into blood. An interesting example is provided by resveratrolglucuronide (R-gluc), which mainly enters the body via MRP3. We have used murine
microsomes to generate radioactive R-gluc and shown that this is a high-affinity
substrate for MRP3. 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 Spectometry. We have identified several potential novel
MRP3 substrates by this approach and anticipate that it may also be helpful to find
substrates of other MRPs.
Borst P, Genest PA. Parasitology:
Switching like for like. Nature 2006;
439:926-927
Borst P, Wielinga P. Pumping Out Drugs:
The Potential Impact of ABC Transporters
on Resistance to Base, Nucleoside, and
Nucleotide Analogs. In: Peters GJ, editor.
Deoxynucleoside Analogs in Cancer
Therapy. Totowa, NJ.: Humana Press,
2006: 109-118
Borst P. How I became a biochemist.
IUBMB Life 2006;58:177-82
Borst P. From cancer cells to trypanosomes
and back again. Cell Mol Life Sci
2006;63:745-54
52
MOLECULAR BIOLOGY
Publications (continued)
Minich T, Riemer J, Schulz JB, Wielinga P,
Wijnholds J, Dringen R. The multidrug
resistance protein 1 (Mrp1), but not Mrp5,
mediates export of glutathione and
glutathione disulfide from brain astrocytes.
J Neurochem 2006; 97:373-384
Zelcer N, Van de Wetering K, De Waart R,
Scheffer GL, Marschall HU, Wielinga PR,
Kuil A, Kunne C, Smith A, Van der Valk M,
Wijnholds J, Oude Elferink RO, Borst P.
Mice lacking Mrp3 (Abcc3) have normal
bile salt transport, but altered hepatic
transport of endogenous glucuronides.
J Hepatol 2006; 44:768-775
MRP4 (ABCC4) and MRP5 (ABCC5) are organic anion transporters with the
unusual ability to transport nucleotide analogs and the cyclic nucleotides cGMP and
cAMP. Using intact transfected cells and vesicular transport assays, we have found
that the affinity of MRP4 and MRP5 for nucleotide analogs and cyclic nucleotides is
low, making a major role in drug resistance or cyclic nucleotide disposition doubtful.
In search for other functions, we found that MRP4 is able to transport some
conjugated bile acids and steroids with high affinity. Interestingly, MRP4 (but not
MRP1, 2 or 3) can also transport prostaglandins E1 and E2, and that transport is
inhibited by some non-steroidal anti-inflammatory drugs, such as indomethacin,
which are also known to block synthesis of these prostaglandins. We are studying
whether MRP4 is a limiting factor in the cellular excretion of endogenously made
prostaglandin using Mrp4 KO mice.
While studying the nature of the transporter(s) responsible for cGMP transport by
murine erythrocytes, we found a contribution by the Breast Cancer Resistance
Protein (ABCG2). This led us to look at resistance against nucleoside analogs in
BCRP-transfected cells. We found a high level of resistance to cladribine, but
unexpectedly this is due to transport of cladribine itself rather than transport of
cladribine-monophospate. This is the first example of a nucleoside analog being
transported by a mammalian ABC-transporter.
Drug resistance in “spontaneous” mouse tumors We have started a new project
in collaboration with Jos Jonkers (NKI-AVL) to study resistance mechanisms in
“spontaneous” tumors arising in genetically modified mice. Initial experiments were
done 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, which encode
drug-transporting P-glycoproteins. In addition to gene expression analysis by
oligoarrays, we developed a reverse transcriptase-multiplex ligation-dependent probe
amplification (RT-MLPA) analysis in collaboration with MRC-Holland to measure
tumor mRNA of selected drug target and drug transporter genes more precisely. Our
results suggest that the available methods to detect Multidrug Resistance (MDR)
caused by P-glycoprotein in clinical material are inadequate.
In contrast to the results obtained with MDR drugs, we have been unable to obtain
cisplatin resistance in this tumor model. As shown in figure V.3 the tumors respond
to each new treatment with cisplatin, but are never fully eradicated. We are studying
the “remnants” from which the tumors regrow to see whether these represent “stem”
cells resistant to cisplatin. In addition we are using this mouse model to test new
anticancer drugs and drug combinations (in collaboration with KuDOS) and we are
also crossing in drug transporter knock out mice.
Figure V.3: Two typical examples of cisplatin responses of Brca1- and p53-deficient mammary tumors.
(A) shows the volume (0.5 x length x width2) of a mammary tumor in a K14Cre; Brca1flox/flox; p53flox/flox
female mouse which was treated with the maximum tolerated dose of cisplatin (6mg/kg i.v., arrows).
The same treatment was given to a syngeneic wild-type mouse into which a Brca1-/-; p53-/- mammary
tumor fragment was transplanted orthotopically (B). Treatment was started when the tumor size was
about 200 mm3. After the initial administration on day 0, additional treatments were given when the
tumor relapsed to 100%.
53
MOLECULAR BIOLOGY
MOUSE MODELS OF BREAST CANCER
The focus of our laboratory is on the genetic dissection of human breast cancer
through the use of genetically engineered mouse models. Thus far, we have
developed models for p53-induced breast cancer, BRCA1- and BRCA2- associated
hereditary breast cancer, and E-cadherin-associated metastatic breast cancer. These
models are used to (i) investigate genotype-phenotype relations in mammary
tumorigenesis; (ii) identify genetic changes underlying breast tumorigenesis;
(iii) study the role of innate and adaptive immunity in breast cancer development;
(iv) perform therapeutic intervention studies.
Group leader Jos Jonkers
Jos Jonkers PhD Group leader
Peter Bouwman PhD Post-doc
Conditional mouse models of BRCA-associated breast cancer In addition to the
previously published mouse model for BRCA2-associated breast cancer (Jonkers et
al., Nat Genet. 2001;29:418) we have established a mouse model of BRCA1-associated
hereditary breast cancer. To this end, we have generated conditional mutant mouse
strains with (K14-Cre induced) epithelium-specific loss of Brca1 and p53. Female mice
of this strain develop significantly faster mammary tumors than females with
epithelium-specific loss of p53 alone, demonstrating collaboration between Brca1
inactivation and p53 loss in mammary tumorigenesis. The Brca1-/-;p53-/- mammary
tumors have similar histomorphologic features as basal-like human breast cancers:
they are highly proliferative, poorly differentiated, ERBB2/HER2- and estrogen
receptor (ER)-negative mammary adenocarcinomas with pushing borders and
increased expression of basal epithelial markers (CK5/6). Brca1-/-;p53-/- mammary
tumors are highly aneuploid and their gene expression signatures resemble those of
human BRCA1-mutated breast cancers. Thus, they display the hallmarks of familial
BRCA1-deficient breast cancer in women. This mouse BRCA1 mammary tumor
model might therefore be helpful in predicting chemotherapeutic responses of
human BRCA1-deficient tumors and basal-like breast cancers with BRCA-like
phenotypes (see below).
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 tissue-specific inactivation of the E-cadherin
tumor suppressor gene. Hereto, we have produced conditional E-cadherin mutant
mice and crossed these to a conditional mouse mammary tumor model based on
epithelium-specific knockout of p53. Combined loss of E-cadherin and p53 causes a
phenotypic change from non-invasive to highly invasive and metastatic mammary
tumors, a morphological switch from ductal to lobular carcinomas and a marked
reduction in tumor latency, showing collaboration between E-cadherin inactivation
and p53 loss in mammary tumorigenesis. The compound mutant female mice
develop mammary tumors with striking resemblance to human ILC, showing
invasion into the surrounding tissue and metastasis to various sites including
gastrointestinal tract and bone. Moreover, loss of E-cadherin suppresses anoikis and
facilitates angiogenesis, thus promoting the growth, survival and dissemination of
tumor cells. These data establish a causal role for E-cadherin in breast cancer
suppression and show that loss of E-cadherin impacts on different stages in breast
oncogenesis, including tumor initiation, invasion and metastasis. We are currently
performing biochemical and functional genetic studies to identify the pathways
downstream of E-cadherin, which mediate the in vitro and in vivo tumor phenotypes
associated with E-cadherin loss.
Array-CGH analysis of mouse mammary tumors
In collaboration with the Wellcome Trust Sanger Institute (Hinxton, UK), we have
developed mouse tile path BAC arrays for comparative genomic hybridization (CGH)
analysis of mouse mammary tumors. In agreement with the published roles of
BRCA1 and BRCA2 in maintenance of genome integrity, CGH analysis revealed
significantly more DNA copy number alterations in Brca1-/-;p53-/- and Brca2-/-;p53-/-
Michiel De Bruin PhD Post-doc
Patrick Derksen PhD Post-doc
Karin De Visser PhD Post-doc
Gilles Doumont PhD Post-doc
Xiaoling Liu PhD Post-doc
Rinske Drost Msc Graduate student
Bastiaan Evers Msc Graduate student
Henne Holstege Msc Graduate student
Hermien Boerhout Technical staff
Eva Kregel Technical staff
Eline Van der Burg Technical staff
Hanneke Van der Gulden Technical staff
Ellen Wientjens Technical staff
54
MOLECULAR BIOLOGY
Publications
Alberici P, De Pater E, Cardoso J,
Bevelander M, Molenaar L, Jonkers J,
Fodde R. Aneuploidy Arises at Early Stages
of Apc-Driven Intestinal Tumorigenesis and
Pinpoints Conserved Chromosomal Loci of
Allelic Imbalance between Mouse and
Human. Am J Pathol 2007; 170:377-387
Boesten LSM, Zadelaar ASM,
Van Nieuwkoop A, Hu L, Jonkers J,
Van de Water B, Gijbels MJJ,
Van der Made I, De Winther MPJ,
Havekes LM, Van Vlijmen BJM.
Macrophage retinoblastoma deficiency leads
to enhanced atherosclerosis development
mammary tumors compared to p53-/- control tumors. We are also using CGH
profiling to identify novel oncogenic mutations that cooperate with loss of BRCA1,
BRCA2, p53 or E-cadherin in mammary tumorigenesis. To find significantly
recurrent regions with DNA copy number aberrations in our array-CGH data sets, we
have developed an objective, statistical method for multi-experiment analysis of arrayCGH data. This method, called KC-SMART (Kernel Convolution – a Statistical
Method for Aberrant Region deTection) uses kernel convolution to generate a Kernel
Smoothened Estimate (KSE) of copy number alterations across the genome,
aggregated over all tumors. The peaks in the KSE are tested against randomly
permutated data to obtain statistically significant aberrant regions. Using this
approach, we have identified in our panels of mouse mammary tumors several
regions with recurrent copy number alterations, including a region on chromosome
10 that is amplified in over 50% of the Brca2-/-;p53-/- tumors. These regions, when
combined with gene expression microarray data, point to various known cancer
genes and several new candidate cancer genes. We are currently validating the
candidate genes within the amplified region on chromosome 10.
in ApoE-deficient mice. FASEB J
2006; 20:953-955
Clark-Knowles KV, Garson K, Jonkers J,
Vanderhyden BC. Conditional inactivation
of Brca1 in the mouse ovarian surface
epithelium results in an increase in
preneoplastic changes. Exp Cell Res 2007;
313:133-145
Derksen PWB, Liu X, Saridin F,
Van der Gulden H, Zevenhoven J, Evers B,
Van Beijnum JR, Griffioen AW, Vink J,
Krimpenfort P, Peterse JL, Cardiff RD,
Berns A, Jonkers J. Somatic inactivation of
E-cadherin and p53 in mice leads to
metastatic lobular mammary carcinoma
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). We are currently investigating whether a similar link
between adaptive and innate immunity contributes to breast cancer development,
utilizing conditional and transgenic mouse models for mammary carcinogenesis.
Similar to human breast cancer lesions, neoplastic mammary glands of these mice
are characterized by leukocyte infiltration. To assess the functional significance of
innate and adaptive immune systems during breast cancer development, these mice
are being crossed with various immune-deficient mice and consequences for chronic
inflammation, angiogenesis, premalignant progression, cancer development and
metastasis formation will be analyzed. We believe that identification of the
mechanisms underlying inflammation-associated cancer development will reveal
drug targets that can be used to design novel therapeutic strategies for arresting
cancer development in humans.
through induction of anoikis resistance and
angiogenesis. Cancer Cell 2006; 10:437-449
Evers B, Jonkers J. Mouse models of
BRCA1 and BRCA2 deficiency: Past lessons,
current understanding and future prospects.
Oncogene 2006; 25:5885-5897
Van Meeteren LA, Ruurs P, Stortelers C,
Bouwman P, Van Rooijen MA,
re JP, Pettit TR, Wakelam MJO,
Saulnier-Blache JS, Mummery CL,
Moolenaar WH, Jonkers J. Autotaxin,
a secreted lysophospholipase D, is
essential for blood vessel formation during
development. Mol Cell Biol 2006;
26:5015-5022
Tumor intervention studies In close collaboration with the groups of Piet Borst and
Sabine Linn, we are utilizing the BRCA1 and E-cadherin mammary tumor models for
preclinical studies. For this purpose we have derived panels of clonal, luciferasemarked tumor cell lines from our “spontaneous” tumor models, and shown that
these cell lines reproduce the disease upon orthotopic grafting in recipient mice.
Together with the spontaneous models, these reagents provide a valuable platform
for validation of candidate drug targets and anticancer drugs in a physiologically
relevant setting. Moreover, synergistic antineoplastic effects of combinations of
targeted and conventional therapeutics may be effectively tested. We are currently
testing a number of cytotoxic drugs and targeted therapeutics on our panels of
Ecad-/-;p53-/- and p53-/- mammary tumor cell lines and the corresponding orthotopic
grafting models.
The central role of BRCA1 and BRCA2 in DNA repair via homologous recombination
(HR) has suggested that BRCA-deficient tumors will be especially sensitive to
chemotherapeutics that induce DNA double-strand breaks. Indeed, intervention
studies performed by Sven Rottenberg in the group of Piet Borst show that
mammary tumors in our BRCA1 model are highly sensitive towards DNAcrosslinking agents such as cisplatin. This chemosensitivity profile is consistent with
in vitro responses of BRCA-deficient cells and clinical responses of BRCA-linked
ovarian cancers, indicating that our BRCA1 tumor model provides a useful preclinical
platform for testing novel therapeutics, such as PARP1-inhibitors, which specifically
target HR-deficient breast cancers.
55
MOLECULAR BIOLOGY
MOLECULAR DISSECTION OF BREAST CANCER BY DIFFERENTIAL DRUG
SENSITIVITY
In the clinic, we only use anticancer drugs selected by clinical trials to perform best
for the whole group of breast cancer patients, whereas little is known about the
molecular mechanisms underlying differential drug sensitivity of breast cancer. The
focus of our research line is to unravel these mechanisms in order to develop tests
that may guide treatment decisions in the clinic and ultimately improve survival. For
this purpose we use several genome-wide approaches and molecular techniques, in
order to dissect the mechanisms that divide clinically well-defined cohorts of breast
cancer patients into resistant and sensitive to a particular drug. In addition, we use
conditional mouse models for breast cancer, and derived clonal tumor cell lines, to
study differential chemosensitivity in a controlled fashion.
Group leader Sabine Linn
Sabine Linn MD PhD Group leader
Michiel De Bruin PhD Post-doc
Jolien Bueno de Mesquita MD Graduate student
Marleen Kok MD Graduate student
Ingrid Van der Heijden Technical Staff
Feasibility of microarray technology as a diagnostic tool in community
hospitals In collaboration with the van de Vijver group, we have assessed whether
microarray technology as a diagnostic tool is feasible in daily clinical practice in
general hospitals. In a pilot study, from 2004 - 2006, we have demonstrated the
feasibility of this technique in general hospitals (see also Division X).
Development of a predictive test for tamoxifen (TAM) resistance in breast
cancer Based on the assumption that breast cancer patients who benefit from1
adjuvant TAM treatment have the same molecular breast cancer subtype as those
patients who did not receive any form of adjuvant systemic treatment, but show long
lasting responses on first line TAM in the metastatic setting, we have started a
retrospective cohort study in over 500 patients who have been treated with TAM,
either in the adjuvant or metastatic setting. Molecular analysis of this series is
ongoing (collaboration with Division VIII).
In collaboration with the Erasmus Medical Center we validated their predictive gene
expression profile for TAM resistance (Jansen et al., J Clin Oncol. 2005; 23:732-40) in
a pilot study of 69 metastatic breast cancer patients.
Molecular mechanisms underlying sensitivity for high dose alkylating agents
We have used Bacterial Artificial Chromosome (BAC) arrays to generate comparative
genomic hybridization (CGH) profiles of primary breast cancers of 31 breast cancer
patients who had been treated with high dose alkylating agents with hematopoietic
stem-cell rescue in the metastatic setting. Ten patients had a progression-free
survival (PFS) of over 24 months, while the others progressed within that time. In
collaboration with the Wessels group we identified a classifier of 31 BAC clones that
correctly classified the patients with a favorable PFS with a sensitivity of 80% and a
specificity of 81%. Figure V.4 shows the heatmap of amplifications and deletions of
the 31 BAC clones for the 31 patients. We are currently validating this CGH classifier
on a larger series.
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. Sofar, growth inhibition
assays with conventional chemotherapeutics did not show any differential
chemosensitivity. Gene expression profiling of all cell lines has uncovered interesting
differences between the two genotypes that may point to drugs active against lobular
breast cancer. These results are used to select additional compounds for further
testing. We will repeat part of the cell line experiments in orthotopic grafting and
spontaneous mouse models, to verify whether the cell lines correctly reflect the in
vivo relative chemosensitivity.
Figure V.4: Heatmap of amplifications and
deletions of 31 BAC clones (horizontal axis)
for 31 patients (vertical axis). On the far
right side the log2 ratios of amplifications
are shown in white and deletions in black,
respectively from 0 to 1 and from 0 to -1. The
last column of the heatmap shows favorable
PFS in white and unfavorable PFS in
black.
56
MOLECULAR BIOLOGY
CHROMATIN GENOMICS
Group leader Bas Van Steensel
Bas Van Steensel PhD Group leader
Lars Guelen PhD Post-doc
Celine Moorman PhD Post-doc
Helen Pickersgill PhD Post-doc
Ulrich Braunschweig MSc Graduate student
Elzo De Wit MSc Graduate student
Frauke Greil MSc Graduate student
Daniel Peric-Hupkes MSc Graduate student
Maartje Vogel MSc Graduate student
Greg Hogan MSc Guest researcher
Ludo Pagie PhD Bioinformatician
Introduction The main goal of our laboratory is to understand how gene expression
programs in higher eukaryotes are regulated. In particular, we focus on mechanisms
of chromatin-mediated gene regulation in the living cell, in the context of the entire
genome. Using Drosophila and mammalian cells as model systems, we develop and
apply new whole-genome approaches to study the structure and composition of
chromatin and the mechanisms of gene regulation.
Over the past years, we developed a novel 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 test thousands of genomic
sequences for in vivo binding of a chromatin protein or transcription factor of
interest. We now routinely use DamID to generate whole-genome maps of protein
binding 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. Gradually, we are building a large database of genomewide binding maps of chromatin proteins and other regulatory proteins. This
database is being analyzed using state-of-the-art bioinformatics approaches to obtain
insight into the interplay between various regulatory proteins.
Francesco Russo MSc Technical staff
Wendy Talhout Technical staff
Transcription factor hotspots In collaboration with the laboratories of Dr. Kevin
White (Yale University) and Dr. Harmen Bussemaker (Columbia University) we have
used DamID in combination with genomic tiling arrays to obtain a highly detailed
view of the binding patterns of a broadly selected set of 14 regulatory proteins,
including transcription factors, co-factors, and chromatin-modifying proteins.
Surprisingly, we found that many functionally unrelated transcription factors all
show strongly overlapping binding patterns, and that the genome contains many
“hotspots” that are targeted by all of these proteins. Several control experiments show
that the strong overlap is not an artefact of the techniques used. Colocalization
hotspots are 1-5kb in size, spaced on average by ~50 kb, and preferentially located in
regions of active transcription. We provide evidence that protein-protein interactions
play a role in the hotspot association of some transcription factors. Colocalization
hotspots constitute a new type of feature in the genome of Drosophila that we intend
to study in more detail in the near future.
Nuclear lamina – genome interactions 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. In close collaboration with the laboratory of Dr. Maarten
Fornerod (Division of Tumor Genetics, NKI), we have used DamID to identify nearly
500 Drosophila genes that interact in vivo with the NL component Lamin. We found
that genes that interact with Lamin are transcriptionally silent, devoid of histone
modifications typical of active chromatin, and late replicating, yet they do not bind
the classical heterochromatin markers HP1 and Su(var)3-9. Interestingly, Lamin
target genes tend to be flanked by very large intergenic regions. This suggests that
large intergenic regions can play a role in the nuclear organization of the genome.
Furthermore, Lamin target genes cluster in the genome, and we provided evidence
that these clusters act as units of co-regulation during development. These results
provide global insights into the organization of the genome in the cell nucleus. We
are currently investigating the dynamics of lamina-genome interactions during
differentiation.
Polycomb domains Polycomb group (PcG) proteins maintain transcriptional
repression of developmentally important genes and have been implicated in cell
proliferation and stem cell self-renewal. In a close collaboration with the laboratory of
Dr. Maarten van Lohuizen (Division of Molecular Genetics, NKI), we used a genomewide approach to map binding patterns of PcG proteins (Pc, esc and Sce) in
Drosophila cells. We found that Pc associates with large genomic regions of up to
approximately 150 kb in size, hereafter referred to as �Pc domains’. Sce and esc
accompany Pc in most of these domains. PcG-bound chromatin is trimethylated at
57
MOLECULAR BIOLOGY
Publications
histone H3 Lys27 and is generally transcriptionally silent. Furthermore, PcG proteins
preferentially bind to developmental genes. Many of these encode transcriptional
regulators and key components of signal transduction pathways, including Wingless,
Hedgehog, Notch and Delta. These results highlight the extensive involvement of
PcG proteins in the coordination of development through the formation of large
repressive chromatin domains.
Choksi SP, Southall TD, Bossing T,
Edoff K, De Wit E, Fischer B,
Van Steensel B, Micklem G, Brand AH.
Prospero Acts as a Binary Switch between
Self-Renewal and Differentiation in
Drosophila Neural Stem Cells. Dev Cell
Heterochromatin Classical heterochromatin is generally believed to be a repressive
type of chromatin. However, this notion is mostly based on studies with artificial
reporter genes; virtually no natural target genes were known. We have mapped HP1
binding sites on chromosome 2 and 4 in Drosophila Kc cells using high-density
oligonucleotide arrays. The resulting high-resolution maps show that HP1 binds
along entire transcription units, except for 5’ regions. Comparison with expression
data shows that most of these genes are actively transcribed. HP1 target genes are
also marked by the histone variant H3.3 and dimethylated histone 3 lysine 4, which
are both typical of active chromatin. Interestingly, H3.3 deposition, which is usually
observed along entire transcription units, is limited to the 5’ ends of HP1-bound
genes. Thus, H3.3 and HP1 are mutually exclusive marks on active chromatin. These
results demonstrate that HP1-chromatin is transcriptionally active and has extensive
links with several other chromatin components.
We also employed DamID to identify four novel heterochromatin proteins in
Drosophila. A yeast-2-hybrid screen indicated that three of these proteins (HP3, HP4,
and HP5) interact directly with HP1, while HP6 in turn binds to each of these three
proteins. Immunofluorescence microscopy and DamID mapping of in vivo binding
sites confirmed that all four proteins are components of heterochromatin. Depletion
of HP1 causes redistribution of these proteins, indicating that HP1 is essential for
their heterochromatic targeting. Finally, mutants of HP4 and HP5 are dominant
suppressors of position effect variegation, demonstrating their importance in
heterochromatic gene silencing. We propose that HP1 acts as a docking platform for
several mediator proteins that contribute to heterochromatin function.
We have adapted the DamID technology for use in human cells and used it to
identify human target genes of heterochromatin proteins. The three HP1 homologs
(HP1a, b and g) show a very similar binding pattern, consistent with their ability to
form heterodimers. Surprisingly, the HP1 proteins bind to most members of a very
large family of genes that encode KRAB domain zinc finger (KRAB-ZNF) proteins.
Because most of these KRAB-ZNF genes in the human genome are located on
Chromosome 19, we constructed a high-density oligonucleotide tiling array with 60mer probes arranged every 200bp along all non-repetitive regions of this 64Mb
chromosome. DamID mapping with these arrays revealed that HP1b covers very
large chromosomal domains (some more than 1Mb in size) that harbor clusters of
KRAB-ZNF genes (Figure V.5). We suggest that heterochromatin has played a key
role in the evolution of this unusually large family of regulatory genes.
2006; 11:775-789
Gilfillan GD, Straub T, De Wit E, Greil F,
Lamm R, Van Steensel B, Becker PB.
Chromosome-wide gene-specific targeting of
the Drosophila dosage compensation
complex. Genes Dev 2006; 20:858-870
Greil F, Moorman C, Van Steensel B.
DamID: Mapping of In Vivo ProteinGenome Interactions Using Tethered DNA
Adenine Methyltransferase. Methods
Enzymol 2006; 410:342-359
Moorman C, Sun LV, Wang J, De Wit E,
Talhout W, Ward LD, Greil F, Lu XJ,
White KP, Bussemaker HJ,
Van Steensel B. Hotspots of transcription
factor colocalization in the genome of
Drosophila melanogaster. Proc Natl Acad
Sci USA 2006; 103:12027-12032
Pickersgill H, Kalverda B, De Wit E,
Talhout W, Fornerod M, Van Steensel B.
Characterization of the Drosophila
melanogaster genome at the nuclear
lamina. Nat Genet 2006; 38:1005-1014
Simonis M, Klous P, Splinter E,
Moshkin Y, Willemsen R, De Wit E,
Van Steensel B, De Laat W. Nuclear
organization of active and inactive
chromatin domains uncovered by
chromosome conformation capture-on-chip
(4C). Nat Genet 2006; 38:1348-1354
Tolhuis B, Muijrers I, De Wit E,
Teunissen H, Talhout W, Van Steensel B,
Van Lohuizen M. Genome-wide profiling of
PRC1 and PRC2 Polycomb chromatin
binding in Drosophila melanogaster.
Figure V.5: DamID binding map of human HP1b (CBX1) along entire chromosome 19 in human MCF7
Nat Genet 2006; 38:694-699
cells. Note that CBX1 forms very large domains, which coincide almost perfectly with clusters of KRABZNF genes.
Vogel MJ, Guelen L, De Wit E,
Peric-Hupkes D, LodГ©n M, Talhout W,
Bioinformatics Bioinformatics tools are essential for the detailed understanding of
our genome-wide datasets (now amounting to millions of datapoints). We are actively
developing new algorithms and approaches for the systematic analysis of the protein
binding patterns that we obtained. In particular, we focus on predicting interactions
between chromatin components by comparative analysis, and on the identification of
multi-gene chromatin domains. 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.
Feenstra M, Abbas B, Classen AK,
Van Steensel B. Human heterochromatin
proteins form large domains containing
KRAB-ZNF genes. Genome Res 2006;
16:1493-1504
58
MOLECULAR BIOLOGY
BIOINFORMATICS AND STATISTICS
Group leader Lodewyk Wessels
Lodewyk Wessels PhD Group leader
Nicola Armstrong PhD Academic staff
Michael Hauptmann PhD Academic staff
Miranda Mandjes PhD Post-doc
The research focus within our group is to develop novel computational approaches to
exploit a wide variety of data sources in order to improve stratification of cancers into
biologically distinct types that are well correlated with outcome and therapy response.
In order to achieve this goal, significant effort is devoted to a better understanding of
disease development and progression by identification of genes and pathways
involved in tumorigenesis. Challenging computational problems that are being
addressed include (i) construction of efficient cross-species mappings to translate
results obtained in mouse model systems to human tumors; (ii) developing
algorithms to efficiently integrate heterogeneous data sources and (iii) extracting
structural pathway information from measured data sources and annotation data
bases.
Carmen Lai MSc Graduate student
Wouter Meuleman MSc Graduate student
Jeroen De Ridder MSc Graduate student
Martin Van Vliet MSc Graduate student
Publications
Armstrong NJ, Mc Peek MS, Speed TP.
Incorporating interference into linkage
analysis for experimental crosses.
Biostatistics 2006; 7:374-386
Armstrong NJ, Brodnicki TC, Speed TP.
Mind the gap: Analysis of marker-assisted
breeding strategies for inbred mouse strains.
Mamm Genome 2006; 17:273-287
Extracting oncogenes and oncogenic pathways from insertional mutagenesis
screens An effective method for identifying candidate oncogenic loci is retroviral
insertion mutagenesis. Within a large set of tumors induced by retroviral infection, a
subset of viral insertions from independent tumors that map to the same genomic
locus is referred to as a Common Insertion Site (CIS). CISs often represent lesions
that have been selected for during tumorigenesis. To automatically detect CISs we
have developed a computational approach, the kernel convolution framework. This
approach finds CISs using a predefined significance level while controlling the
family-wise error and takes bias stemming from preferential viral insertion sites into
account. In contrast to existing approaches, our method operates at any biologically
relevant scale, providing new insights in the behavior of CISs across multiple scales.
To find oncogenic lesions which are collaborating events in tumorigenesis, we
extended the kernel convolution framework to detect the occurrence of multiple
independent insertions within one tumor at a higher frequency than expected by
chance (Fig. V.6). A novel, fast biclustering algorithm which finds subsets of tumors
with similar insertion profiles will also be employed to extract pathway structures
from the insertion data.
Prioritizing candidate oncogenes based on genomic data Gene expression data
of tumor series have been extensively employed to predict outcome and response to
therapy in breast cancer. To uncover the underlying mechanisms that drive these
expression signatures, comparative genomic hybridization and microarray gene
expression data has been collected for a series of breast tumors representing the
intrinsic subtypes. We developed SIRAC, a computational approach which detects
genomic regions that are significantly enriched for BAC clones highly correlated with
a particular subtype. We also developed KC-SMART, a generalization of the kernel
convolution framework to detect genomic regions that are more frequently aberrated
in a set of tumors, than one would expect by chance. In contrast to SIRAC, this
approach is unsupervised, i.e. it does not require a labeling of the tumor set
according to subtypes. Both approaches employ expression data to prioritize genes in
a given aberrated region. Application of KC-SMART to a set of p53 deficient mouse
tumors resulted in the detection of well-known oncogenes such as c-Met as well as
promising new candidates.
Figure V.6: Co-occurence plot for all the
data in the Retroviral Tagged Cancer Gene
Database (RTCGD). The green dots
indicate the insertion co-occurences, the red
dots mark the locations of the common cooccurrences of insertions. The arrows
indicate the gene pairs accociated with a
selection of the top-scoring co-occurerneces,
Knowledge-based approaches for outcome prediction Algorithms which exploit
biological knowledge, such as the functional grouping of genes, could lead to more
accurate predictors and enlarge the possibility of generating novel insights in the
disease. Over-fitting can also be overcome by constructing larger datasets by
collecting related gene expression data sets in a compendium. We employed an
unsupervised approach to derive a set of modules (groups of functionally related
genes with coordinated expression across a subset of tumors). By employing the
module activity as input for training breast cancer outcome predictors, we revealed
functional modules associated with breast cancer outcome. We studied modules
extracted from several compendia: single breast cancer datasets, breast cancer
compendia, and a human cancer compendium. We validated these predictors on
independent data from the same institution and other institutions. Modules derived
from single breast cancer datasets achieve outperform regular gene-based predictors.
59
MOLECULAR BIOLOGY
Publications (continued)
Modules derived from a single breast cancer dataset and a cancer specific
compendium performs better compared to those derived from a human cancer
compendium.
De Ridder J, Uren A, Kool J, Reinders M,
Wessels L. Detecting Statistically
Significant Common Insertion Sites in
Mass spectrometry-based response prediction Poly(ADP-ribose) polymerase
(PARP)-inhibitors in combination with the DNA cross-linker, cisplatin, have recently
been shown to have great potential as a treatment for patients carrying germline
BRCA1 or BRCA2 mutations.
The research questions we address include whether we are able to find markers for
“BRCAness” and PARP-inhibitor treatment response. We are doing so by following
an incremental procedure.
First, we performed a comprehensive comparison of currently used, as well as novel
mass spectrometry normalization approaches involving six mass spectrometry
datasets, three classification approaches and 17 normalization techniques. Based on
the results of this comparison, we constructed a computational workflow to process
mass spectra.
Next, using SELDI-TOF mass spectrometry, we study the proteomic contents of
cell lysates and growth media of both BRCA(1,2)/p53-/- and p53-/- cell lines.
When appropriate biomarkers have been identified, we will also analyze cell lysates
from spontaneous tumors from BRCA1 and BRCA2 deficient mouse models.
Simultaneously, we will study sera from these tumor bearing mice, to investigate the
relationship between the markers obtained from tumor tissue and markers present
in serum. If all these steps have been successfully completed, the approach can be
tested on human tissue from BRCA1 and BRCA2 carriers. In addition to this, we
hope to gain a better understanding of the pathways affected by PARP-inhibitors by
integrating proteomic data with transcriptomic data, yielded by microarray expression
profiling.
Retroviral Insertional Mutagenesis Screens.
Evaluating effects of timing of exposure on cancer risk The effects of timing of
exposure are rarely evaluated in detail in epidemiologic studies of chronic exposures.
Cumulative exposure is usually used as the major exposure metric, while age at
exposure, time since exposure, or intensity of exposure are often ignored. Using
advanced statistical risk modeling, we showed a significantly increased breast cancer
risk from smoking before a first full-term pregnancy, particularly before menarche,
in a large cohort study. These results suggest that the sensitivity of the female breast
to tobacco carcinogens is increased during adolescence and early adulthood but
decreases after first childbirth when most breast tissue has terminally differentiated.
In an analysis of smoking data from ten case-control studies, including cancers of the
lung, bladder, oral cavity, pancreas and esophagus, we demonstrated enhanced
carcinogenic potency of smoking at lower intensities, but narrow ranges for packyears increased uncertainty, precluding definitive conclusions. At higher intensities,
there was a consistent reduced potency effect across studies. Intensity patterns were
comparable across the diverse cancer sites.
Lai C, Reinders M, Van �t Veer L,
PLoS Comput Biol 2006; 2:e166
Fan C, Oh DS, Wessels L, Weigelt B,
Nuyten DSA, Nobel AB, Van �t Veer LJ,
Perou CM. Concordance among geneexpression-based predictors for breast cancer.
N Engl J Med 2006; 355:560-569
Glas AM, Floore A, Delahaye LJMJ,
Witteveen AT, Pover RCF, Bakx N,
Lahti-Domenici JST, Bruinsma TJ,
Warmoes MO, Bernards R, Wessels LFA,
Van’t Veer LJ. Converting a breast cancer
microarray signature into a highthroughput diagnostic test. BMC Genomics
2006; 7:278
Knijnenburg TA, Reinders MJT,
Wessels LFA. Artifacts of Markov blanket
filtering based on discretized features in
small sample size applications. Pattern
Recogn Lett 2006; 27:709-714
Wessels L. A comparison of univariate and
multivariate gene selection techniques for
classification of cancer datasets. BMC
Bioinformatics 2006; 7:235
Linet MS, Hauptmann M, Freedman DM,
Alexander BH, Miller J, Sigurdson AJ,
Morin Doody M. Interventional
radiography and mortality risks in U.S.
radiologic technologists. Pediatr Radiol
2006; 36 Suppl 14:113-120
Van den Broek GB, Balm AJM,
Van den Brekel MWM, Hauptmann M,
Schornagel JH, Rasch CRN. Relationship
Crossover interference in linkage analysis During meiosis, chromosomes
replicate, pair and then synapse. Once pairing is complete, crossing over, the
reciprocal exchange of chromosomal segments among non-sister chromatids begins.
Crossover interference is the non-random placement of the crossover points along
the length of the chromosome. While many models for this process have been
proposed, the majority of genetic mapping studies still ignore the phenomenon of
interference and assume the crossover locations come from a Poisson process. We
have implemented the chi2 model for interference in both simulation studies for
breeding programs and in an extension of the Lander-Green algorithm for genetic
mapping. In simulation studies, this model was shown to be more accurate than the
other methods for estimating genetic distances.
between clinical factors and the incidence of
toxicity after intra-arterial chemoradiation
for head and neck cancer. Radiother Oncol
2006; 81:143-150
Van Houwelingen HC, Bruinsma T,
Hart AAM, Van’t Veer LJ, Wessels LFA.
Cross-validated Cox regression on
microarray gene expression data. Stat Med
2006; 25:3201-3216
60
TUMOR BIOLOGY
VI DIV IS ION OF TU M OR BIOLOG Y
ANTIGEN PRESENTATION BY MHC CLASS I AND MHC CLASS
II MOLECULES
Division head, Group leader Jacques Neefjes
Jacques Neefjes PhD Group leader
Tom Groothuis PhD Post-doc
Christoph Lippuner PhD Post-doc
Marije Marsman PhD Post-doc
Victoria Menendez PhD Post-doc
Veronica Monserrat PhD Post-doc
Alexander Griekspoor MSc Graduate student
Tineke Van den Hoorn MSc Graduate student
Coen Kuijl MSc Graduate student
Joost Neijssen MSc Graduate student
Baoxu Pang MSc Graduate student
Petra Paul MSc Graduate student
Nuno Rocha MSc Graduate student
Izhar Salomon MSc Graduate student
Lennert Janssen BSc Technical staff
Publications
Bergink S, Salomons FA, Hoogstraten D,
Groothuis TAM, De Waard H, Wu J,
Yuan L, Citterio E, Houtsmuller AB,
Neefjes J, Hoeijmakers JHJ, Vermeulen W,
Dantuma NP. DNA damage triggers
nucleotide excision repair-dependent
monoubiquitylation of histone H2A.
Genes Dev 2006; 20:1343-1352
Dantuma NP, Groothuis TAM,
Salomons FA, Neefjes J. A dynamic
ubiquitin equilibrium couples proteasomal
activity to chromatin remodeling. J Cell Biol
2006; 173:19-26
Elliott T, Neefjes J. The Complex Route
to MHC Class I-Peptide Complexes.
Cell 2006; 127:249-251
Griekspoor AC. Single cell biochemistry to
visualize antigen presentation and drug
resistance. Leiden: Leiden University, 2006
Groothuis T, Neefjes J. The ins and outs
MHC class I molecules alert the immune system for intracellular infections or
cellular alterations related to tumour formation. The killer cells in the immune
system can recognize a small fragment of an altered or foreign protein presented in
the context of MHC class I molecules at the cell surface and subsequently respond by
eliminating such cells. This system is central in tumour vaccination studies and we
study the basic machinery.
For successful presentation of fragments of tumour proteins by MHC class I
molecules, first fragments have to be generated and these have to be transferred to
MHC class I molecules after transport over the ER membrane. We have defined most
steps in this process. Proteins are degraded into fragments by the proteasome.
Therefore proteins have to be tagged by a small protein called Ub and the polyUb
form acts as a recognition signal by the proteasome. We have studied the dynamic
nature of the Ub modification in living cells using GFP-tagged Ub. We show that free
mono-Ub is in fact rate limiting and the majority of Ub is observed associated to
histones2 and associated to large protein complexes. Acute need of Ub (following
proteasome inhibition or heat shock) results in more poly-Ub modified proteins, but
the Ubs required for this can not be delivered through the rate limiting small Ub
pool. Instead, histones are de-Ub resulting in altered chromatin structure and
transcription. We are currently studying the details of an analogous event, histone deUb following exposure to topo-isomerase inhibitors like doxorubicin, an often-used
anti-cancer drug. Histone-deUb can be an additional mechanism of tumour cell
growth control. We have studied other factors controlling MHC class I presentation.
One major factor appears to be ionizing radiation that upregulate MHC class I
expression. We have identified the molecular basis for this effect and shown that
tumours are subsequently better-recognized following radiation. This �radioimmunotherapy’ is a new combination therapy for better anti-tumour immune
responses and will be further studied in model systems for further improvements.
MHC class II molecules work complementary to MHC class I molecules by sampling
fragments of proteins degraded in the endocytic track. We have determined and
visualized most steps in this process including the control of motor protein-driven
MHC class II vesicle transport, interactions with intracellular pathogens and
interactions with lysosomal chaperones. We have developed small molecular
compounds that affect intracellular growth of pathogens and identified the molecular
targets. This is the first successful application of �reciprocal chemical genetics’ and in
fact integrates biology, genetics and chemistry to arrive at lead compounds for
defined biological effects and corresponding targets. Similar approaches are followed
to define molecular targets affecting antigen presentation by MHC class I- and MHC
class II molecules.
This year we have developed a new combination therapy combining radio- and
immunotherapy that strongly improves anti-tumour responses. This combination
therapy will be further developed. In addition, we have developed strategies
combining chemistry, biology and genetics to identify novel compounds, define
corresponding targets and define new signalling pathways, in this case in host
responses controlling pathogen growth. This approach will be developed further.
of intracellular peptides and antigen
presentation by MHC class I molecules.
Curr Top Microbiol Immunol
2006; 300:127-148
Groothuis TAM, Dantuma NP, Neefjes JJ,
Salomons FA. Ubiquitin crosstalk
connecting cellular processes. Cell Division
2006; 1:21
Antigen presentation by MHC class I molecules MHC class I molecules present
a sample of the peptides produced intracellularly by the proteasome. But how is the
proteasome degraded? We, in collaboration with Dr Fred van Leeuwen (Div of
Cellular Biochemistry), have established a method to genetically switch colours on
yeast proteasomes and this will be used to establish proteasome half-life. By
combining this with the yeast mutant libraries, we will identify proteins determining
proteasome half-life. Many factors probably determine the efficiency of antigen
presentation by MHC class I molecules. Ionizing radiation induces the level of
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TUMOR BIOLOGY
Publications (c0ntinued)
intracellular peptides by either oxidizing proteins resulting in their degradation or by
activating the mTOR pathway. Radiated cells are better recognized and killed by the
immune system. Other factors could also contribute to MHC class I antigen
presentation. We have generated and directly conjugated monoclonal antibodies
detecting two conformations of MHC class I molecules at the cell surface. This is
combined with selective inactivation of defined proteins by siRNA followed by
automated FACS. Potential targets modifying MHC class I expression, can be used
for isolation of small molecular compounds to manipulate MHC class I expression
and many immune responses.
For a proper cytotoxic T cell response, antigen information has to be delivered to
professional antigen presenting cells like dendritic cells, a process called crosspresentation. We have recently identified gap junctions as being able to transfer such
information in the form of peptides from one cell to its neighbour. However,
apoptosis conditions may be ideal for the induction of cross-presentation. We have
first confirmed whether antigen presentation continues during apoptosis and that
long gap-junction communication persues after apoptosis induction. We are
currently testing whether cells in apoptosis are able to transfer peptides through gap
junctions to healthy neighbouring cells for cross-presentation.
Our approach is to understand the details of MHC class I antigen presentation and,
identify targets and develop methods to improve immune responses to tumour cells.
Groothuis TAM. Manipulations of the
ubiquitin proteasome system and their
effects on antigen presentation. Leiden:
Leiden University, 2007
Herberts CA, Neijssen JJ, De Haan J,
Janssen L, Drijfhout JW, Reits EA,
Neefjes JJ. Cutting edge: HLA-B27 acquires
many N-terminal dibasic peptides:
Coupling cytosolic peptide stability to
antigen presentation. J Immunol
2006; 176:2697-2701
Jordens I, Westbroek W, Marsman M,
Rocha N, Mommaas M, Huizing M,
Lambert J, Naeyaert JM, Neefjes J.
Rab7 and Rab27a control two motor protein
activities involved in melanosomal
transport. Pigment Cell Res 2006;
19:412-423
Antigen presentation by MHC class II molecules and the control of
endocytosis MHC class II molecules meet antigens and their degradation
fragments in the endocytic track followed by transport to the plasma membrane
before presentation of the antigenic fragments to the immune system. We are
studying various aspects of this process. We have used FRET and FLIM technology to
visualize the interaction between MHC class II molecules and the lysosomal
chaperone HLA-DM in living cells. These data revealed that HLA-DM and MHC class
II interacted only in the internal vesicles rather than the limiting membrane of the
late endosomal structures called MIIC. Bacterial-induced phagosomes are devoid of
internal vesicles and we showed (both by FRET and by biochemistry) that MHC class
II molecules fail to acquire antigen in phagosomes. We are currently studying the
molecular basis of this spatial specialization of MIIC with respect to antigen loading
of MHC class II molecules. Using FRET, we have visualized the organization of
tetraspannin networks that also acquire MHC class II and HLA-DM molecules and
determined their redistribution within MIIC structures. This is further studied in
detail. We have design a �reciprocal chemical genetics’ approach to identify leads and
targets that –in this study- determine intracellular growth of pathogens like
Salmonella and M.tuberculosis. Therefore we synthesized chemical kinase inhibitor
homologue libraries to identify a �chemical profile’ and combined this with siRNA
libraries for the human kinome and automated microscopy to identify possible
targets. This revealed the PKB/Akt pathway as controlling intracellular growth of
pathogens acting via a novel pathway that is currently studied in detail. The
�reciprocal chemical genetics’ approach allows rapid identification of lead and targets
and allowed the identification of new signalling pathways downstream of the relevant
anti-cancer target, PKB/Akt. To identify other targets controlling antigen presentation
by MHC class II molecules, we establish a screen where antigen presentation was
followed with two antibodies recognizing two conformations of MHC class II
molecules; the successfully peptide loaded form and the form containing the
invariant chain remnant CLIP. A kinome library was used followed by automated
FACS analyses with the two antibodies. Of the 779 targets tested, only three
reproducibly affected antigen presentation by MHC class II molecules. These are
now tested in more detail to reveal the molecular mechanism.
We have studied the molecular mechanism that control transport of MHC class II
molecules from MIIC to the plasma membrane. We had already identified a Rab7
effector protein called RILP that recruited the dynein motor complex to MIIC preventing
transport to the plasma membrane. We now have shown that the small GTPase Rab7
interacts with two proteins simultaneously: RILP to recruit the dynein motor by
interacting with the stalk p150glued and the oxysterol-binding protein ORP1L that
transfers the Rab7-RILP-dynein motor complex to the general dynein receptor spectrin
b3. Only then, active minus-end driven transport of late endosomes is observed.
Reits EA, Hodge JW, Herberts CA,
Groothuis TA, Chakraborty M,
Wansley EK, Camphausen K, Luiten RM,
De Ru AH, Neijssen J, Griekspoor A,
Mesman E, Verreck FA, Spits H,
Schlom J, Van Veelen P, Neefjes JJ.
Radiation modulates the peptide repertoire,
enhances MHC class I expression, and
induces successful antitumor
immunotherapy. J Exp Med
2006; 203:1259-1271
Figure VI.1: Model for regulation of
microtubule-based dynein motor-driven
minus-end transport of late endocytic
compartments. Active Rab7 localizes to
membranes of late endocytic compartments
and recruits the RILP and ORP1L effectors
to form a tripartite complex. The dyneindynactin motor is recruited to this complex,
via an interaction between RILP and
p150Glued, and subsequently transferred to
the membrane-associated spectrin receptor, a
process facilitated by ORP1L.
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TUMOR BIOLOGY
microRNAs, RNA INTERFERENCE AND CANCER
Group leader Reuven Agami
Reuven Agami PhD Group leader
Mathijs Voorhoeve PhD Senior post-doc
Geetha Achanta PhD Post-doc
Qing Chang PhD Post-doc
Suresh Nair PhD Post-doc
Eitan Zlotorynski PhD Post-doc
Jarno Drost MSc Graduate student
Anja Duursma MSc Graduate student
Martijn Kedde MSc Graduate student
Remco Nagel MSc Graduate student
Carlos Le Sage MSc Graduate student
Josyanne Van Duijse MSc Graduate student
Mariette Schrier PhD Technical staff
Joachim Oude Vrielink BSc Technical staff
Figure VI.2: Identification of miR-372 and
miR-373 that bypass oncogene-induced
senescence. A. A flow-chart of the screen.
Cells transduced with the miR-Lib were
Our main research objective is to understand the cancerous process in humans and
identify essential genes. As functional units, the knowledge obtained on these genes
allows us to design novel therapeutic ways. 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 a vector-based system that mediates suppression of gene
expression through RNA interference (pSUPER). We have successfully applied this
gene-inactivation system to study the importance of tumor-suppressors and DNAdamage checkpoints in protecting humans from cancer. As a result, the pSUPER
RNAi system is very frequently used in the lab to address emerging inquiries. For
example, lately we used pSUPER vectors to characterize a telomerase independent
regulation of ATR by human telomerase RNA (hTR). We also investigated with this
system the Cdc6- dependent effect exerted by p53 on DNA replication. Last, we
recently developed and applied high-throughput pSUPER-RNAi libraries to uncover
novel tumor suppressors and DNA-damage checkpoint genes, and characterized their
role in cancer prevention.
This year we began to study the connection between microRNAs (miRNAs) the new
emerging family of genes and cancer. We developed a novel miRNA expression vector
(miR-Vec), miRNA library (miR-Lib) and a corresponding barcode array (miR-Array).
We used these functional-genetic tools to uncover and describe cancerous miRNAs.
At present, a large body of the group is devoted to the development and usage of
novel genetic approaches to identify and characterize miRNAs involved in cell cycle
control, cell migration and tumor metastasis.
Functional genetic screens identify miR-372&3 as potential oncogenes in
Testicular Germ Cell tumors To identify cancer-related functions of microRNAs
(miRNAs) we performed functional genetic screens. Initially, we constructed miRVec, a retroviral vector for miRNA expression. Based on that vector, we created a
human miRNA expression library (miR-Lib) containing the majority of annotated
human miRNAs (~450), and produced a corresponding microarray (miR-Chip)
containing all miR-Lib inserts. We used these tools to detect miRNAs that bypass
oncogene-induced senescence. In response to mitogenic signals from oncogenes,
primary human cells irreversibly arrest in a phenotype coined senescence. Primary
human cells lacking functional p53 and p16 proteins efficiently escape this arrest and
acquire a transformed phenotype. Importantly, this escape from oncogene-induced
senescence is a prerequisite for full transformation into tumor cells. We found that
the expression of either miR-372 or miR-373 granted cells with tumorigenic growth in
the presence of oncogenic stress, while still harboring wild type p53. This effect was
due to a miR372/3-induced blockade of the ability of the p53 pathway to inhibit cell
cycle dependent kinases (CDK) activities in response to oncogenic stress. This effect
was partly through direct miR-372/3-induced suppression of LATS2 (LArge Tumor
Suppressor gene) expression. Finally, we provided evidence that miR-372&3 are
potential novel oncogenes participating in the development of human Testicular
Germ Cell tumors. We found mutual exclusive expression of miR-372&3 and p53
mutations. For details on our recent work see Voorhoeve et al 2006. Altogether,
beyond demonstrating the potential of miRNAs to support cellular transformation,
our results validated the feasibility and applicability of miRNA-genetic screens and
ascertained our capability to identify relevant miRNA target genes and relevant
cellular pathways. We are now exploiting other miRNA genetic screens to identify
additional cancerous miRNAs.
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.
An RNAi screen identifies PITX1 as a suppressor of RAS activity and human
tumorigenicity In the past, we have developed a vector-based system termed
pSUPER that mediates stable suppression of gene expression through RNAi. With
this system we demonstrated that persistent inactivation of a dominant-RAS
oncogene results in the loss-of tumorigenicity of late-stage cancer-cell lines. We also
studied the tumor-suppressive functions of the human INK4A locus, which encodes
for the p16INK4A and p14ARF genes and is inactivated in many cancers. We showed
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TUMOR BIOLOGY
Publications
that p16INK4A, but not p14ARF, is the major tumor suppressor of the human
INK4A locus.
Recently, a large collection of pSUPER vectors for the knockdown of up to 8000
genes in human cells was assembled in our institute. We used this tool to identify
and characterize novel human-tumor suppressor genes. Activating mutations of RAS
frequently occur in subsets of human cancers, indicating that RAS activation is
important for tumorigenesis. However, a large proportion of these cancers still retain
wild-type RAS alleles, suggesting that either the RAS pathway is activated in a distinct
manner or another pathway is deregulated. To uncover novel tumour-suppressor
genes, we screened the NKI-RNAi library for knockdown constructs that transform
human primary cells in the absence of ectopically introduced oncogenic RAS. We
identified PITX1 whose inhibition induces the RAS pathway and tumorgenicity.
Interestingly, we observed low expression of PITX1 in prostate and bladder tumours
and in colon cancer cell lines containing wild-type RAS. Restoration of PITX1 in the
colon cancer cells inhibited tumorigenicity, in a wild-type RAS dependent manner
(Figure 2). Finally, we identified RASAL1, a RAS-GTPase-activating protein, as a
transcription target through which PITX1 affects RAS function. Thus, PITX1
suppresses tumorgenicity by down-regulating the RAS pathway through RASAL1.
Kedde M, Le Sage C, Duursma A,
Zlotorynski E, Van Leeuwen B,
Nijkamp W, Beijersbergen R, Agami R.
Telomerase-independent Regulation of ATR
by Human Telomerase RNA. J Biol Chem
2006; 281:40503-40514
Le Sage C, Agami R. Immense promises for
tiny molecules: Uncovering miRNA
functions. Cell Cycle 2006; 5:1415-1421
Voorhoeve PM, Le Sage C, Schrier M,
Gillis AJM, Stoop H, Nagel R, Liu YP,
Van Duijse J, Drost J, Griekspoor A,
Zlotorynski E, Yabuta N, De Vita G,
Nojima H, Looijenga LHJ, Agami R.
A Genetic Screen Implicates miRNA-372
and miRNA-373 As Oncogenes in Testicular
Germ Cell Tumors. Cell 2006; 124:1169-1181
Voorhoeve PM, Agami R. Classifying
microRNAs in cancer: The good, the bad
and the ugly. BBA Rev Cancer 2006;
Figure VI.3: Mutual occurrence of miR-372&373 expression
In Press
and p53 mutation in testicular germ cell tumors. A summary
of miR-372 and miR-373 expression as well p53 status in
several TGCT cell lines and primary seminomas (in the latter
only exons 5 to 8 were examined).
Telomerase independent regulation of ATR by human telomerase RNA Using
the pSUPER technology we recently uncovered a novel, telomerase independent,
function of hTR that restrains ATR activity and participates in the recovery of cells
from UV radiation. The human telomerase RNA (hTR) together with the telomerase
reverse transcriptase, hTERT, constitute the core components of telomerase that is
essential for telomere maintenance. While hTR is ubiquitously expressed, hTERT
is normally restricted to germ cells and certain stem cells, but both are often
deregulated during tumorigenesis. We therefore set to investigate the effects of
changes in hTR cellular levels. Surprisingly, inhibition of hTR expression by RNAi
triggered a rapid, telomerase independent, growth arrest that was associated with p53
and CHK1 activation. Moreover, increased expression of hTR neutralized activation of
these DNA damage pathways. The hTR effects were mediated through ATR and were
sufficiently strong to impair ATR-mediated DNA-damage checkpoint responses.
p53-dependent regulation of Cdc6 protein stability controls cellular
proliferation Probably all tumors harbor a certain degree of genomic instability,
underling the tight connection between DNA damage responses (repair and
checkpoint) and tumorigenesis. In the past, we have shown that the DNA-damage
response to ionizing radiation is composed of two processes: initiation and
maintenance. DNA damage causes a fast G1-arrest by rapidly degrading cyclin-D1.
Later, this initial G1-response to DNA damage is maintained and further strengthened
by the stabilization of p53, leading to the induction of p21cip1. Activation of the tumor
suppressor p53 in response to genotoxic stress imposes cellular growth arrest or
apoptosis. We identified Cdc6, a licensing factor of the pre-replication complex
(pre-RC), as a novel target of the p53 pathway. We show that activation of p53 by DNA
damage results in enhanced Cdc6 destruction by the anaphase-promoting complex
(APC). This destruction is triggered by inhibition of CDK2 mediated CDC6
phosphorylation at serine 54. Conversely, suppression of p53 expression results in
stabilization of Cdc6. We show that loss of p53 results in more replicating cells, an
effect that can be reverted by reducing Cdc6 protein levels. Our data suggest that
initiation of DNA replication is regulated by p53 through Cdc6 protein stability.
Figure VI.4: hTR enhances the expression of
fragile sites. (A) Quantification of the
expression of fragile sites in U2OS
transfected with expression vectors for hTR,
shRNA for ATR, and ctrl. The number of
fragile sites expressed in 400 chromosomes is
shown, data are representative of three
independent experiments. (B) Pictures
exemplifying the extent of fragile sites in the
various transfected U2OS cells. Pictures
were made with 1000x magnification.
64
TUMOR BIOLOGY
DYNAMIC INTERACTIONS AT THE NUCLEAR ENVELOPE
Group leader Maarten Fornerod
Maarten Fornerod PhD Group leader
Stijn Heessen PhD Post-doc
Helen Pickersgill PhD Post-doc
Noelia Valle PhD Post-doc
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.
Rafael Bernad-Fernandez MSc Graduate student
Dieuwke Engelsma MSc Graduate student
Jolita Hendriksen MSc Graduate student
Bernike Kalverda MSc Graduate student
Hella Van der Velde BSc Technical staff
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.
Structure-function of the nuclear pore complex The nuclear pore complex (NPC)
conducts macromolecular transport to and from the nucleus and provides a
hydrophobic barrier composed of phenylalanine-glycine (FG) repeats. Nuclear
transport is achieved through permeation of this barrier by transport receptors. So
far, very few FG nucleoporins have been described to be essential for specific
transport pathways: mutation mostly results in a slight inhibition of a subset of
pathways.
One of the most stable interactions between a nucleoporin and a nuclear transport
receptor is the Nup214 interaction with CRM1/exportin 1, which occurs at the
C-terminal FG repeat region. This led to the idea that this interaction is important for
CRM1-mediated nuclear export. We tested this by depletion of Nup214 in cultured
cells. Perhaps surprisingly, no major defect in nuclear export of simple CRM1 cargoes
was observed. In contrast, depletion of Nup214 results in a dramatic reduction of 60S
preribosomal nuclear export. However, the stable CRM1/Nup214 interaction is not
involved in this: the central coiled-coil domain of Nup214 is able to rescue 60S
nuclear export. This suggests that nucleoporin Nup214 plays two distinct roles at the
NPC: it participates redundantly in the hydrophobic barrier with its FG-rich domain,
and plays a non-redundant structural role that is essential for 60S preribosomal
export.
From the inside looking in: chromatin dynamics at the nuclear lamina Since
the late 19th century, studies of interphase nuclei have indicated that the
arrangement of chromosomes within the nucleus is non-random. Many observations
suggest that the nuclear envelope plays a key role in the spatial and functional
organization of chromatin. In close collaboration with the lab of Bas van Steensel
(Dept. of Molecular Genetics), we are analyzing the behaviour of genes in respect to
various nuclear envelope components.
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. We used a genome-wide
approach to identify nearly 500 Drosophila genes that interact in vivo with the NL
component Lam, a B-type lamin.
65
TUMOR BIOLOGY
Publications
Bernad R, Engelsma D, Sanderson H,
Pickersgill H, Fornerod M. Nup214-Nup88
Figure VI.5: Genome-wide analysis of Lamin/
nucleoporin subcomplex is required for
chromatin interactions. 1. Lam binding genes
CRM1-mediated 60 S preribosomal nuclear
identified by Lamin DamID are in dynamic contact
export. J Biol Chem 2006; 281:19378-19386
with the nuclear periphery, as revealed by
fluorescence in situ hybridization FISH. 2. Large
Pickersgill H, Kalverda B, de Wit E,
intergenic regions, transcriptional inactivity and lack
Talhout W, Fornerod M*, Van Steensel B*.
of active histone marks and late replication are
Characterization of the Drosophila
contributing factors in Lam binding, as indicated by
melanogaster genome at the nuclear lamina.
genome-wide comparisons to various chromatin
Nat Genet 2006; 38:1005-14.
factors.
* co-corresponding authors
Genes that interact with Lam are transcriptionally silent, devoid of histone
modifications typical of active chromatin, late replicating, and widely spaced
(Figure VI.5). These factors collectively account for a large proportion of Lamin
binding behavior, suggesting the NL acts as an integrator of different types of
genomic and chromatin features. Lam target genes cluster in the genome, and genes
within each cluster are coordinately activated at specific stages of development. These
results provide global insights into the dynamic organization of the genome at the
NL, and imply that intergenic or “junk” DNA functions in the global organization of
chromatin in the nucleus.
66
TUMOR BIOLOGY
ESTROGEN RECEPTOR AND BREAST CANCER
Group leader Rob Michalides
Rob Michalides PhD Group leader
Mariska Rondaij PhD Senior Post-doc
Wilbert Zwart MSc Graduate Student
Desiree Verwoerd Technical staff
Cristiane Toaldo Technical staff
Publications
Griekspoor A, Margarido TC, Zwart W,
Michalides R. Review of: BRCA1 and cyclin
D1: Gate keepers in hormone responsive
tissues? Breast Cancer Online 2006; 9
Griekspoor AC. Single cell biochemistry to
visualize antigen presentation and drug
resistance. Leiden: Leiden University, 2006
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. To study the molecular mechanism of anti-estrogen resistance, we
measured conformational changes in ERa by fluorescence resonance energy transfer
(FRET). Using an YFP-ER -CFP construct, interaction between the CFP and YFP can
be measured by FRET, where a change in fluorescent ratios implies a conformational
change of the receptor. When anti-estrogens bind the receptor, it adapts an alternative
conformation, which can be visualized using our FRET approach. Using this assay,
we studied the efficacy of anti-estrogens to (in)activate ER under different
experimental conditions, and have found that Protein Kinase A- (Michalides et al,
Cancer Cell 2004) or PAK1-mediated (Rayala et al, Cancer Res, 2006)
phosphorylation of Serine 305 of ERa is responsible for resistance to tamoxifen.
Activation of PAK1, a downstream mediator of Rac, is therefore also involved in
tamoxifen resistance. In addition, we have shown that this phosphorylation of Serine
305 in ERa leads to an altered orientation between ERa and its coactivator SRC-1,
which renders the transcription complex active in the presence of tamoxifen. This
altered orientation is measured by a change in intermolecular FRET between the
C-termini of ERa and SRC-1 and requires interaction between the AF-1 domain at the
N-terminus of ERa and SRC-1. An altered orientation between a transcription factor
and its cofactor due to phosphorylation of a specific site within a tamoxifen-bound
ERa provides a novel mechanism of resistance.
Figure VI.6: Model for resistance to tamoxifen by
PKA or PAK-1 mediated phosphorylation of ERa.
The phosphorylation alters orientation between ERa
and cofactor SRC1 in such a way that RNA
polymerase II is recruited and transcription is
sustained.
Since tumors that are resistant to tamoxifen might still be sensitive to treatment with
other anti-estrogens, we determined a profile of resistance to 10 different antiestrogens based on accumulation of specific modification(s) of ERa by PKA, either or
not in combination with increased expression of cofactors cyclin D1 and SRC-1.
Tamoxifen and EM-652 appeared in this profile as the most sensitive to resistance
development and Fulvestrant and ICI 164.384 as the most stringent anti-estrogens in
this analysis. These FRET findings are supported by transcriptional activation of ER.
This approach reveals modifications in ERa that induce resistance to anti-estrogens
and defines as such conditions for consecutive application of anti-estrogens.
These FRET-based approaches are not directly applicable to clinical specimen, since
they require transfection of life cells. Also, only few cells in a tumor may have
become resistant to tamoxifen, which would easily be undetermined by FRET
techniques. We therefore developed, in collaboration with Upstate Inc, an antibody
that specifically detects the phosphorylated Serine 305 ERa in clinical breast tumor
samples and which could detect tamoxifen resistance in a minority of the tumor cells.
Together with M.Kok and S.Linn, dept of Molecular Pathology, we will evaluate
whether staining with this antibody can predict tamoxifen resistance in breast cancer
patients.
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TUMOR BIOLOGY
CRYO-IMMUNOGOLD ELECTRON MICROSCOPY AND 3D CRYO-ELECTRON
TOMOGRAPHY IS OUR METHODOLOGY TO EMBARK ON AN EXPEDITION
TOWARDS A PSEUDOATOMIC ATLAS OF THE ENDOCYTIC SYSTEM
Supramolecular architecture in an unperturbed cellular context
We now know that assemblies of ten or more proteins carry out almost all processes
in a cell. As it performs its functions, each of these assemblies interacts with several
other large protein complexes. Indeed, the cell can be viewed as a factory that
contains a complex network of interlocking assembly lines, each of which is
composed of a set of large protein machines. In order to understand the machinery
of life and the mechanisms of disease the three-dimensional structures of many
proteins are resolved at an increasing pace by X-ray crystallography and NMR.
However, it is the assembly of proteins into molecular machines and the dynamic
interactions between proteins, which determine their function and activity. The next
great challenge will therefore be to understand the three-dimensional architecture of
cells, in terms of the spatial arrangement of organelle-bound protein complexes, and
to describe quantitatively how this arrangement alters as the cells respond to stimuli.
On a scale of a few nanometers the cell with very dynamic massive supramolecular
assemblies is largely an uncharted territory. Just as high-resolution 3D structures of
macromolecules provide valuable insights into their working, a better understanding
of cellular functions will arise from the ability to visualize supramolecular
architecture in an unperturbed cellular context. Our ultimate goal is to interpret the
structure and dynamics of the molecular networks. We are now working on new
methods to provide 3D maps of cellular components at molecular resolution. There is
currently immense excitement among us and our collaborators, since ongoing
advances in cryo-sectioning of native material at ultra low temperature as well as
cryo-EM instrumentation and computational methods may ultimately make it
possible to obtain resolutions in the range of 3 nm, i.e. potentially high enough to
locate individual proteins in a cell. To arrive at this technology, we are leading an
intense and international group of collaborators that include Helmut Gnaegi
(Diatome, Biel Switzerland), Felix de Haas (FEI electron optics, Eindhoven), Nico
Sommerdijk (University of Eindhoven), Henny Zandbergen (University of Delft),
Peter Frederik (University Maastricht) and Bram Koster (University Leiden).
Group leader Peter Peters
Peter Peters PhD Group leader
Bea Krenn PhD Project manager
Sue Godsave PhD Post-doc
Pekka Kujala PhD Post-doc
Pieter Res PhD Post-doc
Nicole Van der Wel PhD Post-doc
Diane Houben Graduate student
Jason Pierson Graduate student
Erik Bos MSc Technical staff
Hans Jansen BSc Technical staff
Martijn Romeijn BSc Technical staff
Maaike Van Zon BSc Technical staff
Nico Ong Research assistant
Prions
Prion diseases are fatal and presently incurable neurodegenerative diseases caused
by the abnormally folded form (PrPSc) of the cellular prion protein (PrPc).
In collaboration with Stanley Prusiner’s lab in San Francisco, we have been
developing methods to specifically detect PrPSc and PrPc, using cryo-immunogold
EM. Quantification of labeling with an antibody that recognizes only PrPc suggests
that the level and distribution of PrPc is relatively unchanged in prion-infected
hippocampus, while another antibody that recognizes both PrPc and (a subset of)
PrPSc gives increased labeling in the neuropil, both on the plasma membrane and in
early endocytic vesicles of axons and dendrites. Our results of last year support the
idea that PrPc to PrPSc conversion may occur at one of these sites. Synapses may be
an early target of prion disease and synapse numbers are reduced by more than fifty
per cent during the disease incubation period. Interestingly, we have not found any
evidence for PrPSc accumulation at synapses, although PrPC is present.
PrPSc has been reported to exist both in protease-sensitive and relatively insensitive
forms. We have found that a large proportion of the disease-associated labeling
detected in our experiments can be removed by trypsin. The significance of these
different PrPSc forms for disease is unknown, but it is likely that protease sensitive
PrPSc exists in lower aggregation states and it may be the most relevant for both
toxicity and infectivity.
Early stages of prion pathogenesis. In the earliest phase orally acquired PrPSc must cross
the gut epithelium to reach the germinal centres (GCs) of gut associated lymphoid
tissues. We show by microscopical techniques that PrPSc can be detected as a short
transient pulse in follicle associated epithelium (FAE) of Peyer’s patches already one
day after infection. Most PrPSc was found in large endosomes of FAE enterocytes,
but not in the M cells. After crossing the epithelia PrPSc was found accumulating
Figure VI.7: FAE endosomes visualized
by cryo immunogold electronmicroscopy.
Ultrathin cryosection was labeled against
lamp-1 and detected with Protein A
conjugated to 10 nm colloidal gold.
Bar 500 nm.
68
TUMOR BIOLOGY
Publications
Klingenstein R, Löber S, Kujala P,
Godsave S, Leliveld SR, Gmeiner P,
Peters PJ, Korth C. Tricyclic
antidepressants, quinacrine and a novel,
synthetic chimera thereof clear prions by
destabilizing detergent-resistant membrane
compartments. J Neurochem
2006; 98:748-759
Korth C, Peters PJ. Emerging
pharmacotherapies for Creutzfeldt-Jakob
disease. Arch Neurol 2006; 63:497-501
Figure VI.8: Follicle associated epithelium
(FAE) enterocytes have larger endosomes
compared to the enterocytes in the villus.
Dotted lines represent the approximate
location of brush borders both in the FAE
and in the villus. Endosomes of wt mouse
are labeled against late endosomal marker
lamp-1 and the nuclei with dapi.
intracellularly in large vacuoles of macrophage/dendritic type cells in the
subepithelial dome (SED) just underneath the FAE. In the course of infection some
of the DC/macrophages were later found in increasing numbers at the GCs.
In the few following days the PrP expression gradually increased at the surface of
follicular dendritic cells in the GCs of scrapie infected wild type mice, but not in the
non-infected controls, or infected PrPc–deficient mice. Interestingly, increase of PrP
expression on the FDCs was not limited to the GCs of Peyer’s patches, but was
simultaneously also observed in GCs of mesenteric lymph nodes, whereas in the
spleen no changes during the first 21 days post infection were observed. In the
Peyer’s patches finding of PrP together with traces of epithelial membrane marker
A33 suggests that the infectious agent makes it’s journey from gut lumen into the
GCs of Peyer’s patches at least partially membrane bound, probably in association
with membrane domains originating from the FAE. Macrophages/DCs appear to
be strong candidates for assisting the PrP transport from FAE to GC.
Mycobacteria In the last year we discovered that one of the most successful human
bacterial pathogens, Mycobacterium tuberculosis, has a different localization than the
vaccine strain M. bovis BCG used world wide to prevent tuberculosis infections and
bladder cancer. When BCG infects cells the bacteria remain localized in a membrane
enclosed compartment which the bacteria can modify in such a way that it can
survive and replicate. The current dogma is that M. tuberculosis localizes in a similar
compartment that is thought to be optimized for the persistence and replication of
the infectious bacteria. However we have recently established that at later in infection
the bacteria leave such a compartment to enter the cytosol of the host cell.
Here it has access to a nutritious environment in which replication appears to occur
at a higher rate than in the membrane enclosed compartments in which it resides at
the earlier phase of the infection. We have determined which proteins of the
pathogenic M. tuberculosis are facilitating the translocation (patented discovery).
Importantly M. bovis BCG lacks those proteins and consequently remains localized in
the cellular phagosome. This has implications for the recognition by the immune
system. The immune system loads parts of the bacteria (antigens) in specialized
antigen presenting molecules (MHC I, MHC II and CD1) that with the antigen can
trigger T-lymphocytes that then can activate an immune response. These antigenpresenting molecules load antigens in different compartments of a cell. MHC II and
CD1 load their antigens in membrane-enclosed compartments of the endocytic
pathway while MHC I obtains its antigens from the cytosol. M. bovis BCG is unable
to present its antigens via the cytosol as it remains restricted in a membrane-enclosed
compartment. M. tuberculosis however traffics through all the compartments where
loading occurs and thus can trigger immune responses via all classes of antigen
presentation.
In conclusion, we can now explain the discrepancy between the immune response
induced by M. bovis BCG and M. tuberculosis, which was for a long time one of the
most important questions in the field of vaccine development. This at the same time
may give us a tool to improve the BCG vaccine by redirecting the bacteria to the
cytosol. This will be tested with modified BCG strains.
POSTDOCTORAL AFFAIRS
Peter Peters has also been active as dean of postdoctoral affairs. For more details see:
http://www.mekentosj.com/postdocs. Our work and our institute was highlighted in:
Aschwanden C. Professionalizing the postdoctoral experience. Cell. 2006; 124:445-7
and Aschwanden C. Learning to lead. Cell. 2006; 125:407-9.
69
MOLECULAR GENETICS
VI I DI VISION OF MOL E CU LA R
GE NE T ICS
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 stable inherited 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
combinations of collaborating oncogenes and tumor-suppressor genes. Model
organisms comprise Mouse, Drosophila and Zebrafish.
Division head, Group leader Maarten Van Lohuizen
Maarten Van Lohuizen PhD Group leader
Anna Pavlina Haramis PhD Research associate
Elisabetta Citterio PhD Senior Post-doc
Jean Bernard Beaudry PhD Post-doc
Koen Braat PhD Post-doc
Jaap Kool PhD Post-doc
Karim Nacerdine PhD Post-doc
Functional characterization of Pc-G protein complexes Repressive Pc-G proteins and
counteracting Trithorax-group (Trx-G) of nucleosome remodeling factors are involved
in maintenance of proper gene expression patterns during development at the level
of chromatin structure. Pc-G protein complexes control large sets of genes including
Hox gene clusters and the INK4a/ARF tumor suppressor locus. At least two
biochemical distinct evolutionary highly conserved Pc-G protein complexes can be
distinguished. The first (PRC2) contains EzH1/EzH2 (SET domain proteins acting as
Histone H3 methylases), Su(z)12, Eed and histone deacetylases. The second large
complex (PRC1) encompasses Bmi1/Mel18, M33/MPc2, Mph1/Mph2 and Ring1b/
Ring1a together with other more loosely associated proteins is required throughout
development. To study Pc-G function we focus on representative members of PRC1
and PRC2 in gain- and loss-of-function studies in mice, zebrafish and Drosophila cell
lines. In genetic and biochemical experiments we recently identified the Bmi1/
Ring1b heterodimer as an E3 ubiquitin ligase for monoubiquitination of histone H2A
(collaboration with G Buchwald and T Sixma, Division II). 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 cell biological experiments encompassing live cell
microscopy, FRET and FLIP of tagged Pc-G proteins we revealed the highly dynamic
and cell cycle regulated nature of Pc-G proteins in sub-nuclear domains called
Polycomb bodies (collaboration with T Groothuis and J Neefjes, Division VI). In
addition, we demonstrated the requirement for the PRC2 EZH2 methyltransferase
and the DNA methylase DNMT1 to maintain PRC1 proteins properly localized. This
provides new insights in how Pc-G repression is propagated.
A major unresolved question is where and how Pc-G complexes bind to chromatin.
We recently completed 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 B Van Steensel, Division V). Combined with ChIP and unbiased
quantifiable gene onthology analyses this highlights binding of both PRC1 and PRC2
to distinct domains of 10-140 Kb containing В± 400 target genes. These comprise
developmental regulators, transcriptional repressors as well as key stem cell signaling
pathways such as Wnt, Hh, Notch as well as several new distinct functional categories
and provides unprecedented new insight in Pc-G function.
Alexandra Pietersen PhD Post-doc
Anke Sparmann PhD Post-doc
Bas Tolhuis PhD Post-doc
Bart Westerman PhD Post-doc
Sophia Bruggeman MSc Graduate student
Panthea Taghavi MSc Graduate student
Petra Van der Stoop MSc Graduate student
Yme Van der Velden MSc Graduate student
Marleen Blom Technical staff
Danielle Hulsman Technical staff
Sonja Noback Technical staff
Asheeta Prasad Technical staff
Ellen Tanger Technical staff
Hans Teunissen Technical staff
Els Verhoeven Technical staff
Joep Vissers Technical staff
Huub Van Vugt Technical staff
Publications
Bruggeman SWM, Van Lohuizen M.
Controling stem cell proliferation: CKIs at
work. Cell Cycle 2006; 5:1281-1285
Buchwald G, Van der Stoop P, Weichenrieder O,
Perrakis A, Van Lohuizen M, Sixma TK. Structure
and E3-ligase activity of the Ring-Ring complex of
Polycomb proteins Bmi1 and Ring1b. EMBO J
2006; 25:2465-2474
Fujimura YI, Isono KI, Vidal M, Endoh M,
Kajita H, Mizutani-Koseki Y, Takihara Y,
Van Lohuizen M, Otte A, Jenuwein T,
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 neurons in the cerebellum
of Bmi1 knockouts show proliferation defects. We demonstrated that these defects are
due to increased levels of the tumor suppressors p16INK4a and p19ARF, that in turn
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
Deschamps J, Koseki H. Distinct roles of
Polycomb group gene products in
transcriptionally repressed and active
domains of Hoxb8. Development 2006;
133:2371-2381
Haramis AP, Hurlstone A, Van der Velden Y,
Begthel H, Van den Born M, Offerhaus GJA,
Clevers HC. Adenomatous polyposis coli-deficient
zebrafish are susceptible to digestive tract neoplasia.
EMBO Rep 2006; 7:444-449
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MOLECULAR GENETICS
Publications (continued)
Haramis AP, Perrakis A. Selectivity and
promiscuity in Eph receptors. Structure
2006; 14:169-171
Hosokawa H, Kimura MY, Shinnakasu R,
Suzuki A, Miki T, Koseki H,
Van Lohuizen M, Yamashita M,
Nakayama T. Regulation of Th2 cell
development by Polycomb group gene bmi-1
through the stabilization of GATA3.
J Immunol 2006; 177:7656-7664
Oguro H, Iwama A, Morita Y, Kamijo T,
Van Lohuizen M, Nakauchi H. Differential
impact of Ink4a and Arf on hematopoietic
stem cells and their bone marrow
microenvironment in Bmi1-deficient mice.
J Exp Med 2006; 203:2247-2253
Pirrotta V, Van Lohuizen M. Differentiation
and gene regulation. Genomic programs
and differentiation. Curr Opin Genet Dev
2006; 16:443-446
Sparmann A, Van Lohuizen M. Polycomb
and stem/precursor cells. Our recent results highlight the relative importance of
p19ARF and p16INK4a in different cell types.
In addition, using the well-established mammary fat pad transplantation model, we
revealed the essential role of Bmi1 in mammary epithelial precursor cell proliferation
and ductal tree development, again at least in part implicating INK4a/ARF as a
critical target in this developmental cell system. 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 VII.1).
Interestingly, Bmi1 acts in this tumor setting in an Ink4a/ARF-independent manner
on cell adhesion and migration. These results, together with the recently established
role of Bmi1 in hemapoeitic 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 the overexpression of BMI1 in
various tumor types including non-small 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.
silencers control cell fate, development and
cancer. Nat Rev Cancer 2006; 6:846-856
Steele JC, Torr EE, Noakes KL, Kalk E,
Moss PA, Reynolds GM, Hubscher SG,
Van Lohuizen M, Adams DH, Young LS.
The polycomb group proteins, BMI-1 and
EZH2, are tumour-associated antigens.
Br J Cancer 2006; 95:1202-1211
Taghavi P, Van Lohuizen M.
Developmental biology: Two paths to silence
merge. Nature 2006; 439:794-795
Tolhuis B, Muijrers I, De Wit E,
Teunissen H, Talhout W, Van Steensel B,
Van Lohuizen M. Genome-wide profiling of
PRC1 and PRC2 Polycomb chromatin
binding in Drosophila melanogaster. Nat
In vivo and in vitro genetic screens to identify new groups of collaborating oncogenes
or tumor suppressors In close collaboration with A Berns (this Division), J Jonkers
(Division V) and A Bradley (The Sanger Center, 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. We have also developed in vitro suppressor screens, to screen for collaborating
sets of oncogenes and tumor suppressor genes. One such a screen is aimed at
identifying genes contributing to tumor-progression. Hereto, combinations of
predisposing oncogenes such as TBX2, Myc or RasV12 are introduced in MEFs or
conditionally immortalized human primary epithelial cells prior to transduction with
tumor-derived retroviral cDNA libraries or RNAi-hairpin pRETROSUPER libraries.
Transformed clones are subsequently selected in semi-solid medium, and the
relevant genes are isolated using efficient PCR strategies. So far we have isolated
several known and novel candidate oncogenes that cooperate with Myc, one novel
transcription factor that transforms in combination with RasV12 and two RNAi’s
directed at chromatin-regulating genes. The role and mechanism of action of these
new putative oncogenes or tumor suppressors, and their interference with adhesion
and invasion of primary mouse and human cells is under investigation.
Genet 2006; 38:694-699
The zebrafish as a new model to study cancer and development The zebrafish
provides a powerful animal model for genetic analysis of vertebrate embryogenesis,
organ development and disease and is recently beginning to be exploited as a cancer
model. Furthermore, its capacity for forward and reverse genetic analyses, provides a
unique opportunity to uncover novel insights into the molecular genetics of cancer.
We use LKB1-deficient zebrafish as a model to investigate if and how LKB1 function
may cause neoplastic transformation through disruption of epithelial polarization in
a vertebrate organism. In addition, through overexpression and knock-down or
knock-out methodologies we will investigate the roles of Polycomb genes during
embryonic development, stem cell maintenance and cancer in zebrafish.
Figure VII.1: 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 ortholopically transplanted in the forebrain of recipient mice.
71
MOLECULAR GENETICS
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,
expression of multiple oncogenes and tumor suppressor genes can be regulated in a
tissue-specific and spatial-temporal fashion. This permits both a more accurate
modeling of tumorigenesis as it occurs in man as well as the analysis of specific
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 depth.
Group leader Anton Berns
Anton Berns PhD Group leader
Paul Krimpenfort PhD Academic staff
Margriet Snoek PhD Academic staff
Jan Hermen Dannenberg PhD Research associate
Jacqueline Jacobs PhD Research associate
Joan Boren PhD Post-doc
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 lung cancers,
mesotheliomas and prostate cancer. To achieve (sporadic) activation of oncogenes
and inactivation of tumor suppressor genes we use Adeno-Cre virus to switch the
conditional alleles. Subsequently, tumor growth is monitored by noninvasive imaging
using a conditional luciferase reporter.
Joaquim Calbo PhD Post-doc
Hilda De Vries PhD Post-doc
Annemieke IJpenberg PhD Post-doc
Johan Jongsma PhD Post-doc
Martijn Nawijn PhD Post-doc
AndrГ© Bergman MD PhD KWF fellow
Kate Sutherland PhD Post-doc
Anthony Uren PhD Post-doc
Lung tumors We focus on small cell lung cancer (SCLC). 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. Additional small amplicons were
found and the genes in these amplicons are under study. We have established a series
of cell lines from these SCLC that show either neuroendocrine or stem cell like
features. Interestingly, these cell lines show common lesions indicating that they
share a common progenitor. The cell lines with neuro-endocrine features reproduce
SCLC upon orthotopic grafting whereas the cell lines with stem cell features do not.
We are following up several transcriptional regulators that are critical for the
induction for neuroendocrine differentiation. The cell lines appear relative resistant
to cytotoxic drugs. Therefore we are now investigating whether this is also the case
for primary tumor cells that have not been propagated extensively in vitro.
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 the mesothelial lining of the thoracic and
intraperitoneal cavity. Mesotheliomas rarely occurred upon Nf2 inactivation.
Concomitant loss of Ink4a/p19Arf or p53 strongly accelerated mesothelioma
development. We have derived luciferase expressing cell lines from these tumors,
which upon intrathoracic inoculation, reproduce mesotheliomas. We have
investigated whether the nature of the introduced defects (p53 or Ink4a/p19Arf loss)
results in a different resistance profile of the various cell lines. So far we found no
evidence for this. A substantial variation in drug resistance was found. Currently we
are investigating the basis of this.
RenГ©e Van Amerongen PhD Post-doc
Andrej Alendar MSc Graduate student
Jaco Van der Torre MSc Graduate student
Erwin Van Montfort MSc Graduate student
Colin Pritchard MSc Research assistant
Rahmen Bin Ali Technical staff
Ruben Gaasbeek Technical staff
Jules Gadiot Technical staff
Jan Paul Lambooij Technical staff
Natalie Proost Technical staff
Fina Van de AhГ© Technical staff
John Zevenhoven Technical staff
Figure VII.2: Dysfunctional telomeres
impact on DNA damage signaling.
Prostate cancer Overexpression fo Myc is observed in a subset of human prostate
tumors, and is associated with elevated expression levels of Pim kinases. Concurrent
overexpression of Pim1 and cMyc induces a uniform atypical hyperplasia in the
mouse prostate, indicating that Pim kinase activity is rate-limiting for the early stages
of Myc-driven transformation. We are currently testing the effect of Pim-deficiency
on Myc-driven prostate cancer.
Ink4 proteins We have shown previously that loss of Ink4a accelerates carcinogeninduced melanomas and that Ink4a deficiency synergizes with hemizygosity of p19Arf
and p53. We have now disrupted Ink4a, Ink4b and p19Arf together, a situation often
Figure VII.3: Some of requently found
common insertion sites in MuLV-induced
mouse lymphomas.
72
MOLECULAR GENETICS
Publications
Berns A. Cancer biology: Can less be more
for p53? Nature 2006; 443:153-154
Calbo J, Meuwissen R, Van Montfort E,
seen in human tumors. The resulting mice show no developmental abnormalities
but appear highly tumor prone. Mice show a preponderance of squamous cell
carcinomas and mesotheliomas, next to a low incidence of other tumors.
The dramatic tumor predisposition of loss of both Ink4a and Ink4b can most likely
be attributed to a backup function that Ink4b appears to fulfill for Ink4a.
Van Tellingen O, Berns A. GenotypePhenotype Relationships in a Mouse Model
for Human Small-Cell Lung Cancer. Cold
Spring Harb Symp Quant Biol 2005;
70:225-232
De Ridder J, Uren AG, Kool J,
Reinders MJT, Wessels LFA. Detecting
Statistically Significant Common Insertion
Sites in Retroviral Insertional Mutagenesis
Screens. PLoS Comput Biol 2006; 2:e166
Derksen PW, Liu X, Saridin F,
Van der Gulden H, Zevenhoven J, Evers B,
Van Beijnum JR, Griffioen AW, Vink J,
Krimpenfort P, Peterse JL, Cardiff RD,
Berns A, Jonkers J. Somatic inactivation of
E-cadherin and p53 in mice leads to
metastatic lobular mammary carcinoma
through induction of anoikis resistance and
angiogenesis. Cancer Cell 2006; 10:437-449
Huijbers IJ, Krimpenfort P, Chomez P,
Van der Valk MA, Song JY,
Inderberg-Suso EM,
Schmitt-Verhulst AM, Berns A,
Van den Eynde BJ. An inducible mouse
model of melanoma expressing a defined
tumor antigen. Cancer Res 2006;
66:3278-3286
Peeper D, Berns A. Cross-Species
Identification of new oncogenic pathways Proviral insertional mutagenesis
screens in normal and tumor prone mice marks genes instrumental in the
tumorigenesis. Using this approach a large range of oncogenes have been found.
In collaboration with the groups of Maarten Van Lohuizen, Lodewyk Wessels,
Jos Jonkers, and John Hilkens our current focus is saturating proviral insertional
mutagenesis in tumor suppressor knockout mice. With the help of the Sanger Centre
we have sequenced the insertion sites of approximately 1000 tumors. This yielded
close to 600 significant common insertion sites that mark oncogenes and tumor
suppressor genes. Bioinformatical analysis identified a large number of new
potentially haploinsufficient tumor suppressor genes and a vast number of specific
co-mutations suggesting that there are strict requirements for the activation of
cooperating pathways. In addition we continue to study two gene families that were
found in previous screens: Pim and Frat.
Pim family kinases are highly expressed in strong collaboration with Myc in a range
of human malignancies, including prostate carcinoma and lymphomas. Germline
deletion of Pim kinases induces a minimal phenotype, making these kinases
potentially suitable for therapeutic intervention. The redundancy of Pim kinases and
other intracellular signaling pathways in the context of Myc-driven transformation is
being analyzed in both lymphoid and epithelial cell context. In addition, we are
characterizing one of the novel genes with putative remodeling function that was
found in a complementation tagging experiment in Pim1/2 knockout mice.
FRAT proteins (FRAT1-3) bind to and inhibit the GSK3b kinase and have therefore
been implied as critical mediators of canonical Wnt signaling, resulting in the
activation of b-catenin. Indeed, overexpression of FRAT proteins induces b-catenin/
TCF signaling, with FRAT1 and 3 being more potent inducers than FRAT2.
Remarkably, mice, lacking all three frat genes are healthy and fertile and do not
display gross abnormalities. In line with this, Wnt signaling in primary cells of these
tumors appears unaffected. Therefore, Frat is dispensable for mammalian
development and not essential for canonical Wnt-signaling in higher organisms.
We are currently putting emphasis on the role of the FRAT proteins in non-canonical
Wnt signaling, in which the three Frat genes show different activities.
Oncogenomics in Cancer Gene
Identification. Cell 2006; 125:1230-1233
Shakhova O, Leung C, Van Montfort E,
Berns A, Marino S. Lack of Rb and p53
delays cerebellar development and
predisposes to large cell anaplastic
medulloblastoma through amplification of
N-Myc and Ptch2. Cancer Res 2006;
66:5190-5200
Telomeres Loss of telomere function triggers a DNA damage response that results in
apoptosis or growth arrest that depends on both p53 and p16INK4a. In addition,
dysfunctional telomeres result in genome instability due to DNA repair activities that
act on chromosome ends. We are interested in what cellular activities act on
chromosome ends, how telomeres control or protect against these activities and what
the precise nature of the telomere damage signal is. To investigate this we take both a
candidate approach as well as a genome-wide screening approach using cDNA and
RNAi libraries to identify and functionally characterize novel genes with a role in the
telomere damage response. Central in these approaches is the disruption of telomere
function by inhibiting the telomere binding protein TRF2.
Van Amerongen R, Berns A. Knockout
mouse models to study Wnt signal
transduction. Trends Genet 2006;
22:678-689
Histone deacetylases The identification of drug targets whose inhibition will result
in selective killing of tumor cells is highly desirable in new anti-cancer therapies.
Histone deacetylases (HDACs) seem to fulfill the criteria for such drug targets.
Despite promising results obtained in clinical trials using HDAC-inhibitors (HDACi)
many of these drugs are targeting several HDACs and show off-target effects
resulting in undesired toxicities. Using (tumorprone) mice carrying HDAC1 and
HDAC2 conditional knockout alleles we wish to discover anti-tumor relevant HDAC
targets and study HDAC1/2 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.
73
MOLECULAR GENETICS
CELLULAR PROTECTION AGAINST ONCOGENIC
TRANSFORMATION AND METASTASIS
Introduction 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 genome-wide libraries), we have been focusing on two of these events:
bypass of oncogene-induced growth arrest (�premature senescence’, which may limit
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 potential 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.
Group leader Daniel Peeper
Daniel Peeper PhD Group leader
Christophe Desmet PhD Post-doc
Cornelia Hömig PhD Post-doc
Alexandre Prieur PhD Post-doc
Remco Van Doorn MD PhD Clinical fellow
Thomas Geiger MSc Graduate student
Thomas Kuilman MSc Graduate student
Chrysiis Michaloglou BSc Graduate student
Marjon Smit MSc Graduate student
Function-based genomic screens for metastasis genes Metastasis commonly
underlies the malignancy of cancers, representing the principal cause of cancertreatment failure. Tumor colonization is prevented by several physiologic barriers,
including �anoikis’: apoptosis resulting from lack of adhesion. Indeed, acquiring
resistance to anoikis has been proposed to represent a general prerequisite for
survival of metastases during circulation. In an attempt to identify metastasisassociated oncogenes we designed an unbiased, functional genome-wide screen
solely based on anoikis suppression. With this approach, we previously identified
TrkB, a neurotrophic tyrosine kinase receptor, as a potent suppressor of anoikis.
Whereas non-malignant intestinal epithelial cells underwent caspase-associated
anoikis in vitro, this was completely prevented by TrkB, allowing formation of
spheroid aggregates growing in suspension. Mice readily cleared parental cells, but
TrkB-expressing cells formed many lung and heart metastases. Co-expression of
BDNF, the prototypic TrkB ligand, caused highly invasive metastases in virtually
every tissue, with an extremely short latency.
We and others recently identified cancer-associated mutations of TrkB of which we
are currently analyzing the biological significance. We have also begun to identify
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 also performed
a structure-function analysis of TrkB, identifying specific domains involved in anoikis
suppression and metastasis. Finally, we are setting up 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.
Function-based genomic screens to identify novel oncogenes In approximately
30% of all human tumors, the RAS GTPase is mutated at a specific residue
(e.g., G12V), as a result of which it is locked into a constitutively active state.
Surprisingly, rather than proliferating indefinitely upon overexpression of RASV12,
non-malignant cells undergo proliferative arrest. RASV12-induced arrest resembles
�senescence’, which may act as a tumor-suppressing mechanism that protects cells
against oncogenic transformation. We have designed functional genomic screens to
identify novel oncogenes that allow cells to bypass RASV12-induced senescence,
as well as to identify oncogenes that cooperate with RASV12 to achieve full
transformation. This successful approach has led to the independent identification of
a number of new oncogenes, some of which are discussed below.
One of the genes we identified in this manner encodes DRIL1, a transcription factor
of the ARID-domain family of retinoblastoma protein-interacting proteins. We have
observed that DRIL1 immortalizes primary murine fibroblasts, rendering them
unresponsive to by p19ARF, p53, p21CIP1 and p16INK4a. Furthermore, we have observed
that, in contrast to what is seen in mouse cells, DRIL1 induces cell cycle arrest in
primary human fibroblasts. We have carried out a structure-function analysis,
Liesbeth Vredeveld MSc Graduate student
Sirith Douma MSc Technical staff
Figure VII.4: A schematic representation of
structure-function TrkB mutants. Several
mutations were introduced into TrkB, which
were examined for their effects on anoikis
suppression in vitro and tumor formation in
vivo.
74
MOLECULAR GENETICS
Publications
Desmet CJ, Peeper DS. The neurotrophic
receptor TrkB: a drug target in anti-cancer
therapy? Cell Mol Life Sci 2006; 63:755-759
Mooi WJ, Peeper DS. Oncogene-induced
cell senescence - Halting on the road to
cancer. N Engl J Med 2006; 355:1037-1046
Peeper DS, Berns AJ. Cross-Species
Oncogenomics in Cancer Gene
Identification. Cell 2006; 125:1230-1233
Rowland BD, Peeper DS. KLF4, p21 and
context-dependent opposing forces in cancer.
Nat Rev Cancer 2006; 6:11-23
Figure VII.5 : Genome-wide identification
of novel tumor suppressor genes. Primary
human cells were transduced with retrovirus
encoding the mutant, melanoma-associated
BRAFE600 gene. Subsequent changes in the
expression pattern of cellular genes were
used to identify genes antagonizing the
growth-stimulatory action of BRAFE600.
This led to the identification of the
interleukin IL-6, which was upregulated by
BRAFE600. RNA interference of IL-6
abrogated BRAFE600-induced senescence,
causing cells to proliferate.
identifying the domains in DRIL1 that contribute to growth arrest and oncogenic
transformation.
We have also identified a set of cell cycle proteins that are associated with DRIL1induced arrest in human cells.
Oncogene-induced senescence in melanocytic nevi as a melanomasuppressing 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 OncogeneInduced Senescence (OIS) represents a physiological process has long been debated.
Human nevi (�moles’), benign tumours 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).
This raises the question whether nevi undergo BRAFV600E-induced senescence.
In a collaborative study with Drs. Mooi (VUmc, Amsterdam), Soengas (Michigan)
and Shay (Texas), we found that sustained BRAFV600E expression in human
melanocytes induces cell cycle arrest, which is accompanied by the induction of
p16INK4a as well as of a commonly used senescence marker, Senescence-Associated
acidic b-Galactosidase (SA-b-Gal) activity. Validating these results in vivo, congenital
nevi are invariably SA-b-Gal-positive, showing for the first time this classical
senescence-associated marker in a - largely growth-arrested - neoplastic human
lesion. In growth-arrested melanocytes, both in vitro and in situ, we observe a striking
mosaic induction of p16INK4a, suggesting that factors other than p16INK4a also
contribute to protection against BRAFE600-driven proliferation. Finally, nevi do not
appear to suffer from telomere attrition, arguing in favor of an active oncogenedriven senescent process, rather than a loss of replicative potential. Thus, both in
vitro and in vivo, BRAFV600E-expressing melanocytes display classical hallmarks of
senescence, suggesting that OIS represents a genuine protective physiological
process.
Currently, we are using a functional genomic approach to identify novel, p16INK4A
independent, melanocyte-specific pathways whose deregulations contribute to
melanoma. In addition to taking a candidate gene approach, we are combining gene
expression analysis, RNAi and unbiased functional �barcode’ screens. For example,
we postulated that putative tumor suppressors, similar to p16INK4A, are
transcriptionally upregulated during the senescence response. Microarray expression
analysis revealed the induction of various signaling networks that are induced
specifically in BRAFE600-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 BRAFE600-expressing
cells. Similar findings were made for other interleukins, which prompted us to
search for a common denominator of BRAFE600-regulated interleukins. We found
that C/EBPb acts as a major player in this pathway. Consistently, knockdown of
C/EBPb effectively bypassed BRAFE600-induced senescence. Our data suggest that
the C/EBPb-IL6 axis acts as a critical mediator of BRAFE600-induced senescence.
This integrative genomics approach may identify novel tumor suppressors involved
in melanoma and other (BRAFE600-associated) cancers.
75
MOLECULAR GENETICS
GENES INVOLVED IN BREAST CANCER PROGRESSION AND
METASTASIS
Transformation of a normal cell into a fully malignant cell requires several sequential
genetic changes affecting key genes controlling various essential cellular pathways
involved in growth and development. This multistep process of gradual increase in
malignancy is called tumor progression. Complete knowledge of the genes and the
pathways they control is essential for the development of more effective novel
therapeutic strategies. Although a large number of genes that are involved in breast
and other cancers has been discovered, the picture is far from complete. Retroviral
insertional mutagenesis (IM) in mouse models is one of the most efficient tools to
uncover cancer genes. 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 insertional mutagenesis in mouse
models.
Group leader John Hilkens
John Hilkens PhD Group leader
Vassiliki Theodorou MSc Graduate student
Annabel Zwaagstra MSc Graduate student
Mandy Boer Technical staff
Publications
Identification of novel genes involved in mammary tumor progression by
MMTV tagging Mouse mammary tumor virus (MMTV) proviral insertion in the
genomic DNA of murine mammary epithelial cells can activate flanking protooncogenes leading to mammary tumor induction by insertional mutagenesis. The
availability of the almost complete mouse genome sequence and high-throughput
PCR and sequencing technologies has greatly facilitated the identification of
retrovirally-activated oncogenes. By using these technologies, we have searched for
common MMTV integration sites (CIS) in primary tumors of MMTV infected
BALB/c mice and a BALB/c knockout strain that conditionally lacks active p53 in the
mammary gland. A CIS indicates the presence of a nearby oncogene involved in
mammary tumorigenesis.
We have performed a high-throughput retroviral insertional mutagenesis screen in
more than 300 MMTV-induced mammary tumors that developed in both mouse
strains and identified over 50 CIS. In wild type Balb/c mice we identified 33 CIS and
in the p53 deficient mice we found an additional 10 CIS, while combining both
studies led to a further 9 CIS. Although Wnts and Fgfs were frequently affected by
proviral insertions, almost all novel genes are not members of these families.
Approximately 50% of the tagged genes are novel candidate cancer genes not
previously implicated in mammary or other cancers. Interestingly, a comparison of
all our MMTV tagged genes in mammary tumors (including those tagged in only a
single tumor) with all genes tagged by MoMuLV in lymphomas and brain tumors
deposited in the Mouse Retrovirus Tagged Cancer Gene Database showed that both
viruses targeted mostly different genes acting predominantly in distinct pathways.
Indeed, our screen for MMTV common insertion sites reveals a novel repertoire of
candidate oncogenes involved in mammary tumorigenesis.
Rspo-genes and cancer Two genes, Rspo2 and Rspo3, that belong to a novel gene
family, designated R-spondins, were among the most frequently tagged genes, after
the Wnt and Fgf genes, in the mammary tumors. R-spondins belong to a highly
conserved four member gene family. All four Rspo family members encode secreted
molecules with a single thrombospondin type 1 and two furin domains, but the
function of these molecules has not been established. RT-PCR analysis revealed that
approximately 25% of all MMTV induced mammary tumors in BALB/c mice
expressed one of the two former genes. Both genes were also frequently activated in
human breast cancers. Rspo1 and Rspo2 are not expressed in normal resting
mammary gland cells, in contrast Rspo1 is widely expressed in normal tissues
including normal mammary cells and mammary tumors. In murine tumors Rspo2 or
Rspo3 were often co-expressed with Rspo1. Moreover, Rspo2 or 3 were frequently
activated in the same tumors that also overexpressed Wnt and Fgf genes suggesting
that the Rspo genes collaborate with these oncogenes. Preliminary results show that
immortalized normal mouse mammary epithelial cells (HC-11 cells) retrovirally
transduced with Rspo3 cDNA were tumorigenic in nude mice (Figure VII.6),
validating Rspo3 as a novel oncogene.
Hilkens J. Recent translational research:
oncogene discovery by insertional
mutagenesis gets a new boost. Breast Cancer
Res 2006; 8:102-5
Yu LG, Andrews N, Zhao Q, McKean D,
Williams JF, Connor LJ, Gerasimenko OV,
Hilkens J, Hirabayashi J, Kasai K,
Rhodes JM. Galectin-3 Interaction with
Thomsen-Friedenreich Disaccharide on
Cancer-associated MUC1 Causes Increased
Cancer Cell Endothelial Adhesion. J Biol
Chem 2007; 282:773-781
Figure VII.6: HC11 cells (derived from
normal mammary epithelium) retrovirally
transduced with Rspo3 or Wnt1 cDNA or
an empty retroviral construct (designated
HC-Rspo3, HC-Wnt1 and HC-zeo
respectively) were subcutaneously injected
into the flanks of 5 to 7 BALB/c nu/nu mice
per test group. The average latency time
until the tumors reached a size of 1 cm3
(end point) and the standard deviation are
indicated. Rspo3 expression rendered the
HC11 cells highly tumorigenic as compared
to the cells transduced with the empty vector,
however, less tumorigenic than Wnt1
transduced HC11 cells.
76
EXPERIMENTAL THERAPY
VIII D IV IS ION OF
EXP ER IM EN TA L THER A PY
GENES AND PROTEINS INVOLVED IN ANTICANCER DRUG
RESISTANCE AND PHARMACOKINETICS
Division head, Group leader Alfred Schinkel
Alfred Schinkel PhD Group leader
Christian Zimmermann PhD Post-doc
Jurjen Lagas MSc Graduate student
Zeliha Pala MSc Graduate student
Evita Van de Steeg MSc Graduate student
Antonius Van Herwaarden MSc Graduate student
Robert Van Waterschoot MSc Graduate student
Our research focuses on genes and proteins that cause drug resistance 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 approaches for cancer and HIV/AIDS, as well as
pharmacotherapy in a broader sense; ii) increase insights in 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.
Marijn Vlaming MSc Graduate student
Corina Van der Kruijssen MSc Technical staff
Anita Van Esch Technical staff
Els Wagenaar Technical staff
Figure VIII.1: Putative structure of a
prototypic ABC drug transporter.
Figure VIII.2: Hepatobiliary excretion of
Impact of active drug transporters We have a long-standing interest in plasma
membrane proteins of the ATP binding cassette (ABC) transporter family, including
P-glycoprotein (P-gp, ABCB1), MRP2 (ABCC2) and BCRP (ABCG2) (Figure VIII.1).
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 initiated generation of Mrp2 knockout
mice, which are crossed with the existing P-gp and Bcrp1 knockout mice in order
to elucidate the separate and combined contributions of these transporters to
pharmacological, toxicological and physiological functions.
Generation and analysis of Mrp2 knockout mice The ABC transporter and
multidrug resistance protein MRP2 (ABCC2) forms a natural barrier and efflux
system for numerous (conjugates of) drugs, other xenotoxins and many endogenous
compounds. We generated Mrp2 knockout mice, which were viable and fertile but
suffered from mild hyperbilirubinemia due to impaired excretion of bilirubin
monoglucuronides into bile. These mice also had an 80-fold decreased biliary
glutathione excretion and a 63% reduced bile flow. Levels of the multidrug
transporters Mrp3 (Abcc3) in liver and Mrp4 (Abcc4) in kidney of Mrp2-/- mice were
about 2-fold increased. 30 min after oral administration of the food-derived
carcinogens PhIP and IQ, plasma values were 1.9-fold and 1.7-fold higher in Mrp2
knockout mice vs. wild-type mice respectively, demonstrating the role of Mrp2 in
restricting exposure to these compounds. At a high dose of 50 mg/kg of the anticancer drug methotrexate, the plasma exposure after intravenous administration was
1.8-fold higher in Mrp2 knockout mice. In contrast, no clear plasma concentration
difference arose at low dose (1 mg/kg), suggesting that Mrp2 primarily affects
methotrexate (MTX) pharmacokinetics at high plasma concentrations. Using an
Mrp2/P-gp compound knockout mouse, we could further show that hepatobiliary
excretion of doxorubicin is mediated by both P-gp and Mrp2 (Figure VIII.2). The
Mrp2 knockout mouse thus provides a valuable tool for studies of the impact of Mrp2
on behavior of drugs, carcinogens and other xenotoxins, and on liver physiology.
Combination with other transporter knockout mice will further allow systematic
analysis of a plethora of research questions in these areas.
doxorubicin in wild-type (fvb), Mrp2,
Mdr1a/1b, and Mdr1a/1b/Mrp2 knockout
mice. Excretion during 60 min after
intravenous administration of 5 mg/kg
doxorubicin was measured.
Studies of paclitaxel in Mrp2/P-gp compound knockout mice The widely used
anticancer drug paclitaxel is an excellent substrate for P-glycoprotein. We recently
demonstrated that also MRP2 transports paclitaxel and docetaxel in vitro. To
investigate whether MRP2 affects the pharmacokinetics of paclitaxel, we used Mrp2,
77
EXPERIMENTAL THERAPY
Publications
Mdr1a/1b, and Mrp2/Mdr1a/1b combination knockout mice. Disruption of the Mrp2
gene had a clear impact on paclitaxel plasma levels after intravenous administration,
which was quantitatively similar to absence of P-glycoprotein. The area under the
plasma concentration-time curve (AUC) was 1.3-fold higher in both Mrp2 and
Mdr1a/1b knockout mice and 1.7-fold higher in Mdr1a/1b/Mrp2 knockout mice, when
compared to wild-type mice. In contrast to P-glycoprotein, Mrp2 had no significant
role in limiting intestinal absorption of paclitaxel. In Mrp2 knockout mice the oral
AUC was not altered compared to wild-type mice, whereas in Mdr1a/1b knockout
mice a 8.5-fold increase was found, the latter consistent with our previous results.
Interestingly, however, in Mdr1a/1b/Mrp2 knockout mice the oral AUC was 14.2-fold
increased compared to wild-type and 1.7-fold compared to Mdr1a/1b knockout mice.
The impact of Mrp2 on plasma pharmacokinetics could be explained by our finding
that Mrp2 dominates the hepatobiliary elimination of paclitaxel and its principal
metabolites. In Mrp2 knockout mice the hepatobiliary excretion of paclitaxel was
reduced by 80%, whereas excretion of its monohydroxylated metabolites was almost
totally abolished. Therefore, disruption of Mrp2 has a marked impact on paclitaxel
plasma levels after intravenous administration and on plasma levels after oral
administration when absorption is not limited by P-glycoprotein. Our findings may
be of importance for pharmacotherapy, as in current clinical practice taxanes are
administered by intravenous infusion. As a consequence, mutational alterations of
the MRP2 gene in patients may result in increased plasma levels of paclitaxel and
thereby unexpected toxicity.
Kummer W, Wiegand S, Akinci S,
Wessler I, Schinkel AH, Wess J,
Koepsell H, Haberberger RV, Lips KS.
Role of acetylcholine and polyspecific
cation transporters in serotonin-induced
bronchoconstriction in the mouse.
Respir Res 2006; 7:65
Lagas JS, Vlaming ML, Van Tellingen O,
Wagenaar E, Jansen RS, Rosing H,
Beijnen JH, Schinkel AH. Multidrug
resistance protein 2 is an important
determinant of paclitaxel pharmacokinetics.
Clin Cancer Res 2006; 12:6125-6132
Merino G, lvarez AI, Pulido MM,
Molina AJ, Schinkel AH, Prieto JG. Breast
cancer resistance protein (BCRP/ABCG2)
transports fluoroquinolone antibiotics
and affects their oral availability,
pharmacokinetics, and milk secretion.
Drug Metab Dispos 2006; 34:690-695
Bcrp1, P-gp and Mrp2 compound knockout mice We are currently in the process
of generating compound knockout mice for Bcrp1, P-gp and Mrp2. We expect that
these mice will yield very useful tools for studying the separate and combined impact
of these ABC transporters on shared drug and carcinogen substrates. Moreover, both
Bcrp1 and P-gp appear to be expressed in a range of tissue stem cells, and transport
overlapping carcinogenic substrates. Exposure of these compound knockout mice to
such carcinogens may elucidate the impact of the ABC transporters in the protection
against dietary and environmental carcinogens. Finally, we may be able to elucidate
additional physiological functions of these transporters, as some may be obscured by
partial redundancy in the single knockout mouse strains.
Ogasawara M, Yamauchi K, Satoh YI,
Yamaji R, Inui K, Jonker JW, Schinkel AH,
Maeyama K. Recent advances in molecular
pharmacology of the histamine systems:
Organic cation transporters as a histamine
transporter and histamine metabolism.
J Pharmacol Sci 2006; 101:24-30
Van Herwaarden AE, Wagenaar E,
Karnekamp B, Merino G, Jonker JW,
CYP3A transgenic and knockout mice The cytochrome P450 3A (CYP3A) complex
is a major factor in the metabolic breakdown of most drugs used today, and of
various endogenous substrates, but also for the activation and inactivation of many
(pre-)carcinogens. As CYP3A activity shows great 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 study this important system and its
interactions with drug transporters in well-defined models, we are generating CYP3A
transgenic and knockout mice. We have obtained CYP3A4 transgenic mice with
substantial, physiologically relevant and stable CYP3A4 levels in liver, but not in
small intestine. These transgenic mice displayed clearly accelerated kinetics of the
widely used drugs midazolam and cyclosporin A. We further recently obtained mice
with substantial expression of CYP3A4 in the intestine. These mice, together with
additional strains currently under construction, will allow highly informative studies
on drug behavior and (pre)carcinogen susceptibility.
Schinkel AH. Breast cancer resistance
protein (Bcrp1/Abcg2) reduces systemic
exposure of the dietary carcinogens aflatoxin
B1, IQ and Trp-P-1 but also mediates their
secretion into breast milk. Carcinogenesis
2006; 27:123-130
Van Herwaarden AE, Schinkel AH. The
function of breast cancer resistance protein
in epithelial barriers, stem cells and milk
secretion of drugs and xenotoxins. Trends
Pharmacol Sci 2006; 27:10-16
Vlaming MLH, Mohrmann K,
Wagenaar E, De Waart DR,
Oude Elferink RPJ, Lagas JS,
Van Tellingen O, Vainchtein LD,
Rosing H, Beijnen JH, Schellens JHM,
Schinkel AH. Carcinogen and anticancer
drug transport by Mrp2 in vivo: Studies
using Mrp2 (Abcc2) knockout mice.
J Pharmacol Exp Ther 2006; 318:319-327
78
EXPERIMENTAL THERAPY
MECHANISMS AND PREDICTION OF TUMOR RESPONSE TO
RADIATION
Group leader Adrian Begg
We have continued to pursue two lines of research, one aiming at clinical translation
and the other on fundamental aspects of the radiation response. The first line focuses
on expression microarrays, tissue microarrays and cisplatin adducts to predict
therapy outcome. The second involves how the cell handles DNA base damage and
single strand breaks, an aspect of radiation damage that may well provide novel
targets for radiotherapy for cancer.
Adrian Begg PhD Group leader
Frank Hoebers MD Radiation Oncologist
Conchita Vens PhD Post-doc
Sari Neijenhuis MSc Graduate student
Jimmy Pramana MD Graduate student
Christie Vermeulen MSc Graduate student
Ben Floot Technical staff
Ingrid Hofland Technical staff
Manon Verwijs Technical staff
Figure VIII.3: Prediction of outcome after
RadPlat (combined radiotherapy and
cisplatin-based chemotherapy) by gene
expression profiling in advanced head and
neck cancer. A 7-gene profile, found by
supervised hierarchical clustering, correlates
with loco-regional recurrence probability in
the training series (top) and in a separate
validation series (bottom).
Prediction of outcome Microarrays: We are measuring expression profiles of head
and neck cancers prior to treatment, to search for genes which predict outcome in
patients treated with concurrent radiotherapy cisplatin. (collaboration with the NKI
departments of Head and Neck Surgery, Radiotherapy and Pathology). We have now
completed a study on 92 tumors which satisfied the criteria of >50% tumor cells in
the biopsy, high RNA quality and sufficient follow-up time. Amplified and labeled
RNA was hybridized to 35k oligo arrays from our Central Microarray facility. Patients
were split into training and validation series. Supervised hierarchical clustering using
a nearest centroid algorithm produced genes sets significantly associated with
outcome for the endpoints of local control, locoregional control and disease free
survival. These signatures contained 31, 8, and 2 genes, respectively. In the validation
series, only the locoregional control signature remained significantly predictive
(Figure VIII.3). Gene Set Enrichment Analysis (GSEA) on 311 gene sets showed some
were associated with recurrence, but false discovery rates were high. Of interest was
that genes and gene sets correlating with locoregional recurrence were not found in
those correlating with distant metastases, implying locoregional recurrence
signatures predict treatment sensitivity (predictive) rather than being a general
monitor of malignancy (prognostic). Three tissue microarrays have been made from
this patient material, comprising more than 200 patients. To date, these have been
stained with antibodies for 8 candidate markers; 12 more are planned before analysis.
Some markers were chosen from the expression microarray study.
Cisplatin adducts: We are continuing to look at the predictive value of cisplatin-DNA
adducts in buccal cells of patients taken early in their treatment course. We have
applied an immunofluorescence technique developed by us to a series of 85 cervix
cancer patients in a Gynaecology and Oncology Group (GOG) trial of cisplatin plus
concurrent radiotherapy. Pre- and post-treatment samples were obtained during the
first 2 courses of chemotherapy. In the 53 patients having sufficient data from both
first and second courses, high buccal cell adducts were significantly associated with
a worse progression free survival. This is in the opposite direction to that found in
previous lung cancer studies, and not consistent with the notion that buccal cell
adducts are a monitor of integral cisplatin exposure, which should be reflected in
tumor response. Further analyses are continuing.
Mechanisms and modulation of radiosensitivity DNA polymerase beta is known
to play a central role in base excision repair (BER) and single strand break repair
(SSBR). We previously showed that expression of a dominant negative to DNA
polymerase beta (polbetaDN) resulted in radiosensitization of human tumor cell
lines, and also radiosensitizes polbeta-deficient cells, implying interference with
polbeta-independent pathways. In contrast, new data show that expression of the DN
in polbeta deficient cells protects against the alkylating agent MMS. This is consistent
with a dRP lyase activity of the DN, which removes potentially toxic intermediates.
This further implies that removal of dRP groups are not important for ionizing
radiation.
We have now also shown that the polbetaDN does not sensitize cells to H2O2, an
agent producing oxidative base damage and SSB. This indicates that the DN is
a specific sensitizer for ionizing radiation, and further suggests an involvement
in repair of clustered damage, which occurs after radiation but not after H2O2.
We further investigated polbeta’s role by studying polbeta deficient cells. We
previously showed that the short patch polymerase inhibitor methoxyamine (MX)
sensitized only polbeta deficient cells to ionizing radiation. We now show that MX
sensitizes both wildtype and beta deficient cells to H2O2. These and other data
79
EXPERIMENTAL THERAPY
Publications
indicate polbeta is critical for alkylating damage, but not for H2O2, and that both
polbeta and a backup polymerase must act together in the response to ionizing
radiation.
We have shown that confluent cells, in contrast to log phase cells, are hypersensitive
when lacking polbeta. To study the replication dependence of this effect, we irradiated
cells in different cell cycle phases using cells synchronized by mitotic shake-off. DNA
polymerase b deficient cells were found to be more sensitive to ionizing radiation
than wild-type cells in G1, while being equally sensitive after progressing into S.
These data were confirmed using a polbeta-deficient line complemented with
polbeta. This indicates that there is an important S-phase specific repair pathway
after ionizing radiation, which is independent of polbeta, and also confirms polbeta’s
role in the radiation response in G0/G1 cells.
To further characterize possible backup pathways, we are studying two candidate
polymerases, iota and lambda, shown to participate in BER reactions in vitro. Studies
on using both knockout and knock-down strategies for these two polymerases are
continuing. Finally, we are investigating a third translesion synthesis polymerase,
DNA polymerase kappa. So far, we have shown that kappa knockout cells are
sensitive to cisplatin, and we are now investigating its role in ionizing radiation, in
both oxic and hypoxic cells.
A project is continuing to study the role of apoptosis and senescence in a sporadic
tumor model in mice (collaboration with Verheij, Jongsma, Berns; NKI). Experiments
with the original lung tumor model (cre-adenovirus instillation activating oncogenic
K-ras) have stopped for technical reasons, and re-started with a mesothelioma model
with higher tumor incidence and shorter latencies (Nf2-induced tumors, with or
without p53 and/or ink4aARF deficiencies). To date, cells from tumors from each
genotype have shown little radiosensitivity differences in culture. Experiments on
further tumor clones in culture and in animals are underway.
Begg AC. Can the severity of normal tissue
damage after radiation therapy be
predicted? PLoS Medicine 2006; 3:1713-1714
De Schutter H, BarbГ© B, Spaepen M,
Begg AC, Balm A, Gregoire V,
Haustermans K, Mahy P, Poorten VV,
Nuyts S. Microsatellite alterations in head
and neck squamous cell carcinoma and
relation to expression of pimonidazole, CA
IX and GLUT-1. Radiother Oncol 2006;
80:143-150
Goethals L, Debucquoy A, Perneel C,
Geboes K, Ectors N, De Schutter H,
Penninckx F, McBride WH, Begg AC,
Haustermans KM. Hypoxia in human
colorectal adenocarcinoma: Comparison
between extrinsic and potential intrinsic
hypoxia markers. Int J Radiat Oncol Biol
Phys 2006; 65:246-254
Hoebers FJP, Pluim D, Verheij M,
Balm AJM, Bartelink H, Schellens JHM,
Begg AC. Prediction of treatment outcome
by cisplatin-DNA adduct formation in
patients with stage III/IV head and neck
squamous cell carcinoma, treated by
concurrent cisplatin-radiation
(RADPLAT). Int J Cancer 2006;
119:750-756
Sprong D, Janssen HL, Vens C, Begg AC.
Resistance of hypoxic cells to ionizing
radiation is influenced by homologous
recombination status. Int J Radiat Oncol
Biol Phys 2006; 64:562-572
Vermeulen C, Verwijs-Janssen M,
Cramers P, Begg AC, Vens C. Role for
DNA polymerase beta in response to
ionizing radiation. DNA Repair 2007;
6:202-212
Vink SR, Lagerwerf S, Mesman E,
Schellens JHM, Begg AC,
Van Blitterswijk WJ, Verheij M.
Radiosensitization of squamous cell
carcinoma by the alkylphospholipid
perifosine in cell culture and xenografts.
Clin Cancer Res 2006; 12:1615-1622
80
EXPERIMENTAL THERAPY
VASCULAR MEDIATED TISSUE DAMAGE AND THERAPY
Group leader Fiona Stewart
Fiona Stewart PhD Group leader
Improvements in detection and treatment of cancer have lead to increased numbers
of long-term survivors at risk for developing treatment related morbidity. Vascular
injury is the major cause of late radiation morbidity and may seriously impair the
quality of life, or even be life threatening, to cancer patients treated with radiotherapy.
In large vessels this manifests as atherosclerosis, whereas in small vessels it
manifests as telangiectasia. The aim of our studies is to investigate mechanisms
underlying the development and progression of radiation-induced vascular damage
with a view to identifying intervention strategies.
Paul Baas MD PhD Academic staff
Nicola Russell MD PhD Academic staff
Jacqueline Kruse PhD Senior Post-doc
Saske Hoving PhD Post-doc
Martijn Triesscheijn MSc Graduate student
Ben Floot Technical staff
Marjan Ruevekamp Technical staff
Debbie Sprong Technical staff
Johannes Te Poele Technical staff
Figure VIII.4: A: Lawson staining of
irradiated H&N artery (left) and the donor
artery of the same patient (right). B: IMT of
unirradiated and irradiated H&N arteries
corrected for the IMT of the donor arteries
(* p<0.05).
Figure VIII.5: The TGFГџ receptor kinase
inhibitor SB431542 significantly inhibits
radiation-mediated (and TGFГџ induced)
increases in PAI1 mRNA in HMVECs.
Cells were irradiated with a single dose of 0
or 10 Gy in combinations with or without
DMSO or SB431542. Stimulation with
TGFГџ was used as a positive control.
Radiation induced atherosclerosis (in collaboration with Mat Daemen, CARIM,
Maastricht)
Radiation is an independent risk factor in vascular disease after radiotherapy for
Hodgkin’s disease, breast and head and neck cancer. The aim of this work is to
investigate mechanisms of development of radiation-induced atherosclerosis and
to compare the phenotype of the resulting lesions with those in age-related
atherosclerosis. We established a mouse model for studying radiation-induced
atherosclerosis in the carotid arteries of ApoE-/- mice. These mice have increased
cholesterol levels compared with wild type mice and develop atherosclerosis
spontaneously with age. Mice were given single dose (8-14 Gy) or clinically relevant
fractionated irradiation (20 x 2 Gy in 4 weeks) to the neck. Already at 1-4 weeks after
treatment, early atherosclerotic lesions were seen in irradiated carotid arteries but not
in the arteries of control animals. From 22-34 weeks after treatment, there were
significantly more lesions in irradiated carotid arteries (single and fractionated doses)
compared to age matched controls. The results showed that irradiation accelerated
development of macrophage-rich, inflammatory atherosclerotic lesions, prone to
intra-plaque hemorrhage. We are currently evaluating the potential of anti-platelet,
anti-inflammatory and anti-cholesterol drugs (aspirin, nitric oxide-releasing-aspirin,
clopidogrel and atorvastatin) for their potential to inhibit early endothelial cell (EC)
damage and the subsequent development of radiation-induced atherosclerosis.
In parallel clinical studies, we are characterizing radiation-induced vascular damage
in resection material collected from head and neck (H&N) and breast cancer patients
undergoing free-flap reconstructive surgery. Biopsies from H&N cancer patients were
obtained from a branch of the carotid artery (facial artery) at <1 to 26 years after
radiotherapy or surgery. Internal mammary artery biopsies were obtained from breast
cancer patients 1 to 19 years after radiotherapy or surgery. Control biopsies were
obtained from the free flap donor arteries of each patient (radial or deep inferior
epigastric perforator, DIEP, respectively). In the H&N patients we found a 1.7-fold
increase in intima-media thickness (IMT) for the irradiated compared to the
unirradiated arteries, after correction for the IMT of the donor artery from the same
patient (p<0.008) (Figure VIII.4). There was a marked inflammatory component in
the intima and adventitia of some or these irradiated arteries, which was not seen in
donor arteries from the same patients or in arteries from unirradiated H&N cancer
patients. The IMT ratios of the mammarian arteries from breast cancer patients were
generally < 50% of that seen in arteries from H&N cancer patients. Although it is
known that internal mammary arteries are resistant to the development of age-related
atherosclerosis, we saw a trend for increased IMT in irradiated compared to
unirradiated patients (1.4-fold, p=0.09). Further analysis is required to determine
how co-morbidity factors, e.g. age and smoking, as well as the irradiation dose and
time since treatment, affect radiation-induced vascular damage.
Radiation induced telangiectasia (in collaboration with Peter Ten Dijke, LUMC,
Leiden)
Radiation-induced telangiectasia is characterized by abnormal dilated postcapillary
vessels. This compromises the functional integrity of tissues, leading to severe
morbidity (bleeding). It can occur in all tissues, but has been best reported in skin or
rectum after treatment for breast or prostate cancer. Members of TGFb and Notch
families are important regulators of vascular development and perturbations in their
signaling have been implicated in vascular disorders similar to those seen after
radiotherapy. The aim of this study is to investigate the influence of radiation on
81
EXPERIMENTAL THERAPY
Publications
TGFb/Notch signaling in ECs, in relation to the development of telangiectasia in
irradiated tissue.
TGFb activates two distinct type I receptors (ALK1 and ALK5), which have opposite
effects on EC migration and proliferation: ALK1 signaling stimulates, whereas ALK5
inhibits these processes. Irradiation of human microvascular ECs (HMVECs) in vitro
resulted in reduced ALK1 signaling, with decreased production of downstream target
Id1, and stimulation of ALK5 signaling, with increased phosphorylation of Smad2
and production of PAI1. This increased PAI1 expression was, at least partly,
dependent on ALK5 signaling, since exposure of cells to an ALK5 inhibitor almost
completely abolished radiation-induced increase in PAI1 (Figure VIII.5). Irradiation
of ECs also resulted in marked increases in the Notch ligand, Jagged1, and
downstream target Herp2. Herp2 is known to function as a mediator of BMP
signaling in ECs, where it inhibits Id1 expression and promotes degradation of the
protein, thereby antagonizing Id1 induced EC migration. Taken together, the
radiation-induced changes would be expected to inhibit EC migration and
proliferation. We are currently evaluating the functional consequences of radiationinduced changes in TGFb and Notch signaling using assays of cellular migration.
To elucidate long-term effect of ionizing radiation on TGF /ALK and Jagged/Notch
signaling in vivo, mice were irradiated with 16-20 Gy to the kidneys or rectum.
Tissues were harvested at 1 to 30 weeks after irradiation and used for RNA isolation
and quantitative real time PCR or immunohistochemical analyses. Kidney irradiation
resulted in a progressive increase in PAI1, reaching 6-7 fold at 30 weeks. A transient
elevation in Id-1 was found at early time intervals after irradiation (1-10 weeks),
whereas at late time intervals (20-30 weeks), when telangiectasia is established,
Id-1 expression levels returned to control values. In addition, significant increases
in Smad7 and CTGF mRNA were found at 1 week after irradiation, which persisted
during the observation period. These in vivo results are in agreement with in vitro
findings and demonstrated that irradiation shifts the balance between ALK1 and
ALK5 signaling towards a resolution phase (inhibition of migration and
proliferation). It has been postulated that CTGF, a profibrotic factor, enhances the
TGF /ALK5 signaling pathway by transcriptional suppression of inhibitory Smad 7.
Blocking the negative feedback loop provided by Smad 7 might lead to continued
activation of the TGF signaling pathway.
In the irradiated mouse rectum, a reduction of both ALK1 and ALK5 signaling
(mRNA) was found from 10-30 weeks after irradiation. In order to confirm these
changes at the protein level and to determine localisation of the proteins in irradiated
tissues, immunohistochemical stainings are now being performed.
Aalders MCG, Triesscheijn M, Ruevekamp M,
De Bruin M, Baas P, Faber DJ, Stewart FA.
Doppler optical coherence tomography
to monitor the effect of photodynamic therapy
on tissue morphology and perfusion. J Biomed
Optics 2006; 11:044011
Baas P, Triesscheijn M, Burgers S,
Van Pel R, Stewart F, Aalders M.
Fluorescence detection of pleural
malignancies using 5-aminolaevulinic acid.
Chest 2006; 129:718-724
Dorresteijn LD, Stewart FA, Boogerd W.
Stroke As a Late Treatment Effect of
Hodgkin’s Disease. J Clin Oncol 2006;
24:1480
Stewart FA, Heeneman S, Te Poele J,
Kruse J, Russell NS, Gijbels M,
Daemen M. Ionizing radiation accelerates
the development of atherosclerotic lesions in
ApoE-/- mice and predisposes to an
inflammatory plaque phenotype prone to
hemorrhage. Am J Pathol 2006;
168:649-658
Triesscheijn M, Baas P, Schellens JHM,
Stewart FA. Photodynamic therapy in
oncology. Oncologist 2006; 11:1034-1044
Triesscheijn M, Ruevekamp M, Out R,
Van Berkel TJC, Schellens J, Baas P,
Stewart FA. The pharmacokinetic behavior
of the photosensitizer meso-tetra-hydroxyphenyl-chlorin in mice and men. Cancer
Chemother Pharmacol 2007; In Press
Photodynamic Therapy (PDT)
In a separate project we developed a pre-clinical program for optimizing treatment
protocols for clinical use of photodynamic therapy (PDT). PDT is an effective
treatment for small, localized cancers that is now routinely used in the NKI-AVL for
small oral cavity tumors and multiple basal cell carcinomas. Treatment involves
systemic delivery of a photosensitizer followed by local tumor illumination with light
of a specific wavelength, which generates free radicals and singlet oxygen. The
penetration depth of the laser light used to excite the photosensitizer limits damage
to normal tissues. Over the past 10-years we have used animal models to investigate
how sensitizer pharmacokinetics and drug/light scheduling and timing affects the
mechanism of PDT-mediated tumor destruction. Our work has demonstrated that
vascular mediated damage, and not just tumor cell kill, is an essential component of
curative PDT. This has had a major influence on the design of optimal clinical
protocols. Our pre-clinical PDT program has resulted in the successful introduction
and use of clinical PDT for oral cavity and skin tumors. A dedicated PDT unit will
open in December 2006 at the NKI-AVL; laboratory investigations on this topic have
now stopped.
Triesscheijn ML. mTHPC-mediated
photodynamic therapy; from bed to bench
and back again. Utrecht: Universiteit
Utrecht, 2006
Triesscheijn M, Ruevekamp M, Antonini N,
Neering H, Stewart FA, Baas P. Optimizing
Meso-Tetra-Hydroxyphenyl-Chlorin Mediated
Photodynamic Therapy for Basal Cell
Carcinoma. Photochem Photobiol
2006; 82:1686-1690
Van Veen RLP, Nyst H, Rai Indrasari S,
Adham Yudharto M, Robinson DJ, Tan IB,
Meewis C, Peters R, Spaniol S,
Stewart FA, Levendag PC,
Sterenborg HJCM. In vivo fluence rate
measurements during FoscanВ©-mediated
photodynamic therapy of persistent and
recurrent nasopharyngeal carcinomas using a
dedicated light applicator. J Biomed Optics
2006; 11:a041107
82
EXPERIMENTAL THERAPY
PHARMACODYNAMICS OF ANTICANCER DRUGS
The pharmacology group Schellens has a continued 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.
Group leader Jan Schellens
Jan Schellens MD PhD Group leader
Jos Beijnen PhD Academic staff
Irma Meijerman PhD External staff
Serena Marchetti MD External staff
Maarten Deenen MSc Graduate student
Roos Oostendorp MSc Graduate student
Sander Veltkamp MSc Graduate student
Stefan Vink MSc Graduate student
Marije Bolijn Technical staff
Dick Pluim Technical staff
Monique Van Eijndhoven Technical staff
Publications
Appels NMGM, Rosing H, Stephens TC,
Hughes A, Schellens JHM, Beijnen JH.
Absolute quantification of farnesylated Ras
levels in complex samples using liquid
chromatography fractionation combined
with tryptic digestion and electrospray
tandem mass spectrometry. Anal Biochem
2006; 352:33-40
Appels NMGM, Rosing H, Stephens TC,
Schellens JHM, Beijnen JH.
Quantification of farnesylmethylcysteine in
lysates of peripheral blood mononuclear
cells using liquid chromatography coupled
with electrospray tandem mass
spectrometry: Pharmacodynamic assay for
farnesyl transferase inhibitors. Anal Chem
2006; 78:2617-2622
Breedveld P, Pluim D, Cipriani G,
Dahlhaus F, Van Eijndhoven MAJ,
De Wolf CJF, Kuil A, Beijnen JH,
Scheffer GL, Jansen G, Borst P,
Schellens JHM. The Effect of Low pH
on Breast Cancer Resistance Protein
(ABCG2)-Mediated Transport of
Methotrexate, 7-Hydroxymethotrexate,
Methotrexate Diglutamate, Folic Acid,
Mitoxantrone, Topotecan, and Resveratrol
in In Vitro Drug Transport Models.
Mol Pharmacol 2007; 71:240-249
Role of ABC drug transporters in absorption and disposition of anticancer
drugs The research of the group is focused on assessment of the role of the ABC
drug transporters and other transporters on the absorption and disposition of
anticancer drugs. We continued the in vivo studies with the novel lipophilic
camptothecin derivative gimatecan. We identified that this molecule is transported by
BCRP, although at lower affinity than the control drug topotecan. In vivo, area under
the curve (AUC) of gimatecan after oral dosing (2 mg/kg) in Bcrp1-/-/Mdr1a/1b-/(TKO) mice was 1.5-fold higher than in wildtype mice (p<0.001). Intravenous PK
were not significantly different in TKO and wildtype mice. However, CNS
accumulation was significantly increased in wildtype mice upon co-administration of
elacridar. Elacridar did not affect the biotransformation of gimatecan. In TKO mice,
elacridar further increased the systemic exposure after oral administration of
gimatecan, clearly indicating that other drug transporters are involved in the
disposition of the drug.
We continued studies with the orally bioavailable taxane analog BMS-275183 and
established the oral and (i.v.) PK and tissue distribution in vivo in wildtype mice and
in the MDR1 knockout mice (Mdr1a/1b-/-). In vivo, after an oral dose of 10 mg/kg, the
AUC of BMS-275183 was 4665 + 789 (mean + SE; ng.h/ml) in wt and 8761 + 406
(ng.h/ml) in mdra/b KO mice (p<0.01). Co-administration of one dose of 40 mg/kg
of oral pantoprazole did not affect the AUC values in wt mice. CNS concentrations of
BMS in wt mice were below the LLQ of the assay, but were 851 + 442 and 932 + 429
ng/g tissue at 1 and 4 h respectively in the KO mice. Pantoprazole had no significant
effect on CNS concentrations in wt and KO mice. In vitro and in vivo, BMS-275183 is
a good substrate drug for MDR1. In vitro, it is a moderate substrate for MRP2 and not
a substrate for BCRP. It is warranted to further explore the in vivo interaction
between BMS-275183 and PPIs in patients.
Development of resistance to imatinib is observed in many patients. Imatinib has
high oral bioavailability (98%), but preliminary data revealed reduced systemic
exposure to imatinib over time. In previous in vitro and in vivo experiments, we and
others showed that the drug efflux transporters P-glycoprotein (P-gp) and BCRP;
(ABCG2) play an important role in the PK of imatinib. We hypothesized that in
combination with a P-gp/BCRP inhibitor the variability in the oral PK of imatinib
may be further reduced, the tissue distribution improved and PK mechanisms of
clinical resistance to imatinib prevented. We investigated in vivo the absolute
bioavailability in wildtype mice and combined Mdr1a/1b and Bcrp1 knockout mice
(Mdr1a/1b-/-/Bcrp1-/-) and the effect of the Pgp/BCRP inhibitor elacridar on the
absorption and disposition of imatinib and its main metabolite CGP74588 after oral
administration of imatinib.
The absolute oral bioavailability of imatinib in wildtype and Mdr1a/1b-/-/Bcrp1-/- mice
was 71 and 49%, respectively, and 107 and 102 % when elacridar is co-administrated.
The plasma area under the curve (AUC)oral was not significantly different in
Mdr1a/1b-/-/Bcrp1-/- and wildtype mice. The (AUC)i.v. in Mdr1a/1b-/-/Bcrp1-/- mice
was 1.6-fold higher versus wildtype mice (p<0.05). Co-administration of elacridar
increased the (AUC)oral and (AUC)i.v. 3.3- and 2.0-fold, respectively, in wildtype mice
and 2.7- and 1.3-fold in Mdr1a/1b-/-/Bcrp1-/- mice (P<0.001 and P<0.05). This
suggests that besides Mdr1a/1b and Bcrp1 also other drug transporters (e.g. efflux/
uptake transporters) or inhibition of biotransformation are involved in the interaction
between imatinib and elacridar. Oral [14C]imatinib treated wildtype mice resulted in
an overall recovery of 83.2%. Unchanged imatinib and CGP74588 excreted mainly in
the feces 24.8 and 1.3% of the dose, respectively, and urinary excretion accounts for
1.4 and 0.10% of the dose, indicating that imatinib is extensively metabolized in
other metabolites besides CGP74588. The fecal and biliary excretion of unchanged
imatinib was reduced by the absence (5.2 and 8.3-fold, respectively) or inhibition by
83
EXPERIMENTAL THERAPY
Publications (continued)
elacridar (3.3- and 4.0-fold) of Mdr1a/1b and Bcrp1 (P<0.001), and a role for both
Mdr1a/1b and Bcrp1 in these processes was revealed. There is no indication that
elacridar inhibited the biotransformation of imatinib. No significant differences were
found in liver and kidney levels of imatinib to wildtype and Mdr1a/1b-/-/Bcrp1-/- mice
in the presence or absence of elacridar. In vitro studies are ongoing to investigate the
outlined interaction between these two drugs at the level of other efflux/uptake
transporters.
Hoebers FJP, Pluim D, Verheij M,
Balm AJM, Bartelink H, Schellens JHM,
Begg AC. Prediction of treatment outcome
by cisplatin-DNA adduct formation in
patients with stage III/IV head and neck
squamous cell carcinoma, treated by
concurrent cisplatin-radiation
New insights into the pharmacology and toxicology of gemcitabine
Gemcitabine (2’,2’-difluorodeoxycytidine, dFdC), a pyrimidine antimetabolite, is
actively taken up into the cell and intracellularly phosphorylated by deoxycytidine
kinase (dCK) to its monophosphate (dFdCMP) and subsequently to its active
diphosphate (dFdCDP) and triphosphate (dFdCTP) metabolites. Furthermore, dFdC
is converted to 2’,2’-difluorodeoxyuridine (dFdU) by cytidine deaminase (CDA),
which is ubiquitously expressed, such as in liver and intestine. Oral dFdC in a phase I
study resulted in preferential formation of dFdU and incidental severe liver toxicity.
We hypothesized that dFdU is phosphorylated and toxic after liver accumulation
following multiple oral dFdC administration.
We assessed the in vitro toxicity, uptake, and intracellular activation of dFdC and
dFdU in human hepatocellular (HepG2), human lung cancer (A549) and MDCK cells
containing high expression of either the human equilibrative or concentrative type 1
nucleoside transporter (hENT1 and hCNT1). Cytototoxicity was assessed at several
exposure times using SRB and colony assays. The total cellular uptake plus
incorporation into DNA/RNA of radiolabeled dFdC (0-20ВµM) and dFdU (0-1000ВµM)
was determined at different time points. Furthermore, intracellular and extracellular
concentrations of dFdC, dFdU and metabolites were quantified by HPLC-UVscintillation counting. In addition, quantitative RT-PCR was used to determine gene
expression of dCK, CDA, hENT1 and hCNT1.
Compared to A549 cells, HepG2 cells had a 4-fold and 7-fold higher sensitivity to
dFdC and dFdU, respectively. Furthermore, HepG2 cells showed higher expression of
both hENT1 and dCK compared to A549 cells. MDCK cells with hENT1 and hCNT1
overexpression showed increased sensitivity to dFdC and dFdU compared to wildtype cells. dFdC was actively taken up into HepG2 and A549 cells. dFdU was detected
in DNA/RNA. Interestingly, we discovered that dFdU was substrate for hCNT1 but
not for hENT1 and that dFdUTP was formed intracellularly, suggesting
phosphorylation of dFdU and/or dFdUMP. Conclusions: dFdU has high toxicity in
HepG2 cells and is a good substrate for hCNT1. Moreover, dFdUTP is formed
intracellularly and dFdU seems to be incorporated into DNA/RNA. This could be
relevant for the in vivo activity and toxicity of dFdC and dFdU, especially in tissues
with high expression of hCNT1, such as liver and kidney. Currently, in vivo mice
studies are performed to further assess the relevance of dFdU in the activity and
toxicity of dFdC.
(RADPLAT). Int J Cancer 2006;
Pharmacogenetics of fluoropyrimidines About 3-5% of patients treated with
fluorouracil (5-FU) and its oral prodrug capecitabine develop severe hematological
and/or gastrointestinal toxicity, often requiring a long of hospitalization, caused by
DPD-deficiency. DPD (dihydropyrimidine dehydrogenase) is the enzyme responsible
for 85% of the metabolism of 5-FU into the inactive compound FUH2.
Polymorphisms in the gene of DPD, DPYD, is held (partially) responsible for a
decreased activity of the enzyme. The mutation IVS14+1G>A (DPYD*2A) leads to
complete skipping of exon 14, resulting in a truncated protein with major decreased
activity. Patients exhibiting this polymorphism are at great risk of developing severe
fluoropyrimidine-associated toxicity after receiving a full-dose treatment. We
developed a validated, rapid, inexpensive rt-PCR assay, with almost 100% sensitivity
and specificity, for detection of DPYD*2A, which is applicable for routine screening
prior to therapy. We recently identified three heterozygotes for DPYD*2A based on
significant clinical toxicity, which resulted in the death of one of the three patients.
We implemented screening for DPYD*2A in all patients who will start
fluoropyrimidine therapy and prospectively establish a dosing algorithm in these
patients and the cost-effectiveness of this approach.
Vink SR, Lagerwerf S, Mesman E,
119:750-756
Liedert B, Pluim D, Schellens J,
Thomale J. Adduct-specific monoclonal
antibodies for the measurement of cisplatininduced DNA lesions in individual cell
nuclei. Nucleic Acids Res 2006; 34:e47
Veltkamp SA, Alderden-Los C, Sharma A,
Rosing H, Beijnen JH, Schellens JHM.
A pharmacokinetic and safety study of a
novel polymeric paclitaxel formulation for
oral application. Cancer Chemother
Pharmacol 2007; 59:43-50
Veltkamp SA, Thijssen B, Garrigue JS,
Lambert G, Lallemand F, Binlich F,
Huitema AD, Nuijen B, Nol A,
Beijnen JH, Schellens JH. A novel selfmicroemulsifying formulation of paclitaxel
for oral administration to patients with
advanced cancer. Br J Cancer. 2006;
95: 729-34
Veltkamp SA, Hillebrand MJ, Rosing H,
Jansen RS, Wickremsinhe ER, Perkins EJ,
Schellens JH, Beijnen JH. Quantitative
analysis of gemcitabine triphosphate in
human peripheral blood mononuclear
cells using weak anion-exchange liquid
chromatography coupled with tandem
mass spectrometry. J Mass Spectrom.
2006; 41: 1633-42
Schellens JHM, Begg AC,
Van Blitterswijk WJ, Verheij M.
Radiosensitization of squamous cell
carcinoma by the alkylphospholipid
perifosine in cell culture and xenografts.
Clin Cancer Res 2006; 12:1615-1622
Vlaming MLH, Mohrmann K,
Wagenaar E, De Waart DR,
Oude Elferink RPJ, Lagas JS,
Van Tellingen O, Vainchtein LD,
Rosing H, Beijnen JH, Schellens JHM,
Schinkel AH. Carcinogen and anticancer
drug transport by Mrp2 in vivo: Studies
using Mrp2 (Abcc2) knockout mice.
J Pharmacol Exp Ther 2006; 318:319-327
84
EXPERIMENTAL THERAPY
MOLECULAR PATHOLOGY AND MOLECULAR EPIDEMIOLOGY
OF BREAST CANCER
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
Sabine Linn MD PhD Academic staff
Flora Van Leeuwen PhD Academic staff
Senno Verhoef MD PhD Academic staff
Annegien Broeks PhD Post-doc
Dorien Voskuil PhD Post-doc
Marjanka Schmidt PhD Post-doc
Erik Van Beers PhD Post-doc
Britta Weigelt PhD Post-doc
Tim Molloy PhD Post-doc
Molecular profiling for breast cancer classification and therapy response The
classification of breast tumors by the World Health Organization distinguishes 30
different morphological types of breast carcinomas. The rare �special type’ breast
cancers exhibit favorable prognostic features, but are often not recognized and
classified as invasive ductal carcinoma, not otherwise specified (IDC NOS). We
identified by hierarchical clustering of 113 tumors genetically related histological
subtypes, such as medullary and metaplastic carcinomas, but also genetically distinct
types, like micropapillary carcinomas. We show that most special type breast cancers
only belong to one of the molecular intrinsic types. Intrinsic �basal-like’ tumors,
apparently comprise subtypes with an excellent prognosis, such as adenoid cystic
carcinomas. (see Figure VIII.6) Half of the recurrences of ERa+ breast tumors
respond to tamoxifen while the other half is resistant. We validated on a more
advanced whole genome platform and in an independent set of tumors (including
stable disease) a recently identified 81-gene tamoxifen response profile (Jansen et al.,
JCO 2005). The profile is outperforming other parameters in a multivariate response
prediction analysis. Currently, this profile is optimized for clinical use in the adjuvant
setting. The ability to accurately predict tamoxifen treatment outcome would
constitute a significant advance in the management of breast cancer.
Sjoerd Bruin Clinical fellow
Marleen Kok MD Graduate student
Marielle Ruijs MD Graduate student
Alina Vrieling MSc Graduate student
Stella Mook MD Graduate student
Astrid Bosma Technical staff
Arnout Van der Plas Technical staff
Linde Braaf Technical staff
Renske Fles Technical staff
Simon Joosse Technical staff
Renate De Groot Technical staff
Renate Udo MSc Research assistant
Richard Van Hien Technical staff
Radiation-associated breast tumors display a particular gene expression
profile Approximately 90% of the breast carcinomas in women who received
radiation treatment following Hodgkin Lymphoma is estimated to be a result of the
radiation treatment, which makes this series extremely appropriate to determine a
radiation-associated molecular profile. We have used microarray gene expression
profiling to assess specific molecular changes in radiation-associated breast tumors
(BfHL) and unsupervised hierarchical clustering (23 BfHL case- and 19 control breast
tumors) resulted in a clustering of the radiation-associated tumors. A supervised
classification revealed 430 significant genes that can differentiate radiation-associated
breast tumors from control breast tumors. These preliminary results indicate that
radiation-associated tumors can be distinguished from other breast tumors,
indicating that there is a different etiology for radiation-induced breast tumors.
Ferdi Van der Horst Technical staff
Anke Nooijen MSc Research assistant
Genetic determinants of breast cancer risk and breast cancer outcome
Evidence to date strongly suggests that the majority of the familial clustering of
breast cancer is unexplained and, therefore, that many breast cancer susceptibility
genes still remain to be identified. One candidate is the single nucleotide
polymorphism (SNP309) in MDM2. We confirmed that Li-Fraumeni TP53 germline
mutation carriers have earlier onset of cancer if they carry the SNP309. However,
within unselected breast cancer patients, MDM2 SNP309 does not predispose to an
earlier age of onset (study performed within large international collaboration: the
Breast Cancer Association Consortium). Germline mutations in genes of the DNA
damage repair pathway like BRCA1/2, CHEK2 and MDM2, may affect breast cancer
outcome. Our consecutive series of breast cancer patients aged <50 years,
representative for the premenopausal breast cancer population, reveals approximately
4% CHEK2 1100delC carriers who have an increased risk of second breast cancer and
worse long-term recurrence-free survival, which does not seem to be confounded by
other prognostic factors. More research is being performed to explore tumor
characteristics of CHEK2 1100delC and gene-treatment interactions.
Figure VIII.6: Histology. Panel A shows
four invasive ductal carcinomas not
otherwise specified. Panel B shows a of
special type breast cancers: invasive lobular
carcinoma (a), tubular (b) mucinous A
(c), mucinous B (d), neuroendocrine (e),
invasive ductal carcinoma with osteoclastic
giant cells (f), micropapillary (g), apocrine
(h), metaplastic (i), medullary (j) and
adenoid cystic carcinoma (k).
The Insulin-like Growth Factor (IGF) system and its relation to breast and
colorectal cancer Meta-analysis of epidemiological studies has shown that the risk
of several epithelial cancers for individuals with relatively high serum concentrations
of IGF-I. We are evaluating the effects of several dietary strategies to lower
components of the circulating IGF-system, and its association with breast and
colorectal tissue IGF-systems. In a randomized placebo-controlled cross-over trial, we
find that lycopene supplementation does not result in significant changes in serum
IGF-system in pre-menopausal women (n=60) at increased risk of breast cancer.
Isoflavone supplementation in men at increased risk of colorectal cancer (n=37) did
85
EXPERIMENTAL THERAPY
Publications
not affect IGF levels overall. However, our results suggest that this may differ
depending on isoflavone metabolism. The expression of IGF-system components in
normal colorectal biopsies was found to differ markedly between locations in the
colon and rectum and between individuals. The association with serum levels and the
effects of dietary strategies on tissue IGF expression are under study. The proposed
research is expected to provide a better fundamental understanding of whether and
how the IGF-system could be the target of cancer preventive measures.
Bogaerts J, Cardoso F, Buyse M, Braga S,
Loi S, Harrison JA, Bines J, Mook S,
Decker N, Ravdin P, Therasse P,
Rutgers E, Van �t Veer LJ, Piccart M. Gene
signature evaluation as a prognostic tool:
Challenges in the design of the MINDACT
trial. Nat Clin Pract Oncol 2006; 3:540-551
Gene expression profiling to predict the presence of disseminated tumor cell
in breast cancer patients The presence of disseminated tumor cells (DTCs) in the
peripheral blood and/or bone marrow of breast cancer patients is correlated with an
increased risk of metastasis. This work aims to use microarray gene expression
profiling to develop models with the ability to identify primary tumors at high risk of
producing DTCs. Work has already been undertaken in early- and late-stage breast
cancer patient cohorts to estimate the disseminated tumor cell load in the peripheral
blood by QPCR analyses. RNA from primary breast tumors from these patients is
used to interrogate whole-genome microarrays. Bioinformatics techniques will be
used to build a predictive model with the ability to distinguish tumors which are
likely give rise to high numbers of disseminated tumor cells from those that do not.
The ability to predict which tumors are likely to give rise to high numbers of
disseminated tumor cells would be of use in the clinic as it would optimize the
decision-making process for the appropriate application of adjuvant therapy.
Fan C, Oh DS, Wessels L, Weigelt B,
Nuyten DSA, Nobel AB, Van �t Veer LJ,
Perou CM. Concordance among geneexpression-based predictors for breast cancer.
N Engl J Med 2006; 355:560-569
Ruijs MWG, Verhoef S, Wigbout G,
Pruntel R, Floore AN, De Jong D,
Van �t Veer LJ, Menko FH. Late-onset
common cancers in a kindred with an
Arg213Gln TP53 germline mutation. Fam
Cancer 2006; 5:169-174
Ruijs MWG, Schmidt MK, Nevanlinna H,
Clinicopathologic classification and genetic profiling of colorectal metastasis
Specific cases of colorectal carcinoma may have affinity for peritoneal spread in
contrast with more usual lymphatic and haematogenous spread. The NKI-AVL is
a center for treating this peritoneal spread with hyperthermic intraperitoneal
chemotherapy, first as an experimental but now as an established treatment in this
clinical context, although with many side effects. Using histology, gene expression
profiling and CGH analysis we will try to predict response to this treatment.
Furthermore, we will try to find the molecular and histological characteristics that
discriminate tumours with peritoneal spread from those with the more usual
lymphatic and haematogenous metastasis.
Tommiska J, Aittomaki K, Pruntel R,
Verhoef S, Van �t Veer LJ. The singlenucleotide polymorphism 309 in the MDM2
gene contributes to the Li-Fraumeni
syndrome and related phenotypes. Eur J
Hum Genet 2006; 15:110-114
Schmidt MK, Tollenaar RA, De Kemp SR,
Broeks A, Cornelisse CJ, Smit VT,
Peterse JL, Van Leeuwen FE, V
an �t Veer LJ. Breast Cancer Survival and
Genome-wide chromosome analysis in hereditary breast cancer (PI Petra
Nederlof ) We have shown that BRCA1 and BRCA2 tumors show specific
chromosomal changes, and that these somatic aberrations 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. In collaboration with Dr. P Devilee (Leiden
University), we analyzed a series of 62 breast tumors from high-risk BRCA1/2
negative breast cancer only (HBC) families. Within these BRCAx families, different
subgroups of tumors were identified on the basis of their aCGH profile. In a series of
40 breast tumors from high-risk BRCA1/2 negative breast and ovarian cancer
(HBOC) families, only three tumors showed a BRCA1-like profile, one was proven to
be a splice-site mutant by RNA analysis and one patient showed BRCA1 inactivation
through promoter hypermethylation. In addition, so-called unclassified variants were
tested, and showed that aCGH profiling could classify them as BRCA1, which is clear
additional proof of significance of the variation.
Tumor Characteristics in Premenopausal
Women Carrying the CHEK2*1100delC
Germline Mutation. J Clin Oncol
2007; 25:64-69
Van Beers EH, Joosse SA, Ligtenberg MJ,
Fles R, Hogervorst FBL, Verhoef S,
Nederlof PM. A multiplex PCR predictor
for aCGH success of FFPE samples.
Br J Cancer 2006; 94:333-337
Van Beers EH, Nederlof PM. Array-CGH
and breast cancer. Breast Cancer Res
2006; 8:210
Van Houwelingen HC, Bruinsma T,
Molecular characteristics of uterine corpus cancers subsequent to use of
tamoxifen for breast cancer (PI Petra Nederlof ) Long-term use of tamoxifen
(TAM) after breast cancer is associated with an unexplained increased risk of uterine
corpus cancer. We analyzed FFPE endometrioid, serous and carcinosarcomas tumor
samples after long-term exposure (≥3 years) to TAM and TAM- (age- and morphology)
matched control patients by 1Mb array-CGH. Results show that there is no
correlation between TAM use and the number and type of genomic aberrations
detected. Endometrioid tumors show fewer aberrations than the other tumor types,
and frequently show MSI. Fresh-frozen tissue of 170 patients has been collected and
RNA isolated from these samples will be hybridized on 30K oligo-microarrays to
study differentially expressed genes in TAM+ and TAM- tumors.
Hart AAM, Van �t Veer LJ, Wessels LFA.
Cross-validated Cox regression on
microarray gene expression data. Stat Med
2006; 25:3201-3216
86
EXPERIMENTAL THERAPY
MOLECULAR ANALYSIS OF BREAST CANCER
Group leader Marc Van de Vijver
Our lab is studying breast cancer, with the aim to understand the genetics of the
development of these tumors and use this knowledge for the clinical management of
breast cancer patients. Breast cancer is a heterogeneous disease and based on
morphological and genetic characteristics, various subtypes can be identified. The
main aim of our research is to refine the classification of breast cancer; to achieve this
goal, we are combining histological examination, genetic analysis and gene
expression profiling of tumors and correlate these features to clinical parameters.
Marc Van de Vijver MD PhD Group leader
Harry Bartelink MD PhD Academic staff
Hans Peterse MD Academic staff
Sjoerd Rodenhuis MD PhD Academic staff
Emiel Rutgers MD PhD Academic staff
Juliane Hannemann MSc Graduate student
Hugo Horlings MD Graduate student
Bas Kreike MD Graduate student
Dimitry Nuyten MD Graduate student
Hans Halfwerk Technical staff
Stephanie Hundscheid Technical staff
Petra Kristel Technical staff
Lennart Mulder Technical staff
Publications
Abdullah-Sayani A, Bueno de Mesquita
JM, Van de Vijver MJ. Technology insight:
Tuning into the genetic orchestra using
Molecular subtypes of breast cancer and amplification of Topoisomerase II :
predictive role in dose intensive adjuvant chemotherapy Benefit from
chemotherapy treatment in breast cancer patients is determined by the molecular
make-up of the tumor. In a retrospective analysis of patients treated in a randomized
clinical trial comparing high dose chemotherapy with standard chemotherapy. In
addition, the topoisomerase IIa amplification status was determined by FISH and
CISH. 411 of the 753 tumours (55%) were classified as luminal-like, 137 (18%) as
basal-like and 205 (27%) as HER2 amplified. The 137 basal-like tumors were defined
as having no expression of ER and HER2; 98 of them did express EGFR and/or
cytokeratin 5/6. The luminal-like tumors had a significantly better recurrence free
and overall survival than the other two groups. From the 194 HER2 positive tumors,
47 (24%) were shown to harbor an amplification of TOP2A. Patients with a HER2amplified tumor randomized to the high-dose therapy arm did worse than those in
the conventional treatment arm, possibly caused by the lower cumulative
anthracycline dose in the high-dose arm. The tumors with a TOP2A amplification
contributed hardly to this difference, suggesting that TOP2A amplification is not the
cause of the steep dose-response curve for anthracyclines in breast cancer. The
difference of the cumulative dose of only 25% between the treatment arms was
possibly insufficient to yield a survival difference.
microarrays - Limitations of DNA
microarrays in clinical practice. Nat Clin
Pract Oncol 2006; 3:501-516
Adler AS, Lin M, Horlings H,
Nuyten DSA, Van de Vijver MJ, Chang
HY. Genetic regulators of large-scale
transcriptional signatures in cancer.
Nat Genet 2006; 38:421-430
Chi JT, Wang Z, Nuyten DSA,
Rodriguez EH, Schaner ME, Salim A,
Wang Y, Kristensen GB, Helland A,
Predicting the response to specific forms of systemic therapy An ongoing
research project investigates whether the gene expression profile of the primary
breast tumor can predict response to specific regimens of neoadjuvant chemotherapy.
Gene expression data of the primary tumor before chemotherapy are now available
from 63 patients and recruitment of patients is ongoing. 27% of the patients showed
a (near) pathologic complete remission, 40% of the patients had a partial remission
and 33% of the patients did not respond to chemotherapy. Based on gene expression
profiling, we identified the molecular subtypes of breast cancer in our data. Basal-like
and ERBB2-like tumors responded frequently to chemotherapy, whereas the luminal
tumors were often resistant to treatment. We are exploring the predictive value of
several gene expression signatures identified by others; and are using supervised
classification approaches to find chemotherapy response classifiers.
BГёrresen-Dale A-L, Giaccia A,
Longaker MT, Hastie T, Yang GP,
Van de Vijver MJ, Brown PO. Gene
expression programs in response to hypoxia:
Cell type specificity and prognostic
significance in human cancers.
PLoS Medicine 2006; 3:395-409
Hannemann J, Velds A, Halfwerk JBG,
Kreike B, Peterse JL, Van de Vijver MJ.
Classification of ductal carcinoma in situ
by gene expression profiling. Breast Cancer
Res 2006; 8:R61
Classification of ductal carcinoma in situ by gene expression profiling Ductal
carcinoma in situ (DCIS) is characterized by the intraductal proliferation of
malignant epithelial cells. Several histological classification systems have been
developed, but assessing the histological type/grade of DCIS lesions is still
challenging, making treatment decisions based on these features difficult. To obtain
insight into the molecular basis of the development of different types of DCIS and its
progression to invasive breast cancer, we have studied differences in gene expression
between different types of DCIS and between DCIS and invasive breast carcinomas.
Gene expression profiling using microarray analysis has been performed on 40 in
situ and 40 invasive breast cancer cases. DCIS cases were classified as well (n=6),
intermediately (n=18) and poorly (n=14) differentiated type. Of the 40 invasive breast
cancer samples, 5 samples were grade I, 11 samples were grade II and 24 samples
were grade III. Using two-dimensional hierarchical clustering, the basal-like type,
ERB-B2 type, and the luminal type tumors originally described for invasive breast
cancer could also be identified in DCIS. Using supervised classification, we identified
a gene expression classifier of 35 genes, which differed between DCIS and invasive
breast cancer; a classifier of 43 genes could be identified separating between well and
poorly differentiated DCIS samples.
87
EXPERIMENTAL THERAPY
Publications
Do specific DNA copy number changes regulate prognostic gene expression
signatures in breast cancer? Our goal is to identify candidate functional regulators
of expression signatures by comparing genome-wide DNA copy number with gene
expression data. We want to test if these candidate functional regulators can act as
potential prognostic and/or predictive markers in breast cancer patients. In order to
reach this goal we carried out Comparative Genomic Hybridization (CGH) in 67
human breast carcinomas for which gene expression data and clinical data were
available. Statistical analysis linked distinct regions of DNA copy numbers alterations
to different prognostic gene expression signatures. For example: the previously
identified poor prognosis 70 gene signature is associated with gain of 3q and the 70
genes good prognosis profile with loss at 16q; the activated wound signature is
associated with gain at 8q and 12q; genomic grade 3 is associated with genetic gain at
3q, 8q; basal type tumors show losses at 4p, 5q, 14q, 15q and a gain of 6p, 10p and
12p; HER2 positive breast tumors gain at 17q (where the HER2 gene is located), but
no specific additional alterations.
These findings help establish a link in the interaction between genetic changes and
gene expression profiles that determine tumor cell behavior in breast cancer.
Hannemann J, Kristel P, Van Tinteren H,
Bontenbal M, Van Hoesel QGCM,
Smit WM, Nooij MA, Voest EE,
Van Der Wall E, Hupperets P, De Vries
EGE, Rodenhuis S, Van de Vijver MJ.
Molecular subtypes of breast cancer and
amplification of topoisomerase IIa:
Predictive role in dose intensive
adjuvant chemotherapy. Br J Cancer 2006;
95:1334-1341
Kreike B, Halfwerk H, Kristel P, Glas A,
Peterse H, Bartelink H, Van de Vijver MJ.
Gene expression profiles of primary breast
carcinomas from patients at high risk for
local recurrence after breast-conserving
therapy. Clin Cancer Res 2006;
Gene expression profiles of primary breast carcinomas from patients at high
risk for local recurrence after breast conserving therapy Several risk factors for
local recurrence of breast cancer after breast conserving therapy (BCT) have been
identified. The identification of additional risk factors would be very useful in guiding
optimal therapy and also improve understanding of the mechanisms underlying local
recurrence. We used cDNA microarray analysis to identify gene expression profiles
associated with local recurrence.
Gene expression profiles were obtained from 50 patients who underwent BCT. Of
these 50 patients 19 developed a local recurrence; the remaining 31 patients were
selected as controls as they were free of local recurrence at least 11 years after
treatment. For 9/19 patients also the local recurrence was available for gene
expression profiling. Unsupervised and supervised methods of classification were
used to classify patients in groups corresponding to disease outcome and to study the
overall gene expression pattern of primary tumors and their recurrences.
No significant differences in gene expression between primary breast cancer tumors
in patients with or without local recurrence after breast conserving therapy were
identified. Furthermore, analyses of primary tumors and the paired local recurrences
show a preservation of the overall gene expression pattern in the local recurrence,
even after radiotherapy.
From this work we conclude that there are no major differences in the gene
expression profile of tumors that do or do not develop a local recurrence
development, but that it is likely that subtle differences are present. To identify such
subtle differences, larger tumor series will need to be studied. We have therefore
expanded our study group to 170 primary breast carcinomas by collaborating with
several other hospitals (J. Foekens, Erasmus Medical Center; P. Bult (UMC St.
Radboud) and S. Veltkamp, Amstelland Hospital, Amstelveen).
12:5705-5712
Sotiriou C, Wirapati P, Loi S, Harris A,
Fox S, Smeds J, Nordgren H, Farmer P,
Praz V, Haibe-Kains B, Desmedt C,
Larsimont D, Cardoso F, Peterse H,
Nuyten D, Buyse M, Van de Vijver MJ,
Bergh J, Piccart M, Delorenzi M. Gene
expression profiling in breast cancer:
Understanding the molecular basis of
histologic grade to improve prognosis.
J Natl Cancer Inst 2006; 98:262-272
Figure VIII.7a:
Figure VIII.7b:
Survival curves: Recurrence-free and overall survival among
Forest Plot: Correlation of the recurrence free
the HER2-expressing, ER-expressing and the basal-like
survival in these subgroups with response to
subtype of invasive breast cancer.
conventional anthracycline-based or alkylating
high-dose chemotherapy, respectively.
In this Forest-plot, the closed squares represent
the point estimate of the effect of high-dose
chemotherapy (HD) vs conventional-dose
therapy (CONV) within that particular
subgroup (with the size of the square
corresponding to its variance) and the lines
correspond to the 99% confidence interval
of that effect. The open figure at the bottom
shows the overall effect of high-dose
chemotherapy vs CONV for this particular
cohort of patients.
88
RADIOTHERAPY
Division head, Group leader Harry Bartelink
Berthe Aleman MD Academic staff
Harry Bartelink MD PhD Academic staff
Jose Belderbos MD Academic staff
Iain Bruinvis PhD Academic staff
EugenГЁ Damen PhD Academic staff
Roel De Boer PhD Academic staff
Luc Dewit MD PhD Academic staff
Rick Haas MD Academic staff
Guus Hart MSc Academic staff
Wilma Heemsbergen MSc Academic staff
Edwin Jansen MD Academic staff
Joos Lebesque MD PhD Academic staff
Corrie Marijnen MD PhD Academic staff
Harry Masselink MD Academic staff
Ben Mijnheer PhD Academic staff
IX D IV IS ION OF R A D IOTHER A PY
Our previous research results, leading to the development of a Cone Beam CT guided
linear accelerator and the creation of software for image guided radiotherapy (IGRT),
are now in full operational use in our and many other clinics. These developments
have led to new approaches in several tumor sites, for example adaptive radiotherapy
for prostate cancer. Here, treatment plans for individual patients are refined based
upon repetitive imaging of prostate and normal tissues during the first week of
treatment. Another example is frameless stereotactic radiotherapy for lung cancer: by
localizing the tumor just prior to each daily treatment fraction, a very high radiation
dose can be delivered safely in a few fractions to the tumor process. A complimentary
development is in vivo dosimetry with electronic portal imaging devices, which
allows us to check for each individual patient the proper delivery of the radiation
dose. This is especially important since more complex and sophisticated treatments
are being introduced in the clinic with intensity-modulated radiotherapy (IMRT).
These developments have already led to better results in improving the cure rate: for
example in prostate cancer and breast cancer our own trials demonstrated that by
delivering a higher radiation dose, a better local control is achieved. At the same time
in prostate cancer it was shown that the toxicity could be reduced with these
approaches. Safe execution of these complex techniques is, however, hardly possible
without the aforementioned developments in patient positioning and quality
assurance.
Luc Moonen MD PhD Academic staff
Coen Rasch MD PhD 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
Jacqueline Theuws MD PhD Academic staff
Bart Van Bunningen MD Academic staff
Marcel Van Herk PhD Academic staff
Marcel Verheij MD PhD Academic staff
Corine Van Vliet-Vroegindeweij PhD
Academic staff
Fritts Wittkämper PhD Academic staff
Conny Vrieling MD PhD Academic staff
Nina Bijker MD PhD Temporary staff
Monique Bloemers MD Temporary staff
Research aimed at combined modality treatment with new novel agents is ongoing to
maximally profit from the progress with sophisticated radiotherapy with IMRT and
IGRT. Translational research is focused at discovery of new targets; novel agents
directed against these targets are now brought into the clinic. Especially, improving
the results of radiation by increasing the amount of apoptosis by novel agents is
explored extensively. To predict treatment outcome and monitor the response during
therapy, several approaches are investigated in the clinic. Examples of successful
strategies to predict treatment outcome are: in vivo annexin V scintigraphy to
demonstrate the amount of apoptosis after radiation, or measuring cisplatin DNA
adducts in patients receiving cisplatin-based chemoradiotherapy. Recently, a clinical
trial has been completed comparing the intra-arterial versus intra-venous
administration of cisplatin concomitant with radiation for head- and neck tumors,
aiming at delivering a higher dose of the cisplatin drug into the tumors without
unacceptable side effects. Finally, a large multicenter randomized phase III trial has
been launched this year investigating the role of post-operative chemoradiotherapy in
gastric cancer.
Gerben Borst MD Temporary staff
Desiree Van den Bongard MD Temporary staff
Patricia De Haan MD Temporary staff
IMAGE ACQUISITION AND PROCESSING
Frank Hoebers MD Temporary staff
Anja Betgen, Jose Belderbos, Josine De Bois, Eugene Damen, Jurjen Van Dijk, Joop Duppen,
Joost Knegjens MD Temporary staff
Michel Frenay, Joris Van der Geer, Frank Hoebers, David Jaffray1, Rianne de Jong, Simon Van Kranen,
Bas Kreike MD Temporary staff
Vannesa Mexner, Danny Minkema, Tonnis Nuver, Jasper Nijkamp, Erik-Jan Rijhorst, Tezontl Rosario,
Dimitry Nuyten MD Temporary staff
Floris Pos, Christoph Schneider, Monique Smitsmans, Roel Steenbakkers, Joost Voogt,
Vera Opperdijk MD Temporary staff
Corine Van Vliet-Vroegindeweij, Marnix Witte, Jochem Wolthaus, Lambert Zijp, Coen Rasch,
Floris Pos MD Temporary staff
Joos Lebesque, Peter Remeijer, Jan-Jakob Sonke, Marcel Van Herk
Roel Steenbakkers MD Temporary staff
Jan-Jakob Sonke PhD Post-doc
Leah McDermott Ba/Bs Graduate student
Agnieszka Olszewska MSc Graduate student
Marco Schwarz MSc Graduate student
Monique Smitsmans MSc Graduate student
Anja Betgen Technical staff
Bob Brand Technical staff
Josine De Bois Technical staff
The continuing remodeling of the department necessitated a temporary displacement
of the research group to the 3rd floor. With the increased distance the communication
between the clinic and research is more difficult. In the next year the final housing
should be finished and we are eager to renormalize the situation. This year saw a
large increase in the clinical application of the cone-beam CT (CBCT) guided
radiotherapy: now available on four machines in our department. The software used
in this equipment is for a large part developed in our department, and is now in use
in over 100 departments worldwide. Our leading role in this field was recognized by
Joop Duppen Technical staff
Michel Frenay Technical staff
1
University of Toronto/Princess Margaret Hospital, Ontario, Canada
89
RADIOTHERAPY
invitations for lectures and teaching courses and also in the form of our chairman
receiving the Regaud award at the 2006 ESTRO. With the increased clinical
application there is also a hugely increased data-production. There is now around
3TB cone beam data stored. The help of our research developers was invaluable to
manage this flow.
First clinical results of an adaptive radiation scheme using CBCT for prostate
cancer An adaptive scheme for prostate cancer radiotherapy based on kV cone-beamCT (CBCT) images obtained in the first six treatment days is in large-scale use. In
this way, better estimates of the average prostate position and rectum shape are
obtained, and the PTV margin was safely reduced from 10 to 7 mm. Weekly CBCTscans, used for patient set-up correction, are also used to verify that the prostate is
inside the new PTV. So far, 46 patients were treated with our adaptive treatment
scheme. For 92% of the CBCT-scans a successful grey-value match was obtained, the
other scans were discarded. Only in a few cases the weekly scans show a marginal
miss (1 mm) of the CTV. We estimate that our scheme reduces rectal toxicity by 30%.
This is the first routine clinical application of soft tissue image guidance for the
prostate using kV CBCT. Contrary to adaptive schemes that use implanted markers,
our method is non-invasive and improves localization of the rectum, as well as the
prostate.
The influence of a dietary protocol on automatic prostate localization in
cone-beam CT scans The success rate of the automatic prostate registration
process described above depends on the image quality of the CBCT scans. The image
quality of these scans is deteriorated when gas in the rectum moves during CBCT
acquisition. We have evaluated the impact of a dietary protocol on the presence of
moving gas and automatic grey value registration. To that end we analyzed 271 scans
of 21 patients that were subjected to the dietary protocol (DP data set) and 190 scan of
18 patients that were not subjected to the protocol (NDP data set). The percentage of
scans with feces, gas and moving gas were significantly reduced from 67%, 61% and
45% in the NDP data set to 47%, 30% and 26% in the DP data set (p<0.001). The
success rate of 3D gray-value registration improved significantly from 83% in the
NDP to 92% in the DP data set (p<0.001). This suggests that a dietary protocol is also
important to reduce intra-fraction prostate motion during treatment delivery.
Therefore, the dietary protocol is currently prescribed to all our prostate cancer
patients, independent of the imaging protocol (CBCT or EPID) used. (Figure IX.1a
and 1b)
Figure IX.1: The impact of a large, moving gas pocket during cone-beam computed tomography (CBCT)
image acquisition on the CBCT reconstruction result. (a) Projection images (PI) showing a large, moving
gas pocket (bright structure) emerging in the rectum in a time span of 17 s at a position where the prostate
would be projected in the projection images. (b) The reconstruction result, showing the streak artifacts in a
transverse slice of the CBCT scan.
High precision CBCT guided hypofractionated radiotherapy We implemented
high dose hypofractioned regimes for solitary brain metastases and small lung
tumors with on-line patient position verification and correction using CBCT and
investigated the accuracy of these techniques. First, a CBCT scan was acquired and
registered to the planning CT (using bone for the head, and soft tissue for the lung)
Tom Minderhoud Technical staff
Kenneth Pengel Technical staff
Maddalena Rossi Technical staff
RenГ© Tielenburg Technical staff
Peter Van de Ven MSc Technical staff
Lambert Zijp Technical staff
90
RADIOTHERAPY
to check the patient setup. Deviations larger than 1 mm were corrected by a manual
couch shift and verified by a 2nd CBCT. The stability of the patient position was
checked by an additional scan after treatment. Patient stability was excellent and no
errors larger than 2 mm were observed in the follow-up scans for the brain. For the
lung cancer patients, however, larger shifts were observer, probably due to instability
of the matting used for patient comfort. By using on-line CBCT we can correct for
setup errors and verify the patient’s position during treatment, thereby providing a
similar accuracy and safety as stereotactic techniques but more efficient and patient
friendly. However, patient stability during hypofractionated lung radiotherapy must
be improved.
Figure IX.2: Schematic overview of
systematic (arrows) and random (ellipses)
base-line deviations and their locations
found in a group of 32 lung cancer patients.
Analysis of baseline shifts in lung cancer patients With four-dimensional (4D)
CT it is possible to measure the tumor trajectory and mean position relative to the
bony anatomy. A single 4DCT scan, however, captures just a snapshot movie-loop of
the patient’s respiratory motion pattern. We investigated the variability of respiratory
motion over the course of radiotherapy using repeat 4D CBCT scans. It was found in
a group of 32 patients that the shape of the tumor trajectory (averaged over the course
of 4D CBCT acquisition taking approximately 4 min) hardly changes over the course
of treatment (1 mm SD). The center of mass of the trajectory, on the other hand,
exhibited substantially baseline shifts, i.e., variations relative to the bony anatomy.
Systematic deviations were 1 mm (LR), 3 mm (CC) and 2 mm (AP) and similar
random deviations 1 mm (LR), 3 mm (CC) and 3 mm (AP). These baseline shifts
should be incorporated in an appropriate PTV margin or be corrected for using softtissue imaging and guidance. (Figure IX.2)
Anatomical variability of head and neck cancer patients Head and neck cancer
patients in our clinic receive radiotherapy while immobilized by a 5-point mask.
Geometrical deformations of the anatomy due to patient setup were analyzed. In
CBCT scans of 15 patients (7 scans per patient), local rigid body registration was used
to measure displacements of each bone from CBCT to planning CT. To separate
setup error and deformation, vertebrae C3-C5 were used a reference. Deformations
far away from the reference are often larger than the setup errors of C3-C5. Causes of
these deformations are bending if the neck and instability of the mandibula in
patients using a biteblock. In conclusion, head and neck cancer patients often show
anatomical variability that cannot be corrected with a simple couch shift. Careful
registration and selection of the reference point is required, as well as adjustment of
the margins, to avoid underdosage of the target and overdosage of organs at risk.
(Figure IX.3)
Figure IX.3: Local geometrical uncertainties
(AP, CC) after corrections for patient setup
vertebrae C3-C5 taken as correction point.
Effect of respiratory monitoring on tumor motion 4DCT is used to decrease
geometrical uncertainties induced by respiratory tumor motion. During 4DCT, we
use a thermocouple mounted on a standard oxygen mask to register the respiratory
cycle. We investigated the impact of this monitoring system on tumor amplitude and
mean position by applying it during 4DCBCT acquisitions for routine set-up
verification. The respiration signal required for 4DCBCT reconstruction was directly
obtained from the CB projection data making the oxygen mask unnecessary. Half the
scans were performed with the oxygen mask in place. There was no significant
difference in the mean tumor position between the 4D CBCT scans with or without
the respiratory monitoring system. Tumor amplitude, however, increased on average
with 29% (9-64%) for scans with the oxygen mask. We conclude that the use of this
monitoring system has an impact in tumor amplitude, potentially leading to an
overestimation of treatment margins.
Software development for image-guided radiotherapy The implementation of
on-line corrections based on soft tissue imaging of lung tumors required a method
for inline 4D reconstruction, i.e. while the scan is acquired. This is problematic as
the phase-sorting algorithm interpolates over many projection images to estimate the
motion. To solve this problem, a new algorithm was developed in which the
projection images are temporarily cached until sufficient neighbors are available. In
practice this mean that a small fraction of projection images (50 out of 1000) must be
processed after acquisition is complete. The delay between image acquisition and
91
RADIOTHERAPY
Publications
availability of the 4D data is, however only a few seconds. We also implemented
automatic grey-value based registration of the lung tumor in each phase of the 4D
CBCT scan. This means that currently on-line corrections for lung treatments
applying full 4D analysis are feasible. The technique has just been introduced
clinically.
Image acquisition and processing
Keall PJ, Mageras GS, Balter JM,
Emery RS, Forster KM, Jiang SB,
Kapatoes JM, Low DA, Murphy MJ,
Analysis of inter-patient variability in setup motion It is generally assumed that
the statistics of geometric errors differs from patient to patient. We developed a
method to estimate the distribution variability based on analysis of a large patient
population and investigated the effect of detected variability on population based
PTV-margins. Setup data of 470 prostate cancer patients (11 fractions per patient),
were analyzed for random setup errors. The SD of the SD per patient contains the
real distribution variability diluted by “measurement error in the SD” due to the
limited number of samples. After correction is applied for this “measurement error”,
the true variability is about 25% of the SD. This only slightly exceeds the SD
measurement error and is therefore difficult to detect for individual patients. Interpatient distribution variability does lead to larger PTV-margins, because the range of
dose blurring becomes patient dependent. Assuming normality and the same SD
variability in random and systematic errors, the margin for systematic errors
increases from 2.5SD to about 2.8SD, maintaining the same margin for random
errors.
Murray BR, Ramsey CR, Van Herk MB,
Vedam SS, Wong JW, Yorke E. The
management of respiratory motion in
radiation oncology report of AAPM Task
Group 76. Med Phys 2006; 33:3874-3900
Koshani R, Balter JM, Hayman JA,
Henning GT, Van Herk M. Short-term and
long-term reproducibility of lung tumor
position using active breathing control
(ABC). Int J Radiat Oncol Biol Phys 2006;
65:1553-1559
Lotz HT, Pos FJ, Hulshof MCCM,
Van Herk M, Lebesque JV, Duppen JC,
Remeijer P. Tumor motion and
Modeling tumour control probability including geometrical uncertainty For
over 500 high-risk prostate cancer patients the delineated CTV (prostate + SV) and
rectum contours were available, as well as the dose distribution from the treatment
planning system. We simulated many treatment histories by sampling the
distributions of systematic and random set-up errors and organ motion, translating
and rotating the CTV volume relative to the dose. The expected TCP (Webb model)
was estimated and fitted with the clinical data. The effect of the geometrical
uncertainties is a slight reduction of the estimated inter-patient variability in radiosensitivity. This is the first TCP model fit, using a moving CTV as tumor, instead of a
static PTV. Future work will focus on the impact of inhomogeneous clonogen
distributions and incorporation of rectum distention subgroup data.
deformation during external radiotherapy of
bladder cancer. Int J Radiat Oncol Biol
Phys 2006; 64:1551-1558
Ruan D, Fessler JA, Balter JM, Sonke JJ.
Exploring breathing pattern irregularity
with projection-based method. Med Phys
2006; 33:2491-2499
Steenbakkers RJHM, Duppen JC,
Van Herk M, Rasch CRN. In response to
Dr. Rodriguez de Dios. Int J Radiat Oncol
The effect of undersampling pathology on safety margins for microscopic
disease To accurately define the clinical target volume for radiotherapy, pretreatment imaging is be correlated with histopathology. In clinical practice, however,
only a limited number of histopathological slices can be analyzed. In this study, we
investigate the influence of this undersampling on the accuracy of detecting
microscopic disease spread. The microscopic disease is assumed to consist of islets
spread randomly around a spherical tumor, with a probability that decreases
exponentially with distance from the gross tumor. Possible undersampling due to
limited number of slices is calculated for various values of the GTV size, the number
and the size of the microscopic islets, and the number of microscopic slices analyzed.
For instance, using 10 slices, the maximum distance found would on average be only
60% of the actual distance (i.e. 0.84 cm). We conclude that pathology findings of
microscopic spread around tumors must be corrected for possible undersampling
effects. Otherwise, the margin for microscopic disease may be seriously
underestimated.
Figure IX.2: Schematic overview of systematic (arrows) and random (ellipses) base-line deviations and their
locations found in a group of 32 lung cancer patients.
Biol Phys 2006; 65:1276-1277
Steenbakkers RJHM, Duppen JC, Fitton I,
Deurloo KEI, Zijp LJ, Comans EFI,
Uitterhoeve ALJ, Rodrigus PTR,
Kramer GWP, Bussink J, De Jaeger K,
Belderbos JSA, Nowak PJCM,
Van Herk M, Rasch CRN. Reduction of
observer variation using matched CT-PET
for lung cancer delineation: A threedimensional analysis. Int J Radiat Oncol
Biol Phys 2006; 64:435-448
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Publications (continued)
DOSIMETRY
Dosimetry
Leah McDermott, Anton Mans, Jan-Jakob Sonke, Joep Stroom, Marcel Van Herk, Markus Wendling,
Ben Mijnheer
Bloemen-van Gurp EJ, Minken AWH,
Mijnheer BJ, Dehing-Oberye CJG,
Lambin P. Clinical implementation of
MOSFET detectors for dosimetry in electron
beams. Radiother Oncol 2006; 80:288-295
McDermott LN, Wendling M, Sonke JJ,
Van Herk M, Mijnheer BJ. Anatomy
changes in radiotherapy detected using
portal imaging. Radiother Oncol 2006;
79:211-217
Portal dosimetry Portal imaging devices (EPIDs) are now used in our department
for the dosimetric verification of external beam, megavoltage radiation therapy – both
pre-treatment and in vivo. Many studies have reported dosimetric characteristics of
these devices. Some studies ascribed a non-linear signal to gain ghosting and image
lag. Other reports, however, state the effect is negligible. This study compared the
signal-to-monitor unit (MU) ratio for three different brands of EPID systems. The
signal was measured for a wide range of monitor units (5-1000), dose-rates and beam
energies. All EPIDs exhibited a relative under-response for beams of few MUs; giving
4 to 10% lower signal-to-MU ratio relative to that of 1000 MUs as shown in Figure
IX.4 this under-response is consistent with ghosting effects due to charge trapping.
McDermott LN, Wendling M,
Van Asselen B, Stroom J, Sonke JJ,
Van Herk M, Mijnheer BJ. Clinical
experience with EPID dosimetry for
prostate IMRT pre-treatment dose
verification. Med Phys 2006; 33:3921-3930
Figure IX.4: Signal-to-MU ratios for Elekta,
McDermott LN, Nijsten SMJJ, Sonke JJ,
Varian and Siemens a-Si EPIDs, averaged
Partridge M, Van Herk M, Mijnheer BJ.
over 2 to 3 dose-rate settings for different
Comparison of ghosting effects for three
energies, with 1 or 2 detectors for each brand.
commercial a-Si EPIDs. Med Phys 2006;
All points are normalised at 1000 MUs.
33:2448-2451
Different scintillators employed by different
brands will exhibit slight variation in ghosting
Van Elmpt WJC, Nijsten SMJJ, Schiffeleers
effects; however there is a consistent under-
RFH, Dekker ALAJ, Mijnheer BJ, Lambin
response for fields of fewer MUs for all three
P, Minken AWH. A Monte Carlo based
brands.
three-dimensional dose reconstruction
method derived from portal dose images.
Med Phys 2006; 33:2426-2434
Wendling M, Louwe RJW, McDermott LN,
Sonke JJ, Van Herk M, Mijnheer BJ.
Accurate two-dimensional IMRT
verification using a back-projection EPID
dosimetry method. Med Phys 2006;
33:259-273
In order to investigate the feasibility of replacing pre-treatment with in vivo EPID
dosimetry for IMRT prostate cancer treatments, dose distributions of 75 patient plans
were reconstructed from EPID images, inside a patient or phantom. Planned and
EPID dose values were compared at the isocentre and in 2D using the g index
(3%/3mm). The number of measured fractions required to detect the same errors as
pre-treatment verification was determined, and the time required to perform both
methods was compared. Average planned and EPID isocentre dose values agreed
within 1% for both pre-treatment verification and 5 in vivo fractions. An alert was
raised for 10 out of these 75 pre-treatment checks, however, with no clinically relevant
errors. Multiple in vivo fractions were combined by assessing gamma images
comprising median, minimum and low pixel values of 1 to 5 fractions. Eleven plans
had discrepancies using the low gamma values of 3 fractions. Additional time for pretreatment checks was ~2.5 h per plan. For in vivo dosimetry, only an additional 15 min
+ 10 min/fraction were required. It can be concluded that in vivo EPID dosimetry is
an efficient alternative to pre-treatment verification for prostate IMRT treatments.
The number of in vivo fractions required to replace pre-treatment verification is a
balance between accurate detection and workload. Checking three fractions in vivo
appears to be optimal, in order to distinguish systematic errors from random errors,
with very little additional time required for verification.
The g-evaluation method is a tool by which dose distributions can be compared in a
quantitative manner combining dose-difference and distance-to-agreement criteria.
One major disadvantage is, however, its long computation time, which especially
applies to the comparison of 3D dose distributions. In this study, we present a fast
algorithm for a full 3D g-evaluation at high resolution. Both the reference and
evaluated dose distributions are first resampled on the same grid. For each point of
the reference dose distribution, the algorithm searches for the best point of
agreement according to the g method in the evaluated dose distribution, which can
be done at a sub-voxel resolution. Speed, computer memory efficiency, and high
93
RADIOTHERAPY
Publications (continued)
spatial resolution are achieved by searching around each reference point with
increasing distance in a sphere. The smaller the sample step size and the larger the
differences between the dose distributions, the longer the g-evaluation takes. Two
clinical examples were investigated using 3% of the maximum dose as dosedifference and 0.3 cm as distance-to-agreement criteria. For 0.2 cm grid spacing, the
change in g indices was negligible below a sample step size of 0.02 cm. Comparing
the full 3D g-evaluation and slice-by-slice 2D g-evaluation for these clinical examples,
the g indices improved by searching in full 3D space, with the average g index
decreasing by at least 7%.
Treatment planning
Schwarz M, Van der Geer J, Van Herk M,
Lebesque JV, Mijnheer BJ, Damen EMF.
Impact of geometrical uncertainties on 3D
CRT and IMRT dose distributions for lung
cancer treatment. Int J Radiat Oncol Biol
Phys 2006; 65:1260-1269
TREATMENT PLANNING
Van Asselen B, Schwarz M,
Jose Belderbos, Suzanne Van Beek, Martijn Eenink, Marcel Van Herk, Katrien De Jaeger1,
Vliet-Vroegindeweij C, Lebesque JV,
Annemarie. Lakeman-Groot, Emmy Lamers, Joos Lebesque, Ben Mijnheer, Tom Minderhoud,
Mijnheer BJ, Damen EMF. Intensity-
Anke Van Mourik, Coen Rasch, Christoph Schneider, Marco Schwarz, Jan-Jakob Sonke,
modulated radiotherapy of breast cancer
Corine Van Vliet-Vroegindeweij, Maddalena Rossi, Jochem Wolthaus, Eugene Damen
using direct aperture optimization.
Radiother Oncol 2006; 79:162-169
Evaluation of different strategies for integration of 4D CT scans into the treatment
planning of lung cancer patients. For 45 patients with lung tumors, tumor size and
tumor motion were assessed from 4D CT scans. The average tumor volume was
40 В± 47 cm3, while the average tumor motion was 1.9 В± 1.3 mm, 7.2 В± 5.5 mm, and
3.1 В± 2.3 mm in left-right, cranial-caudal, and anterior-posterior direction, respectively.
The GTV was delineated in the first phase of the 4D scan by an experienced
radiation-oncologist. The delineation was copied to the other phases by applying the
transformation matrix resulting from a phase-to-phase tumor match. Three different
strategies to incorporate tumor motion and setup uncertainties into the PTV were
evaluated: 1) the concept of Internal Target Volume (ITV); 2) gating at the exhale
phase of the breathing cycle; 3) the use of a mid-ventilation scan. For each approach,
the GTV to PTV margin was calculated using the recipe of van Herk: margin =
2.5S + 0.7s, where S and s are the standard deviations of systematic and random
errors, respectively. The resulting PTV volumes were compared to the conventional
approach, i.e. without using a 4D CT scan.
The ITV approach resulted in an average increase of the PTV volume with 5.5%
В± 4.5%. Gating and the mid-ventilation concept resulted in an average decrease of
PTV volume with 12.7% В± 10.8%, and 8.9% В± 8.6%, respectively.
Based on the results it was concluded that the ITV concept leads to grossly
overestimated PTV volumes compared to the mid-ventilation and gating approaches
and may therefore lead to an increased probability of normal tissue complications.
Motion Estimation in 4D CT scans for assessment of internal motion, image
enhancement and construction of the optimal CT scan for treatment planning
Currently we use a mid-ventilation CT scan (MidV CT) for treatment planning of
lung tumors. This MidV CT is reconstructed from a 4D CT scan and represents the
tumor and surrounding lung tissue in its average position over the breathing cycle.
However, by choosing the MidV CT frame from the 4D data set, a geometrical error
( eh ) with respect to the mean position is introduced due to hysteresis in the tumor
motion. To overcome this problem, software was developed to warp each frame of the
4D CT scan and project all structures on their time-weighted mean position. The
software uses an adaptation of an existing motion estimation method. The resulting
CT scan depicts the tumor in its exact time-averaged position and yields less noisy
images since the transformed CT frames are averaged (see Figure IX.5).
The influence of heterogeneity corrections on the dose distribution of
stereotactic treatment of lung tumors A protocol for stereotactic irradiation of
medically inoperable stage I NSCLC has been introduced, closely following the
RTOG 0236 phase II trial. The RTOG protocol required dose calculation without
correction for tissue heterogeneity. Since this is not the current clinical practice in
our institution, we investigated the influence of heterogeneity corrections on the
prescription dose and the dose to organs at risk. The planning CT scans of 8 patients
with NSCLC were used to compare different radiation plans. The IMRT plans were
1
Catharina Ziekenhuis, Eindhoven, The Netherlands
Wolthaus JWH, Schneider C, Sonke JJ,
Van Herk M, Belderbos JSA, Rossi MMG,
Lebesque JV, Damen EMF. Mid-ventilation
CT scan construction from four-dimensional
respiration-correlated CT scans for
radiotherapy planning of lung cancer
patients. Int J Radiat Oncol Biol Phys
2006; 65:1560-1571
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RADIOTHERAPY
Figure IX.5: Results of motion estimation software. The upper row shows in column 1-3 coronal
reconstructions of phase 0, 3, and 7 of the original 4D CT scan (10 phases). The last column shows the
average over all phases. The middle row shows the deformation fields from phase 3 and 7 to phase 0.
The lower row shows the result of deforming phase 3 and 7 to phase 0 (column 2 and 3). The last column
of the lower row shows the average of the deformed images, illustrating the reduction in image noise that
can be achieved by this method.
optimized in Pinnnacle with direct machine parameter optimization (DMPO, 10-20
beams, 1-2 segments per beam), with and without heterogeneity correction in the
dose calculation. Dose prescription for the homogeneous plans (i.e. calculated
without heterogeneity correction) was according to RTOG 0236 criteria, i.e. 60 Gy
(20 Gy per fraction) was prescribed to the isodose surface encompassing 95% of the
PTV volume. For the heterogeneous plans (i.e. calculated with heterogeneity
correction), dose calculation was performed while maintaining the monitor units of
the homogeneous plan. Compared to the homogeneous plan, heterogeneity
correction resulted on average in a lower dose to the PTV: average = 54.9 Gy (range
46.6 to 61.7 Gy). Due to decreased attenuation and broadening of the beam
penumbra in low-density lung tissue, the relative maximum dose (Dmax) in the PTV
was higher for the heterogeneous plans (i.e. the dose in the PTV was less
homogeneous), while dose fall-off outside the PTV was less steep. The latter is
reflected in a higher value of V20 for lung.
Intensity-modulated versus conformal radiotherapy of parotid gland tumors:
potential impact on hearing loss The purpose of this study was to design and
evaluate an IMRT class solution in order to prevent hearing loss due to irradiation of
parotid gland tumors. Twenty patients with parotid gland cancer were retrospectively
planned with two different techniques using the original planning target volume
(PTV). First a conventional technique using a wedged beam pair was applied yielding
a dose distribution conformal to the shape of the PTV. Next an IMRT technique with
pre-defined optimization constraints was designed. A dose of 66 Gy in the PTV was
given at the ICRU dose prescription point. Dose distributions were compared with
respect to PTV coverage and dose to organs at risk (OAR).
With similar PTV coverage, the dose to OARs was lower in the IMRT plans. The
mean volume of the middle ear receiving a dose higher than 50 Gy decreased from
66.5 to 33.4%. The mean dose in the oral cavity decreased from 19.4 Gy to 16.6 Gy.
The auditory system can be spared if the distance between the inner ear and the PTV
is 0.6 cm or larger, and if the overlap between the middle ear and the PTV is smaller
than 10%. The maximum dose in the spinal cord was below 40 Gy in all treatment
plans. It can be concluded that the auditory system as well as the oral cavity can be
spared with IMRT.
95
RADIOTHERAPY
Publications (continued)
Evaluation of a single-isocenter technique for axillary radiotherapy in breast
cancer A planning study was performed in 20 breast cancer patients, comparing a
conventional AP-PA technique with a single-isocenter technique for irradiation of the
axilla. The target volume of the axillary treatment encompassed the periclavicular and
axillary lymph node areas. The lymph node areas and the organs at risk (OAR, i.e.,
the esophagus, spinal cord, brachial plexus, and lung) were delineated on CT scans.
The conventional technique consisted of an AP-PA field with a transmission plate
placed in the AP beam. The single-isocenter technique consisted of AP-PA fields
using a gantry rotation of + or -20 degrees and a medial AP segment. Dose
distributions were analyzed with respect to target coverage and dose to OARs. The
field borders and humeral shielding were re-defined based on the 3D anatomical
references. Adapting the humeral shielding reduced the irradiated volume by 19%
and might contribute to a reduction of the incidence of arm edema and impairment
of shoulder function. With similar PTV coverage and maximum dose to the brachial
plexus, the maximum dose in the esophagus and spinal cord was reduced by more
than 50% using the single-isocenter technique. Moreover, the single-isocenter
technique allowed a fast and easy treatment preparation and reduced the execution
time considerably (with approximately 10 minutes per fraction).
Studies of radiation response in tumors and
organs at risk
Belderbos JSA, Heemsbergen WD,
De Jaeger K, Baas P, Lebesque JV. Final
results of a Phase I/II dose escalation trial
in non-small-cell lung cancer using threedimensional conformal radiotherapy. Int J
Radiat Oncol Biol Phys 2006; 66:126-134
Heemsbergen WD, Peeters STH,
Koper PCM, Hoogeman MS, Lebesque JV.
Acute and late gastrointestinal toxicity after
radiotherapy in prostate cancer patients:
Consequential late damage. Int J Radiat
Oncol Biol Phys 2006; 66:3-10
Heemsbergen WD, Hoogeman MS,
STUDIES OF RADIATION RESPONSE IN TUMORS AND ORGANS AT RISK
Witte MG, Peeters S, Incrocci L,
Paul Baas, Jose Belderbos, Michel Dielwart1, Katrien De Jaeger2, Guus Hart, Wilma Heemsbergen,
Lebesque JV. Increased risk of biochemical
Mischa Hoogeman3, Peter Koper3, Stephanie Peeters, Wim Van Putten3, Annerie Slot4,
and clinical failur for prostate patients with
Joos Lebesque
a large rectum at radiotherapy planning:
results from the Dutch trial of 68 Gy
Prostate cancer: The Dutch dose escalation randomized trial of 68 Gy versus 78 Gy
with 669 prostate cancer patients showed a significantly improved freedom from
failure in patients treated to 78 Gy, at the cost of a very modest increase in
gastrointestinal toxicity. Different aspects of this toxicity were studied in detail.
versus 78 Gy. Int J Radiat Oncol Biol Phys
2007; In press
Nuver T, Hoogeman MS, Remeijer P,
Van Herk MB, Lebesque JV. An adaptive
Localized volume effects for late rectal and anal toxicity after radiotherapy for
prostate cancer In this analysis 641 patients from the Dutch dose escalation
randomized trial (68 Gy vs. 78 Gy) were included. Toxicity was scored with adapted
RTOG/EORTC criteria and five specific complications. The variables derived from
DVH of anorectal, rectal and anal wall were: % receiving ≥ 5 to 70 Gy (V5-V70),
maximum (Dmax) and mean dose (Dmean). The anus was defined as the most caudal
3 cm of the anorectum. Statistics were done with multivariate Cox regression models.
Median follow-up was 44 months.
Anal dosimetric variables were associated with RTOG/EORTC grade ≥ 2 (V5-V40,
Dmean), and incontinence (V5-V70, Dmean). Bleeding correlated most strongly with
anorectal V55-V65, and stool frequency with anorectal V40 and Dmean. Use of
steroids was weakly related to anal variables. No volume effect was seen for RTOG/
EORTC grade ≥ 3 and pain/cramps/tenesmus.
off-line procedure for radiotherapy of
prostate cancer. Int J Radiat Oncol Biol
Phys 2007; In press
Peeters STH, Heemsbergen WD,
Koper PC, Van Putten WL, Slot A,
Dielwart MF, Bonfrer JM, Incrocci L,
Lebesque JV. Dose-response in radiotherapy
for localized prostate cancer: results of the
Dutch multicenter randomized phase III
trial comparing 68 Gy of radiotherapy with
78 Gy. J Clin Oncol 2006; 24:1990-1996
Peeters STH, Lebesque JV,
Rectal bleeding, fecal incontinence and high stool frequency after conformal
radiotherapy for prostate cancer: normal tissue complication probability
modeling This study included 468 prostate cancer patients participating in the above
mentioned trial. We fitted the probability of developing late toxicity within 3 years
(rectal bleeding, high stool frequency and fecal incontinence) with the original, and a
modified LKB-model, in which a clinical feature (e.g. history of abdominal surgery)
was taken into account by fitting subset specific TD50’s. The ratio of these TD50’s is
the dose-modifying factor for that clinical feature. Dose distributions of anorectal
(bleeding and frequency) and anal wall (fecal incontinence) were used.
The modified LKB-model gave significantly better fits than the original LKB-model.
Patients with a history of abdominal surgery had a lower tolerance to radiation than
patients without previous surgery, with a dose-modifying factor of 1.1 for bleeding,
and of 2.5 for fecal incontinence. The dose-response curve for bleeding was
Heemsbergen WD, Van Putten WLJ,
Slot A, Dielwart MFH, Koper PCM.
Localized volume effects for late rectal and
anal toxicity after radiotherapy for prostate
cancer. Int J Radiat Oncol Biol Phys 2006;
64:1151-1161
Peeters STH, Hoogeman MS,
Heemsbergen WD, Hart AAM,
Koper PCM, Lebesque JV. Rectal bleeding,
fecal incontinence, and high stool frequency
after conformal radiotherapy for prostate
cancer: Normal tissue complication
probability modeling. Int J Radiat Oncol
1 Zeeuws Radiotherapeutic Institute, Vlissingen, The Netherlands
2 Catharina Ziekenhuis, Eindhoven, The Netherlands
3 Erasmus MC-Daniel Hoed den Cancer Center, Rotterdam, The Netherlands
4 Radiotherapeutic Institute Friesland, Leeuwarden, The Netherlands
Biol Phys 2006; 66:11-19
96
RADIOTHERAPY
Publications (continued)
Breast cancer
Antonini N, Jones H, Horiot JC,
Poortmans P, Struikmans H,
Den Bogaert WV, Barillot I, Fourquet A,
Jager J, Hoogenraad W. Effect of age and
radiation dose on local control after breast
conserving treatment: EORTC trial 2288110882. Radiother Oncol 2006; In Press
Bijker N, Meijnen P, Peterse JL,
Bogaerts J, Van Hoorebeeck I, Julien JP,
Gennaro M, Rouanet P, Avril A,
Fentiman IS, Bartelink H, Rutgers EJ.
approximately two times steeper than that for frequency, and three times steeper than
that for fecal incontinence
Acute and late gastrointestinal toxicity after radiotherapy in prostate cancer
patients: consequential late damage We studied whether there was a direct
relationship between acute and late GI toxicity for the patients in the trial. We defined
three outcomes for acute reactions: 1) maximum RTOG (Radiation Therapy Oncology
Group) acute toxicity, 2) maximum acute mucous discharge (AMD) and 3) maximum
acute proctitis. At multivariate analysis, AMD and acute proctitis were strong
predictors for overall late toxicity, �intermittent bleeding’ and �incontinence pads’
(p≤0.01). For �stools ≥ 6/day’ all three were strong predictors. No significant
associations were found for �severe bleeding’ and �use of steroids’. The predictive
power of the dose parameters remained at the same level or became weaker for most
late endpoints. These results suggest a significant consequential component in the
development of late GI toxicity.
Breast-conserving treatment with or without
radiotherapy in ductal carcinoma-in-situ:
ten-year results of European Organisation
for Research and Treatment of Cancer
randomized phase III trial 10853--a study
by the EORTC Breast Cancer Cooperative
Group and EORTC Radiotherapy Group.
J Clin Oncol 2006; 24:3381-3387
Lung
Final results of a phase I/II dose escalation trial in non-small cell lung cancer
using three-dimensional conformal radiotherapy A phase I/II trial was
performed including inoperable non-small cell lung cancer patients. Patients were
irradiated once or twice daily, with 2.25 Gy per fraction and a fixed overall treatment
time of 6 weeks. According to the relative mean lung dose (rMLD) 5 risk groups were
defined. The rMLD was defined as the ratio of the MLD and the NTD normalized
prescribed dose. The rMLD boundaries of these risk groups were: group 1, 0.0-0.12;
group 2, 0.12-0.18; group 3, 0.18-0.24; group 4, 0.24-0.31; and group 5, 0.31-0.40.
The dose was escalated with 6.75 Gy after a follow-up of 6 months without DLT.
Eighty-eight patients included had tumor stage I or II in 53%, IIIA in 31% and IIIB in
17%. The maximum tolerated dose (MTD) was not achieved in the lowest risk group
(reached dose 94.5 Gy). For risk groups 2 and 3 the MTD was 81 Gy. The 74.3 Gy was
safe for group 4. In group 5 the starting dose level of 60.8 Gy was evaluated and safe.
In 2 patients (5%) an isolated nodal relapse occurred. The dose delivered did have an
impact on failure-free interval (FFI) and overall survival (OS), especially for small
tumor volumes in multivariable analysis.
In conclusion, dose escalation is safe up to 94.5 Gy in 42 fractions in 6 weeks in
patients with a MLD ≤ 13.6 Gy. Higher doses are associated with a better FFI and OS
for smaller tumor volumes. Involved-field irradiation results in a low percentage of
isolated nodal relapses.
BREAST CANCER
Ninja Antonini, Liesbeth Boersma1, Eugene Damen, Guus Hart, Heather Jones, Bas Kreike,
Ben Mijnheer, Nicola Russell, Laura Van ’t Veer, Marc Van De Vijver, Conny Vrieling, Harry Bartelink
Figure IX.6: Rish on local recurrence by age.
The 10 years result of an EORTC trial were analyzed to investigate the long term
impact of a boost radiation dose of 16 Gy on local control, risk of developing fibrosis
and overall survival for patients with stage I and II breast cancer who underwent
lumpectomy and whole breast irradiation as part of their breast conserving therapy
(BCT). In this trial 5318 patients with microscopically complete excision followed by
whole breast irradiation of 50 Gy were randomized between a boost dose of 16 Gy
(2661 patients) and no boost dose (2657 patients) and follow-up for a median of 10.8
years. The median age was 55 years. Local recurrence was reported as the first failure
in 278 with boost vs. 165 patients without boost; at 10 years, the cumulative incidence
of local recurrence was 10.2% vs. 6.2 % for the no boost and the boost group,
respectively (P<0.0001). The hazard ratio of local recurrence was 0.59 (0.46 – 0.76)
in favour of the boost, with no statistically significant interaction per age group. The
absolute risk reduction at 10 year per age group was the largest in patients 40 years
or less 23.9% to 13.5% (P=0.0014). Severe fibrosis was statistically significantly
(P< 0.0001) increased in the boost group with 10-year rate of 4.4% versus 1.6% in
the no boost group (P<0.0001). Survival at 10 years was 82% in both arms. We
concluded that a boost irradiation of 16 Gy improved local control in all age groups at
1
MAASTRO Clinic, Maastricht, The Netherlands
97
RADIOTHERAPY
Publications (continued)
the cost of a small increase of severe fibrosis, while no difference in survival was
observed.
Mechanisms, modulation and prediction of
radiation-induced cell death
MECHANISMS, MODULATION AND PREDICTION OF RADIATION-INDUCED
CELL DEATH
Den Dulk M, Verheij M, Cats A,
Jannie Borst, Daphne De Jong, Rick Haas, Frank Hoebers, Marina Kartachova, Jeffrey Klarenbeek,
Jansen EPM, Hartgrink HH,
Menno Van Lummel, Jan Schellens, Renato ValdГ©s Olmos, Wim Van Blitterswijk, Inge Verbrugge,
Van de Velde CJH. The essentials of
Stefan Vink, Esther Wissink, Shuraila Zerp, Harry Bartelink, Marcel Verheij
locoregional control in the treatment
of gastric cancer. Scand J Surg 2007;
Our line of research continues to focus on (1) the identification of novel targets and
agents to increase the cytotoxic effect of radiation and on (2) the validation of new
endpoints to quantify and predict the efficacy and toxicity of new combination
therapies. The ultimate goal is to translate these strategies from the lab into the
clinic.
95:242-247
Hoebers FJP, Pluim D, Verheij M,
Balm AJM, Bartelink H, Schellens JHM,
Begg AC. Prediction of treatment outcome
by cisplatin-DNA adduct formation in
Alkyl-lysophospholipids (ALPs) ALPs such as Perifosine represent a first group of
compounds that have become available for clinical use along this approach. These
synthetic anti-tumor agents are known to accumulate in dynamic, sphingomyelin
(SM) and cholesterol-enriched plasma membrane microdomains, also known as
“lipid rafts”. Once taken up via these membrane portals, ALPs interfere with lipid
metabolism, inhibit survival signaling and stimulate apoptosis induction. In
combination with radiation, ALPs cause a synergistic apoptotic effect and reduce
clonogenic cell survival. Besides these radiosensitizing properties, ALPs show
inhibition of cell cycle progression and potent anti-angiogenic effects in vitro. In a
separate project (in collaboration with W Van Blitterswijk, Division III) we investigate
mechanisms of cellular ALP 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 ALPs 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 ALP. 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. The common factor of this “multi-stress resistance” is
a defect in SM synthesis by downregulation of the enzyme SMS1. The functional
consequences, however, are different for the various apoptotic stimuli and are topic of
current investigation.
Following their in vitro characterization, ALPs were subsequently tested in vivo. In a
mouse xenograft KB squamous cell carcinoma model the combination of oral
Perifosine and radiation resulted in complete and sustained tumor regression.
Histopathological examination demonstrated a prominent apoptotic tumor response.
These in vitro and in vivo observations constituted the basis for a clinical phase I
study that was completed in 2004. This study defined the MTD, toxicity profile and
optimal scheduling of the combined treatment consisting of oral administration of
Perifosine and radiotherapy in patients with locally advanced inoperable solid
tumors. Next, we initiated a multicenter randomized placebo-controlled phase II
study in locally advanced NSCLC, randomizing patients between radiation + placebo
and radiation + daily Perifosine.
patients with stage III/IV head and neck
squamous cell carcinoma, treated by
concurrent cisplatin-radiation
(RADPLAT). Int J Cancer 2006;
119:750-756
Horsman MR, Bohm L, Margison GP,
Milas L, Rosier JF, Safrany G, Selzer E,
Verheij M, Hendry JH. Tumor
radiosensitizers - Current status of
development of various approaches: Report
of an International Atomic Energy Agency
meeting. Int J Radiat Oncol Biol Phys
2006; 64:551-561
Jansen EPM, Saunders MP, Boot H,
Oppedijk V, Dubbelman R, Porritt B,
Cats A, Stroom J, ValdГ©s Olmos R,
Bartelink H, Verheij M. Prospective study
on late renal toxicity following postoperative
chemoradiotherapy in gastric cancer. Int J
Radiat Oncol Biol Phys 2007; In Press,
Corrected Proof
Kartachova MS, ValdГ©s Olmos R,
Haas RLM, Hoebers FJP,
Van den Brekel MW, Van Zandwijk N,
Herk MV, Verheij M. Mapping of
treatment-induced apoptosis in normal
structures: 99mTc-Hynic-rh-annexin
V SPECT and CT image fusion. Eur J Nucl
Med Mol Imaging 2006; 33:893-899
Van de Velde CJH, Verheij M, Cats A.
Other radiosensitizers in preclinical development In a separate project with
J Borst (Division IV) we study the interaction between the death receptor ligand
TRAIL and radiation. TRAIL induces apoptosis in a wide variety of tumor tissues, but
lacks normal tissue toxicity in preclinical animal models. Although of interest as a
therapeutic modality in itself, TRAIL is also an attractive candidate for combination
therapy, 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. For proof of principle,
we use lymphoid cells as model system, because these cells effectively die by
apoptosis upon treatment with either radiation or TRAIL. In both EВµ myc and Jurkat
cells, we demonstrated combined (additive to synergistic) effects of TRAIL and
radiation, which were independent of the p53 status of the cells. In Jurkat cells, the
Gastric cancer: a personal view. Adv
Gastrointest Canc 2006; In press
Van der Luit AH, Budde M, Zerp S,
Caan W, Klarenbeek JB, Verheij M,
Van Blitterswijk WJ. Resistance to alkyllysophospholipid-induced apoptosis due to
downregulated sphingomyelin synthase 1
expression with consequent sphingomyelinand cholesterol-deficiency in lipid rafts.
Biochem J 2007; 401:541-549
98
RADIOTHERAPY
Publications (continued)
Vink SR, Schellens JHM, Beijnen JH,
Sindermann H, Engel J, Dubbelman R,
Moppi G, Hillebrand MJX, Bartelink H,
Verheij M. Phase I and pharmacokinetic
study of combined treatment with perifosine
and radiation in patients with advanced
solid tumours. Radiother Oncol 2006;
80:207-213
Vink SR, Lagerwerf S, Mesman E,
Schellens JHM, Begg AC, Van Blitterswijk
WJ, Verheij M. Radiosensitization of
squamous cell carcinoma by the
apoptotic response to radiation or TRAIL is blocked by Bcl-2 (indicating dependence
on the mitochondrial signalling pathway). Interestingly, upon treatment of the cells
with radiation, TRAIL can effectively induce apoptosis independent of the
mitochondrial pathway. This results in synergistic cell kill upon combined treatment.
The effectiveness of the combined treatment modality was subsequently evaluated in
vivo in collaboration with Dr. Henning Walczak, DKFZ, Germany. Jurkat T cells
overexpressing Bcl-2 were grown as xenografts in mice and treated with radiation,
IZ-huTRAIL i.p and the combination. Tumor growth delay was measured by caliper
and luciferin-based in vivo bioluminescence. While radiation or TRAIL alone caused
significant tumor growth delay, the combination was much more effective as tumors
regressed and showed no regrowth (Figure IX.7). Normal tissue toxicity measured as
changes in body weight and in liver enzymes, was minimal. These experiments are
currently expanded with another death receptor ligand (FasL) and using spontaneous
solid tumor mouse models for mesothelioma.
alkylphospholipid perifosine in cell culture
and xenografts. Clin Cancer Res 2006;
12:1615-1622
Wissink EHJ, Verbrugge I, Vink SR,
Schader MB, Schaefer U, Walczak H,
Borst J, Verheij M. TRAIL enhances
efficacy of radiotherapy in a p53 mutant,
Bcl-2 overexpressing lymphoid malignancy.
Radiother Oncol 2006; 80:214-222
Figure IX.7: Inhibition of tumor growth by radiation (IR) and/or TRAIL. Sequential bioluminescence
images of animals treated with vehicle (control) or with IR + TRAIL.
Figure IX.8: Chemical structure of racemic
Gossypol, a small molecule inhibitor of antiapoptotic Bcl-2 family members.
Sequential bioluminescence images of animals treated with vehicle (control)
or with IR + TRAIL
In collaboration with Drs. Lippman and Yang from the University of Michigan 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
and Bcl-2. Gossypol binds to the BH3 domains of Bcl-2, Bcl-XL, and possibly other
family members, and induces apoptosis. Because overexpression of these antiapoptotic molecules confers radioresistance and is often associated with poor
prognosis, inhibition of Bcl-2/Bcl-XL represents a promising strategy to increase
tumor response to radiation. A panel of human leukemic and head and neck cancer
cell lines has been selected based on their expression pattern of the most relevant
Bcl-2 family members. Gossypol was found to induce apoptosis in a time- and dosedependent fashion and to increase radiation-induced cell death. Isobolographic
analysis revealed a synergistic interaction between radiation and Gossypol in these
cell systems. In addition, activation of the pro-apoptotic SAPK/JNK pathway appeared
to play an important role in this response as blockade of this signaling cascade by
kinase inhibitor or overexpression of a dominant negative mutant of c-Jun (TAM-67)
abrogated Gossypol-induced apoptosis. (Figure IX.8)
Prediction of tumor response (1) In vivo apoptosis imaging. To evaluate whether
early treatment-induced apoptosis 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 (ValdГ©s 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
99
RADIOTHERAPY
Publications (continued)
radiotherapy and/or chemotherapy. A total of 65 patients underwent TAVS before
and early after the start of treatment. A statistically significant correlation was found
between the increase in annexin V tumor uptake on the post-treatment scan and
tumor response. These results indicate that TAVS might be useful as a non-invasive
predictive test for treatment outcome.
(2) Cisplatin-DNA adducts. Levels of cisplatin-DNA adducts have been shown to
correlate with treatment outcome in patients with NSCLC. Quantification of adduct
levels may provide a valuable tool to select patients for cisplatin-based combined
modality treatment. In collaboration with the Division VIII (Begg and Schellens) we
measure levels of cisplatin-DNA adducts in tumor and normal tissue obtained from
different patient populations treated with cisplatin-based chemoradiation. In patients
with stage IV head and neck cancer treated by conventional i.v (100 mg/m2),
supradose i.a (150 mg/m2), or daily low dose i.v. (6 mg/m2) cisplatin-based
chemoradiation (RADPLAT) and in cervical cancer patients treated by 40 mg/m2
cisplatin-based chemoradiation adduct levels have been measured. Samples for
cisplatin-DNA adduct determination were obtained from 77 patients. Normal tissue
samples were taken from all patients, tumor biopsies from 28 patients. Adduct-levels
in tumor were 2.5-5 times higher than those in WBC for all 4 treatment regimes
(p < 0.001). Analysis by schedule showed that increasing doses of cisplatin resulted
in higher levels of GG-adducts in primary tumor (Spearman rank-correlation,
r = 0.70, p < 0.001). For normal tissue (WBC and buccal cells) a similar pattern was
seen for the i.v. schedules. However, the i.a. 150 schedule resulted in decreased GGadduct formation in normal tissue compared to the i.v. 100 schedule, indicative of
more systemic exposure to cisplatin after i.v. cisplatin compared to selective highdose i.a. cisplatin administration. In multivariate analysis the relative contribution of
cisplatin-dose and mode of administration (i.v. vs i.a.) were calculated separately. The
level of GG-adducts was higher with higher cisplatin-dose (increase about 1.5% per
mg cisplatin; p < 0.001). Adduct-levels in normal tissue were lower after i.a.
administration than expected from this dose effect (by about 65 - 70%; p < 0.001).
The effect of i.a. administration on primary tumor adducts showed a non-significant
effect compared to i.v. treatment. In a subgroup of 35 head and neck cancer patients
treated with conventional i.v. or supradose i.a. cisplatin-based chemoradiation, high
tumor GG-adduct levels predicted better disease free survival than low adduct levels.
Combination of radiotherapy and
chemotherapy
Van den Broek GB, Balm AJM,
Van den Brekel MWM, Hauptmann M,
Schornagel JH, Rasch CRN. Relationship
between clinical factors and the incidence of
toxicity after intra-arterial chemoradiation
for head and neck cancer. Radiother Oncol
2006; 81:143-150
Van den Broek GB, Rasch CRN,
Pameijer FA, Peter E, Van den Brekel
MWM, Balm AJM. Response measurement
after intraarterial chemoradiation in
advanced head and neck carcinoma:
Magnetic resonance imaging and
evaluation under general anesthesia?
Cancer 2006; 106:1722-1729
Zuur CL, Simis YJW, Lansdaal PEM,
Rasch CRN, Tange RA, Balm AJM,
Dreschler WA. Audiometric patterns in
ototoxicity of intra-arterial cisplatin
chemoradiation in patients with locally
advanced head and neck cancer. Audiol
Neurootol 2006; 11:318-330
99mTc
Hynic-rh-Annexin V scintigraphy (TAVS) imaging is a non-invasive technique
to demonstrate apoptosis in vivo. It was previously shown that the degree of Annexinuptake correlated with response to therapy in lymphoma patients (Kartachova et. al,
2004). We now used this technique in patients with head and neck squamous cell
carcinoma, treated with concurrent cisplatin-based chemoradiotherapy and wanted to
evaluate patterns of Annexin-uptake in normal tissue and tumor. TAVS was
performed before and within 48 hours after the 1st course of cisplatin-based
chemoradiation. SPECT data were co-registered to planning CT-scan. A complete set
of these data was available for 13 patients. Regions of interest (ROI) were delineated:
primary tumor, involved lymph nodes and salivary glands. The radiation-dose at
follow-up TAVS was calculated for each ROI. Annexin-uptake was determined in each
ROI, and the difference between follow-up and baseline TAVS represented the
absolute Annexin-uptake: Delta-uptake (О”U). Annexin V scintigraphy showed a
radiation-dose-dependent uptake in parotid glands, indicative of early apoptosis
during treatment. The interindividual spread in Annexin-uptake in primary tumors
could not be related to differences in treatment schedule or tumor volume, but the
Annexin-uptake in tumor and lymph nodes were closely correlated. This effect might
represent a tumor-specific apoptotic response.
Other
Adler AS, Lin M, Horlings H,
Nuyten DSA, Van de Vijver MJ, Chang HY.
Genetic regulators of large-scale
transcriptional signatures in cancer.
Nat Genet 2006; 38:421-430
Aleman BMP, Van den Belt-Dusebout AW,
De Bruin ML, Van’t Veer MB,
Baaijens MH, De Boer JP, Hart AA,
Klokman WJ, Kuenen MA, Ouwens GM,
Bartelink H, Van Leeuwen FE. Late
cardiotoxicity after treatment for Hodgkin’s
lymphoma. Blood 2006;[Epub ahead of
print]
COMBINATION OF RADIOTHERAPY AND CHEMOTHERAPY
Baas P, Belderbos JSA, Senan S, Kwa HB,
Berthe Aleman, Jose Belderbos, Henk Boot, Edwin Jansen, Corrie Marijnen, Annemiek Cats,
Van Bochove A, Van Tinteren H,
Floor Van Leeuwen, Marcel Verheij, Harry Bartelink, Coen Rasch
Burgers JA, Van Meerbeeck JP. Concurrent
chemotherapy (carboplatin, paclitaxel,
Head and neck cancer Cisplatin and irradiation for inoperable head and neck
cancer: a multi-institutional trial comparing intra-arterial with intravenous cisplatin
chemo-radiation was closed early November 2004. A total of 240 patients with stage
III/IV head and neck cancer were included. Median follow-up was 20 months. No
etoposide) and involved-field radiotherapy
in limited stage small cell lung cancer:
A Dutch multicenter phase II study.
Br J Cancer 2006; 94:625-630
100
RADIOTHERAPY
Publications (continued)
Belderbos JSA, Heemsbergen WD,
difference was found in the loco-regional recurrence rate between the treatment arms
(Figure IX.9).
De Jaeger K, Baas P, Lebesque JV. Final
results of a Phase I/II dose escalation trial
Figure IX.9: Locoregional control as first
in non-small-cell lung cancer using three-
event by treatment arm.
dimensional conformal radiotherapy. Int J
Radiat Oncol Biol Phys 2006; 66:126-134
Bijker N, Meijnen P, Peterse JL,
Bogaerts J, Van Hoorebeeck I, Julien JP,
Gennaro M, Rouanet P, Avril A,
Fentiman IS, Bartelink H, Rutgers EJ.
Breast-conserving treatment with or without
radiotherapy in ductal carcinoma-in-situ:
ten-year results of European Organisation
for Research and Treatment of Cancer
randomized phase III trial 10853--a study
by the EORTC Breast Cancer Cooperative
Group and EORTC Radiotherapy Group.
J Clin Oncol 2006; 24:3381-3387
Boyle P, Yasantha Ariyaratne M,
Barrington R, Bartelink H, Bartsch G,
Berns A, De Valeriola D, Dinshaw KA,
Eggermont AM, Gray N, Kakizoe T,
Singh Karki B, Kaslar M, Kerr DJ,
Khayat D, Khuhaprema T, Kim IH,
Martin-Moreno J, McVie G, Park JG,
Philip T, Ringborg U, Rodger A, Seffrin JR,
Semiglazov V, Soo KC, Sun Y, Thomas R,
Tursz T, Veronesi U, Wiestler O, Yoo KY,
The effect of the intra-arterial versus intravenous regimen on loss of hearing was
measured. The aim of the study was to determine the difference between the
treatment arms and to determine prognostic factors. Earlier study only demonstrated
that the largest predictive factor for hearing loss was the pretreatment hearing loss
for intra-arterial chemo-radiation treated patients. The mean hearing loss at 4 kHz
was 20dB. There was a 10 dB (15 versus 25) difference in hearing loss at 4 kHz
between the treatment arms favoring the intra-arterial arm, the mean dose to the
inner ear was 14 Gy. Prediction of hearing loss remained predominantly determined
by the pre-treatment audiogram (figure IX.10). For 101 patients treated with radiation
alone the hearing damage due to radiation was measured by comparing pre- and
posttreatment audiograms. The median dose to the inner ear was 11.4 (0.2 to 69 Gy).
The hearing loss at 1,2-4 kHz was dependent on dose to the target and pretreatment
hearing loss. For the 153 ears receiving <25 Gy no damage was measured. For the 22
ears receiving 26 to 35 Gy the mean damage was 5.5 dB. For the 19 ears receiving 3669 Gy the mean damage was 9 dB. Hearing loss due to chemo-radiation is
predominantly determined by pretreatment hearing loss, thereafter cisplatin
(intravenous more than intra-arterial) and thereafter by the radiation dose.
Zatonski W, Zhao P. Tobacco: deadly in
any form or disguise. Lancet 2006;
367:1710-1712
Chi JT, Wang Z, Nuyten DSA,
Rodriguez EH, Schaner ME, Salim A,
Wang Y, Kristensen GB, Helland A,
BВ°rresen-Dale AL, Giaccia A,
Longaker MT, Hastie T, Yang GP,
Van de Vijver MJ, Brown PO. Gene
expression programs in response to hypoxia:
Cell type specificity and prognostic
significance in human cancers. PLoS
Figure IX.10: Hearing loss for Intra-arterial chemoradiation (left) versus intravenous chemoradiation
Medicine 2006; 3:395-409
(right) after each administration of cisplatin.
De Bock GH, Van der Hage JA, Putter H,
Bonnema J, Bartelink H, Van de Velde CJ.
Isolated loco-regional recurrence of breast
cancer is more common in young patients
and following breast conserving therapy:
Long-term results of European Organisation
for Research and Treatment of Cancer
studies. Eur J Cancer 2006; 42:351-356
Gastroenterology Combined radiotherapy and chemotherapy (CRT) has improved
treatment outcome and organ preservation in a variety 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 designed two 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, that was finished recently, 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.
We are currently testing cisplatin in a weekly schedule in this combination. In the
second trial radiotherapy is combined with escalating doses of capecitabine only. In
this study, with a dose of 1000 mg/m2 the MTD is not reached and accrual
continues.
101
RADIOTHERAPY
Publications (continued)
Until now, we have treated over 100 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 longterm 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. This has
prompted us to introduce 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 designed a large international
phase III study (CRITICS) which has been granted by the Dutch Cancer Society
(CKTO) and has been approved by the protocol review board, in which patients with
operable gastric cancer will receive preoperative chemotherapy, surgery and then
randomized between continuation of chemotherapy or postoperative CRT in a
schedule based on the above-mentioned phase I-II studies.
Deurloo EE, Klein Zeggelink WFA,
Teertstra HJ, Peterse JL, Rutgers EJT,
Muller SH, Bartelink H, Gilhuijs KGA.
Contrast-enhanced MRI in breast cancer
patients eligible for breast-conserving
therapy: Complementary value for
subgroups of patients. Eur Radiol 2006;
16:692-701
Kreike B, Halfwerk H, Kristel P, Glas A,
Peterse H, Bartelink H, Van de Vijver MJ.
Gene expression profiles of primary breast
carcinomas from patients at high risk for
In locally advanced rectal cancer, preoperative CRT with 5FU has demonstrated
superiority in downsizing, downstaging and local recurrence rates compared to
radiotherapy alone. Capecitabine, an oral prodrug of 5FU, mimics infusional 5FU
and has provided similar response rates with acceptable acute toxicity. In the last year,
we treated 43 patients with preoperative concurrent capecitabine (825 mg/m2 bid,
day 1-33) and radiotherapy, because of a locally advanced tumor. A retrospective
analysis of 33 patients demonstrated that this treatment was feasible with minor
acute toxicity and promising efficacy (TRG ≥ 2 in 64%). Serious peri-operative
toxicity occurred in 10%, mainly following a low anterior resection. To further
optimize the effect of CRT on rectal tumors, without increasing the toxicity, a new
study has been initiated with an antibody against the VEGF receptor (bevacizumab).
For patients with a locally advanced rectal carcinoma, preoperative radiotherapy will
be combined with capecitabine (825 mg/ m2 bid, day 1-33) and bevacizumab
(Avastin®, 5 mg i.v. on day –14, 1, 15, 29). Surgery will be performed 6-8 weeks after
the last chemoradiotherapy. This combination has shown to be effective in a phase I
study, performed in the US. The primary goal of this study is to evaluate the
feasibility, with special attention for the surgical complications. So far, 11 patients
have been included in this multicenter study. An interim analysis for surgical
complications will take place after the first 15 APR patients have been included.
When the procedure seems feasible in these first 15 patients, another 45 patients will
be included.
local recurrence after breast-conserving
therapy. Clin Cancer Res 2006;
12:5705-5712
Nuyten DSA, Kreike B, Hart AAM,
Chi JTA, Sneddon JB, Wessels LFA,
Peterse HJ, Bartelink H, Brown PO,
Chang HY, Van de Vijver MJ. Predicting a
local recurrence after breast-conserving
therapy by gene expression profiling. Breast
Cancer Res 2006; 8:R62
Sotiriou C, Wirapati P, Loi S, Harris A,
Fox S, Smeds J, Nordgren H, Farmer P,
Praz V, Haibe-Kains B, Desmedt C,
Larsimont D, Cardoso F, Peterse H,
Nuyten D, Buyse M, Van de Vijver MJ,
Bergh J, Piccart M, Delorenzi M. Gene
expression profiling in breast cancer:
Understanding the molecular basis of
histologic grade to improve prognosis. J Natl
Cancer Inst 2006; 98:262-272
Van den Belt-Dusebout AW, Nuver J,
De Wit R, Gietema JA,
Ten Bokkel Huinink WW, Rodrigus PT,
Schimmel EC, Aleman BMP,
Van Leeuwen FE. Long-term risk of
cardiovascular disease in 5-year survivors of
testicular cancer. J Clin Oncol 2006;
24:467-475
102
MEDICAL ONCOLOGY
X D IV IS ION OF M ED ICA L
ON COLOG Y
CLINICAL PHARMACOLOGY OF ANTICANCER DRUGS
Jos Beijnen, Jan Schellens, Henk Boot, Annemieke Cats, Helgi Helgason, Alwin Huitema,
Bastiaan Nuijen, Hilde Rosing, Wim Ten Bokkel Huinink
Division head, Group leader Sjoerd Rodenhuis
Sjoerd Rodenhuis MD PhD Head
Joke Baars MD PhD Academic staff
Paul Baas MD PhD Academic staff
Evert Bais MD Academic staff
Background and objectives The Division of Pharmacology is involved in research
in different phases of anticancer drug development. These include: I) Pharmaceutical
formulation, II) Bioanalytical method development and implementation in PK
studies and III) Early clinical phase I/II studies. A new research line was initiated
which is supportive care of patients with breast cancer (IV).
Jos Beijnen PhD Academic staff
Willem Boogerd MD PhD Academic staff
Henk Boot MD PhD Academic staff
Wieneke Buikhuisen MD PhD Academic staff
The research activities of the subdivision of Pharmacology of the department of
Medical Oncology, the department of Pharmacy & Pharmacology and the department
of Experimental Therapy (group Schellens) are closely integrated.
Sjaak Burgers MD PhD Academic staff
Annemieke Cats MD PhD Academic staff
Jan Paul De Boer MD PhD Academic staff
Helga Droogendijk MD Academic staff
Bert De Gast MD PhD Academic staff
John Haanen MD PhD Academic staff
Helgi Helgason MD PhD Academic staff
Alwin Huitema PhD Academic staff
Agnes Jager MD 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 stafff
Herman Neering MD PhD Academic staff
Bastiaan Nuyen PhD Academic staff
Hilde Rosing PhD Academic stafff
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
I. Pharmaceutical formulation
Formulation research and clinical manufacture of small molecule experimental
anticancer agents has continued with the compounds EO-9 (EOquinTM), Kahalalide
F, Imexon, and C1311 (SymadexTM). All compounds are currently in worldwide
clinical trials in patented formulations designed by us. Recently, a formulation
project with the anti-metastatic ruthenium complex NAMI-A was initiated. The
clinical manufacture (thalidomide tablets) or development (5-MI tablets) of oral
formulations of anticancer agents has continued. A pharmaceutical development
program of oral taxanes has been initiated. The Amsterdam BioTherapeutics Unit
(AmBTU) is now fully operational for the manufacture of a GMP-grade DNAplasmid. 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 the upcoming phase I clinical trial in
our institute. Also, a research program in collaboration with Utrecht University has
been initiated, aimed at the pharmaceutical development of non-viral vectors
(e.g. lipoplexes and polyplexes) for improvement of DNA-plasmid transfection.
For this, an in vitro human skin screening model is being developed in close
collaboration with the Division of Immunology. The ultimate aim of this program is
the translation of selected, promising vectors to the clinic. By the end of 2006, the
clean room facilities of the AmBTU have been expanded in order to enable
GMP-preparation of T-lymphocytes for T-cell receptor gene therapy.
Academic staff
Jaap Van der Sande MD PhD Academic staff
Annette Van der Velden MD Academic staff
Marchien Van der Weide MD PhD
Academic staff
Roel Van Gijn PhD Academic stafff
Rianne Van Maanen PhD Academic staff
Nico Van Zandwijk MD PhD Academic staff
David Boss Graduate student
Elke Brouwers Graduate student
Carola Damen Graduate student
Maarten Deenen Graduate student
Corine Ekhart Graduate student
Judith Engwegen Graduate student
Suzanne Frankfort Graduate student
Claudia Heijens Graduate student
Marie-Christine Gast Graduate student
Robert Jansen Graduate student
Markus Jörger Graduate student
Ron Keizer Graduate student
Stijn Koolen Graduate student
Roos Oostendorp Graduate student
II. Bioanalytical method development and implementation in PK studies
A new laboratory dedicated to the bioanalysis of samples using Liquid
Chromatography coupled with tandem quadrupole Mass Spectrometry (LC-MS/MS)
has been built in the department of Pharmacy & Pharmacology. In total, 5 LC-MS/MS
systems have been transferred to this laboratory.
A new assay has been validated for gemcitabine and its (phosphorylated) metabolites
to support a clinical study with the gemcitabine prodrug LY2334737. A quantitative
assay for the determination of the bioreductive alkylating aziridinyl-quinone EO9 and
its metabolites EO5a and EO9-Cl in urine has also been validated. The drug is
administered intravesically for the treatment of superficial bladder cancer. We also
found a new potential metabolite, EO9-OH. The structure of this compound remains
to be elucidated.
E7389 is a synthetic analogue of the natural product halichondrin B and it is
currently being developed as an anti-cancer agent. For the bioanalysis of E7389, the
compound is extracted from plasma and an LC-MS/MS method has been validated
for the quantitative analysis.
We continued to measure drug levels to support several clinical trials in and outside
the Institute. This concerns clinical studies with: topotecan, ES-285, trabectedin,
kahalalide F, D-24851, paclitaxel, and docetaxel. In addition, clinical trials with the
farnesyl transferase inhibitor lonafarnib are supported.
103
MEDICAL ONCOLOGY
Trastuzumab is a humanized monoclonal antibody and inhibits the proliferation of
human tumor cells that over express HER2. For the determination of trastuzumab
concentrations in human serum, an Enzyme-Linked Immuno Sorbent Assay (ELISA)
has been described in literature. Our laboratory is involved in the pharmaceutical
characterisation of trastuzumab and the bioanalysis of the drug using LC-MS/MS.
III. Early clinical phase I/II studies
III-1 Novel approaches to improve oral bioavailability
We previously demonstrated that the oral applicability of commonly used anticancer
drugs is limited by affinity for one or more of the ABC transporters expressed in the
epithelial layer of the gastro-intestinal tract, especially P-glycoprotein (Pgp; ABCB1),
Breast Cancer Resistance Protein (BCRP; ABCG2), MRP2 (ABCC2) and/or affinity
for cytochrome P450 3A (CYP3A). This year we have shown that the systemic
exposure to oral docetaxel can be significantly improved by co-administration of oral
docetaxel and one single oral dose of 100 mg of ritonavir. The systemic exposure of
100 mg oral docetaxel is comparable to exposure after iv administration of the same
dose. The phase I study with the novel orally applied taxane derivative BMS-275183,
revealed significant interpatient variability in pharmacokinetics. The oral
bioavailability is low and variable (mean and SD are 24 В± 11%). Based on our
observation that the co-administration of pantoprazole (a benzimidazole proton
pump inhibitor) and BMS-275183 resulted in a significant two-fold increase in the
systemic exposure to BMS-275183 and also in an increase of clinical toxicity, we
embarked on a drug-drug interaction study with BMS-275183 and esomeprazole.
We continued development of oral formulations of paclitaxel in capsules. [Supported
by a six-year program grant of the Dutch Cancer Society]
Susanne Quaak Graduate student
Rob Ter Heine Graduate student
Ly Tran Graduate student
Liia Vainchtein Graduate student
Joost Van den Berg Graduate student
Jolanda Van den Hoven Graduate student
Sabien Van der Schoot Graduate student
Annemieke Van Winden Graduate student
Anthe Zandvliet Graduate student
Carolien Alderden Technical staff
Valerie Doodeman Technical staff
Abadi Gebretensae Technical staff
Michel Hillebrand Technical staff
Ciska Koopman-Kroon Technical staff
Lianda Nan-Offeringa Technical staff
MariГ«t Ouwehand Technical staff
Mathijs Tibben Technical staff
III-2 Pharmacology (PK/PD, ADME, mass balance) of novel anticancer drugs
Currently, we perform 22 phase I/II, pharmacological and proof of concept studies.
We recruited close to 200 new patients in these studies in 2006. Thirteen of the
current phase I studies are two- or multicenter studies. Representative examples are
described below.
Marja Roelvink Clinical trial manager
Sandra Adriaansz Nurse practitioner
Annelies Boekhout Nurse practitioner
Ria Dubbelman Nurse practitioner
Joke Foekema Nurse practitioner
Marjo Holtkamp Nurse practitioner
a. Studies with cell cycle inhibitors
The phase I study with AZD5438, given orally four times per day on one day every
week has been completed. The starting dose was 4 times 10 mg, which could safely
be increased to 4 times 90 mg. At this dose-level, asthenia and general malaise
developed. As major toxicities and wide pharmacokinetic variability have been
observed at other schedules, the development of the drug has been discontinued.
Marianne Keessen Nurse practitioner
Maria Kuiper Nurse practitioner
Henk Mallo Nurse practitioner
Annemarie Nol Nurse practitioner
Albert Olijve Nurse practitioner
Margaret Schot Nurse practitioner
Jana Van der Sar Nurse practitioner
b. PK/PD support of a phase I study with oral gemcitabine
Gemcitabine is an attractive candidate for oral application. The phase I study with
oral gemcitabine every other day for 21 days followed by one week of rest was
completed. One patient with metastatic leiomyosarcoma which had been rapidly
progressive on doxorubicine as well as on ifosfamide therapy, showed tumor
regression followed by disease stabilization for three years. She had to be taken off
study because of moderate, but reversible, liver dysfunction. Clinical and laboratory
studies continue with a novel prodrug formulation of gemcitabine, which is not
extensively metabolized presystemically.
Marja Voogel-Fuchs Nurse practitioner
Publications
Aalders MCG, Triesscheijn M,
Ruevekamp M, De Bruin M, Baas P,
Faber DJ, Stewart FA. Doppler optical
coherence tomography to monitor the
c. Studies with angiogenesis inhibitors
The three phase I studies with the orally available angiogenesis inhibitors E7080, Bay
57-9352 and AZD2171, that were started last year are ongoing. The main mechanism
of action of these three molecules is inhibition of VEGFR 1 and 2. In dose-escalating
studies, E7080 is employed as single agent and Bay 57-9352 in combination with
irinotecan and capecitabine. AZD2171 is given at a fixed dose in a randomized
study with or without upfront antihypertensive medication to test whether the
best treatment for hypertension induced by this molecule is either upfront
co-administration of antihypertensives or treatment when hypertension develops
during treatment. All three studies have revealed hypertension and proteinuria as the
main side-effects. Significant antitumor activity has been observed in all three
studies.
effect of photodynamic therapy on tissue
morphology and perfusion. J Biomed
Optics 2006; 11:044011
Aleman BMP, Van den Belt-Dusebout AW,
De Bruin ML, Van �t Veer MB,
Baaijens MH, De Boer JP, Hart AA,
Klokman WJ, Kuenen MA, Ouwens GM,
Bartelink H, Van Leeuwen FE. Late
cardiotoxicity after treatment for Hodgkin’s
lymphoma. Blood 2006; [Epub ahead of
print]
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Publications (continued)
Appels NMGM, Tung KO, Rosing H,
Schellens JHM, Beijnen JH. A rapid and
simple HPLC-UV method for the
determination of inhibition characteristics
of farnesyl transferase inhibitors. Biomed
Chromatogr 2006; 20:161-165
Appels NMGM, Rosing H, Stephens TC,
Hughes A, Schellens JHM, Beijnen JH.
Absolute quantification of farnesylated Ras
d. Other phase I studies
We embarked on a three-center phase I study with the orally available irreversible
HER-1, -2 and -4 inhibitor PFS-00299804. This is a first-in-man study initiated in
our institute. The dose-level of 0.5 mg/day could be increased to 60 mg/day for
21 days q day 28. Moderate gastro-intestinal toxicity was observed.
A first-in-man phase I study with an orally available PARP1 (poly(ADP-ribose)
polymerase-1) inhibitor (KU36-92) was initiated. The study is especially of interest for
patients with BRCA2 mutated tumors. Two significant tumor regressions were
observed in BRCA2 mutated breast and ovarian cancer patients. PD studies in white
blood cells, skin biopsies and tumor biopsies revealed up to 60 % inhibition of
PARP1 activity, compatible with active drug levels in these cells and tissues.
levels in complex samples using liquid
chromatography fractionation combined
with tryptic digestion and electrospray
tandem mass spectrometry. Anal Biochem
2006; 352:33-40
Appels NMGM, Rosing H, Stephens TC,
Schellens JHM, Beijnen JH. Quantification
of farnesylmethylcysteine in lysates of
peripheral blood mononuclear cells using
liquid chromatography coupled with
electrospray tandem mass spectrometry:
Pharmacodynamic assay for farnesyl
transferase inhibitors. Anal Chem 2006;
78:2617-2622
Baas P, Belderbos JSA, Senan S, Kwa HB,
Van Bochove A, Van Tinteren H,
Burgers JA, Van Meerbeeck JP. Concurrent
III-3 Advanced data analysis employing population analysis by NONMEM
Non-linear mixed effects modeling (NONMEM) has been applied for population
pharmacokinetic and pharmacodynamic data analysis. Using this technique, the
complex interaction between capecitabine and indisulam, as observed in a phase I
study, was described. It was shown that the observed excessive hematological
toxicity of the combination was caused by time-dependent inhibition of indisulam
elimination by capecitabine. Subsequently, the developed pharmacokinetic/
pharmacodynamic model was used in a simulation study to establish a feasible
regimen for the combination of these two drugs.
Population pharmacokinetic analyses were performed to investigate the influence of
relevant SNPs in metabolic enzymes and drug transporters on the disposition of
several (experimental) anticancer agents, such as irinotecan, docetaxel, epirubicine,
indisulam, thiotepa and cyclophosphamide. By linking these pharmacokinetic
models to models describing the dose-limiting hematological toxicity of these agents,
the influence of the polymorphic forms on treatment outcome could be assessed. For
instance for indisulam, it was shown that considerable dose reductions are necessary
for safe administration of this drug in patients with polymorphic alleles of CYP2C9
and 2C19.
chemotherapy (carboplatin, paclitaxel,
etoposide) and involved-field radiotherapy
in limited stage small cell lung cancer:
A Dutch multicenter phase II study.
Br J Cancer 2006; 94:625-630
Baas P, Triesscheijn M, Burgers S,
Van Pel R, Stewart F, Aalders M.
Fluorescence detection of pleural
malignancies using 5-aminolaevulinic acid.
Chest 2006; 129:718-724
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. A diagnostic real-time PCR assay was developed, suitable for highthroughput screening of this mutation in order to prevent severe 5-FU-related
toxicity. Recently, we identified three heterozygotes for DPYD*2A that showed severe
toxicities, resulting in the death of one of the three patients. We now consider the
assessment of this SNP as the standard of care in all patients with planned
fluoropyrimidine therapy and we have finalized a protocol with the aim to show that
adaptive dosing in DPYD*2A patients is cost-effective.
Baas P, Van �t Hullenaar N, Wagenaar J,
Kaajan JPG, Koolen M, Schrijver M,
Schlösser N, Burgers JA. Occupational
asbestos exposure: How to deal with
suspected mesothelioma cases - The Dutch
approach. Ann Oncol 2006; 17:848-852
Belderbos JSA, Heemsbergen WD,
De Jaeger K, Baas P, Lebesque JV. Final
III-5 Proteomics
We aim to identify and prospectively validate unique protein profiles in serum of
patients with breast cancer, colorectal cancer and other solid malignancies that can be
used as biomarkers to predict treatment outcome (antitumor activity and toxicity)
after (adjuvant) chemotherapy, surgery and/or radiotherapy. The ProteinChip
SELDI-TOF MS system (Ciphergen) is used for this purpose in our group. We have
performed proteomics studies in breast cancer, colorectal cancer (CRC) and renal
cancer (RCC).
results of a Phase I/II dose escalation trial
in non-small-cell lung cancer using threedimensional conformal radiotherapy. Int J
Radiat Oncol Biol Phys 2006; 66:126-134
Breast cancer: In a group of 114 breast cancer patients, we have identified the
haptoglobin alpha-1 protein to be predictive for the recurrence-free survival after
surgery and adjuvant chemotherapy. The expression of this protein was consecutively
proven to be dependent on the three major haptoglobin phenotypes. Haptoglobin
phenotype appeared to be highly predictive of recurrence free survival (global Log
Rank test; p=0.01, test for trend; p= 0.0025). Cross-institute validation studies
revealed that, although the haptoglobin phenotype initially showed a significant
relation with recurrence free survival in high-risk primary breast cancer patients, this
significant relationship was not found upon expansion of the study population to
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Publications (continued)
n=432. However, at explorative analysis in specific subgroups of patients, a
significant relation of haptoglobin phenotype on recurrence free survival again was
observed. A prospective study is ongoing in which serum and tissue is collected from
patients diagnosed with either primary breast cancer or benign breast diseases and
from healthy women undergoing breast reducing surgery.
Belderbos J, Uitterhoeve L,
Van Zandwijk N, Belderbos H,
Rodrigus P, Van de Vaart P, Price A,
Van Walree N, Legrand C, Dussenne S,
Giaccone G, Koning C, on behalf of the
In 2006, we started a collaboration with the Department of Molecular Biology in the
investigation of the effects of PARP-inhibitors, which are potential drugs against
breast cancer. To this end, cultured BRCA1- and BRCA2-deficient murine cell lines as
well as control cell lines are grown. We are currently analyzing the protein content of
both the cultured cells and the culture medium to investigate the influence of PARP
inhibitors on cell behavior. Results permitting, the effects of PARP inhibitors will
also be investigated in mouse models and in human cell lines. Microarray analysis
will be performed, which allows comparisons between our findings on the proteomic
and the genomic levels.
EORTC LCG and RT Group. Randomised
trial of sequential versus concurrent chemoradiotherapy in patients with inoperable
non-small cell lung cancer (EORTC 0897222973). Eur J Cancer 2007; 43:114-121
Beumer JH, Beijnen JH, Schellens JHM.
Mass balance studies, with a focus on
anticancer drugs. Clin Pharmacokinet
2006; 45:33-58
Colorectal cancer: Using a previously developed SELDI ProteinChipВ® protocol, we
assessed changes in protein expression in patients with advanced CRC treated with
first-line oxaliplatin (130 mg/m2 day 1, q 21)-capecitabine (1000 mg/m2 d1-14, q21).
By comparing t=0 sera from these patients with those of healthy persons, we
identified the same discriminating peaks in this prospective analysis as in previous
retrospective ones. Moreover, some of these proteins (e.g. apolipoprotein A-I) were
found to change during chemotherapy depending on response. We were also able to
discriminate responders from non-responders based on the t=0 protein profile.
A prospective study is ongoing (currently n=400) in which serum and tissue is
collected from patients undergoing colonoscopy. This will yield a control group of
persons without polyps and tumor, a group with adenomatous polyps and a group
with (mostly early-stage) CRC. An analytical protocol will be set up for analysis of
tissue lysates and serum. Tissue protein profiles of these three groups will be
compared and analyzed for discriminating proteins.
Beumer JH, Rademaker-Lakhai JM,
Rosing H, Hillebrand MJX, Bosch TM,
Lopez-Lazaro L, Schellens JH, Beijnen JH.
Metabolism of trabectedin (ET-743,
YondelisTM) in patients with advanced
cancer. Cancer Chemother Pharmacol 2007;
Published online: 20 September 2006
Beumer JH, Buckle T, Ouwehand M,
Franke NEF, Lopez-Lazaro L,
Schellens JHM, Beijnen JH,
Van Tellingen O. Trabectedin (ET-743,
Yondelisв„ў) is a substrate for P-glycoprotein,
but only high expression of P-glycoprotein
Renal cancer: The stability and reproducibility of previously obtained SELDI-TOF MS
protein profiles for RCC were confirmed one year after the original analyses and they
were subsequently validated with an independent method, i.e. ELISA. As only one
protein cluster (serum amyloid- and fragments) showed biomarker potential in these
analyses, we developed a new protocol for RCC serum protein profiling with
SELDI-TOF MS to yield more biomarker candidates. Several new protein peaks were
shown to discriminate patients from healthy controls in two populations from
different institutes. The identity of these proteins is currently under investigation.
The prognostic potential of our RCC serum protein profiles was also evaluated and a
model was constructed that could reliably distinguish short-term from long-term
survivors (sensitivity and specificity 70-80%), independent of the commonly used
prognostic factor models (collaboration with the UMC Utrecht).
confers the multidrug resistance phenotype.
Invest New Drugs 2007; 25:1-7
Bex A, Kerst M, Mallo H, Meinhardt W,
Horenblas S, De Gast GC. Interferon
alpha 2b as medical selection for
nephrectomy in patients with synchronous
metastatic renal cell carcinoma: A
consecutive study. Eur Urol 2006; 49:76-81
Boekhout AH, Beijnen JH, Schellens JHM.
Symptoms and treatment in cancer therapyinduced early menopause. Oncologist 2006;
In conclusion, we have identified the haptoglobin phenotype to be predictive for
recurrence free survival after surgery and adjuvant chemotherapy in specific
subgroups of breast cancer patients. In renal cancer, we found prognostic serum
protein profiles as well and in colorectal cancer we found protein profiles predictive
of response as well as chemotherapy-induced changes in protein profiles. The
research that is performed in collaboration with several departments of the NKI will
likely result in a better understanding of the molecular development of cancer. We
expect that such proteomic analyses will benefit patients with breast cancer and CRC
and possibly other tumors by improving prediction of treatment outcome and
possibly early detection.
11:641-654
Boogaerts M, Oberhoff C,
Ten Bokkel Huinink W, Nowrousian MR,
Hayward CRW, Burger HU. Epoetin beta
(NeoRecormonВ«) therapy in patients with
solid tumours receiving platinum and nonplatinum chemotherapy: A meta-analysis.
Anticancer Res 2006; 26:479-484
Bosch TM, Meijerman I, Beijnen JH,
IV. Supportive care studies in patients with breast cancer
We initiated a single center study to identify the best therapeutic intervention in
female patients with premature menopausal complaints induced by treatment for
breast cancer. It concerns a prospective double-blind randomized placebo-controlled
intervention study with placebo, venlafaxine (EfexorВ®) and clonidine hydrochloride.
The primary endpoint is reduction in intensity and number of flushes. Secondary
Schellens JHM. Genetic polymorphisms of
drug-metabolising enzymes and drug
transporters in the chemotherapeutic
treatment of cancer. Clin Pharmacokinet
2006; 45:253-285
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Publications (continued)
Bosch TM, Huitema ADR,
endpoints are effect on sexual dysfunction and side-effects of therapy. The total study
population is 100 and a total of 39 patients has been included.
Doodeman VD, Jansen R, Witteveen E,
Smit WM, Jansen RL, Van Herpen CM,
Soesan M, Beijnen JH, Schellens JHM.
CLINICAL IMMUNOTHERAPY AND TARGETED THERAPY
Pharmacogenetic screening of CYP3A and
John Haanen, Gijsbert De Gast, Willem Boogerd, Henk Mallo
ABCB1 in relation to population
pharmacokinetics of docetaxel. Clin Cancer
Res 2006; 12:5786-5793
Bosch TM, Doodeman VD, Smits PHM,
Meijerman I, Schellens JHM, Beijnen JH.
Pharmacogenetic screening for
polymorphisms in drug-metabolizing
enzymes and drug transporters in a
Dutch population. Mol Diagn Ther 2006;
10:175-185
Bosch TM, Deenen M, Pruntel R,
Smits PHM, Schellens JHM, Beijnen JH,
Meijerman I. Screening for polymorphisms
in the PXR gene in a Dutch population.
The clinical immuno- and targeted therapy group focuses on the treatment of
melanoma and renal cell carcinoma patients. In the past years, a series of
immunotherapy trials have been conducted, including peptide and TLR agonist
vaccination trials, cytokine-based immunotherapy and concurrent chemo- and
immunotherapy trials. Recently, due to the development of small molecule receptor
tyrosine kinase inhibitors (RTKI) the group has participated in several trials in which
these RTKI’s have been studied as single agent therapy or in combination with
chemotherapy. Based on the exciting results, investigator-inititated studies have been
designed to explore the combination of small molecules and cytokines, such as IL-2
in patients with metastatic disease.
Melanoma
A phase I clinical study has been performed in stage IV melanoma patients with a
multiple peptide vaccine. Although no clinical responses were observed
immunological efficacy was detected (Figure X.1).
Eur J Clin Pharmacol 2006; 62:395-399
Figure X.1: Peripheral blood samples from patient 1
Bottomley A, Gaafa R, Manegold C,
and 2 after MART-1 peptide vaccination in
Burgers S, Coens C, Legrand C,
Montanide ISA 51, GM-CSF and tetanus toxoid.
Vincent M, Giaccone G, Van Meerbeeck J.
Short-Term Treatment-Related Symptoms
and Quality of Life: Results From an
International Randomized Phase III Study
of Cisplatin With or Without Raltitrexed in
Patients With Malignant Pleural
Mesothelioma: An EORTC Lung-Cancer
Group and National Cancer Institute,
Canada, Intergroup Study. J Clin Oncol
2006; 24:1435-1442
Brandon EFA, Sparidans RW, Guijt KJ,
Löwenthal S, Meijerman I, Beijnen JH,
Schellens JHM. In vitro characterization of
the human biotransformation and CYP
reaction phenotype of ET-743 (YondelisВ«,
TrabectidinВ«), a novel marine anti-cancer
drug. Invest New Drugs 2006; 24:3-14
Brandon EFA, Sparidans RW,
Van Ooijen RD, Meijerman I,
Lopez-Lazaro L, Manzanares I,
Beijnen JH, Schellens JHM. In vitro
characterization of the human
biotransformation pathways of aplidine,
a novel marine anti-cancer drug.
Invest New Drugs 2007; 25:9-19
Participations in three phase III clinical studies have been initiated:
Randomized, placebo-controlled multicenter trial for second-line treatment of
unresectable stage III or IV melanoma patients. In this study a total of six patients
were enrolled. Patients were randomized to receive carboplatin/paclitaxel
chemotherapy either in combination with placebo or with sorafenib. Sorafenib, a
small molecule RTKI, was originally developed as a B-Raf inhibitor. B-Raf kinase is
mutated in the majority of melanomas. It is involved in the Ras-Raf-MEK-MAPK
pathway which is important for cell proliferation. Iinhibition of B-Raf kinase appears
to be a rational target for this patient population.
We further participate in two phase III clinical trials in which a fully human
monoclonal antibody against �cytotoxic lymphocyte antigen-4’ (CTLA4) is the
study drug. Blockade of CTLA4, a molecule expressed on activated T lymphocytes
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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, either as a single agent or in combination with vaccination.
Due to a more vigorous immune system, the typical side effects of these drugs are socalled immune breakthrough events, such as immune colitis, dermatitis, uveitis,
vitiligo, pancreatitis and hypophysitis. These syndromes can be managed by
administration of topical or systemic corticosteroids.
The first of these studies is a randomized multicenter phase III trial of first line
treatment for metastatic melanoma. Patients are randomized between standard
chemotherapy (dacarbazine) and ticilimumab. The clinical endpoint of this study is
overall survival.
The second trial is a randomized placebo-controlled multicenter trial of second line
treatment for metastatic melanoma. 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, ipilimumab only or the combination.
Brandon EFA, Bosch TM, Deenen MJ,
Levink R, Van Der Wal E,
Van Meerveld JBM, Bijl M, Beijnen JH,
Schellens JHM, Meijerman I. Validation of
in vitro cell models used in drug metabolism
and transport studies; Genotyping of
cytochrome P450, phase II enzymes and
drug transporter polymorphisms in the
human hepatoma (HepG2), ovarian
carcinoma (IGROV-1) and colon
carcinoma (CaCo-2, LS180) cell lines.
Toxicol Appl Pharmacol 2006; 211:1-10
Brandsma D, Voest EE, De Jager W,
Bonfrer H, Algra A, Boogerd W, Korse T,
Reijneveld JC, Verbeek MM, Rijkers G,
Renal cell carcinoma In the renal cell carcinoma program we closely collaborate
with Axel Bex from the urology-oncology group and with Florry Vyth-Dreese who
supervises the monitoring of clinical immunotherapy studies (in collaboration with
the Clinical Laboratory, headed by Wim Nooijen). The development of investigatorinitiated study to define the optimal time point for tumor nephrectomy in metastatic
renal cell carcinoma patients is under way.
We have also 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 a prominent role in the
pathogenesis of sporadic clear cell renal cell cancer, inhibition of kinase activity of
their receptors appeared to be a rational target in this patient population. Indeed,
phase II studies showed impressive objective clinical responses and durable stable
disease in patients treated with single agent sunitinib. In total we enrolled over 50
patients in this program and we observed objective clinical responses in a substantial
number of patients, and durable stable disease lasting over 12 months in others. The
overall results are being analyzed in collaboration with investigators from the Free
University Medical Center.
Taphoorn MJB. CSF protein profiling using
Multiplex Immuno-assay: A potential new
diagnostic tool for leptomeningeal
metastases. J Neurol 2006; 253:1177-1184
Brandsma D, Taphoorn MJB, De Jager W,
Bonfrer H, Algra A, Reijneveld JC,
Boogerd W, Korse T, Verbeek MM,
Rijkers GT, Voest EE. Interleukin-8 CSF
levels predict survival in patients with
leptomeningeal metastases. Neurology 2006;
66:243-246
Breedveld P, Beijnen JH, Schellens JHM.
Use of P-glycoprotein and BCRP inhibitors
to improve oral bioavailability and CNS
penetration of anticancer drugs. Trends
Pharmacol Sci 2006; 27:17-24
BREAST CANCER THERAPY AND RESPONSE PREDICTION
Breedveld P, Pluim D, Cipriani G,
Sjoerd Rodenhuis, Sabine Linn, Joke Baars, Jos Beijnen, Jan Paul De Boer, Corine Ekhart,
Dahlhaus F, Van Eijndhoven MAJ,
Juliane Hannemann, Helgi Helgason, Marjo Holtkamp, Alwin Huitema, Marc Van de Vijver,
De Wolf CJF, Kuil A, Beijnen JH,
Jan Schornagel
Scheffer GL, Jansen G, Borst P,
Schellens JHM. The Effect of Low pH
The objective of this program is to develop and improve potentially curative therapy
for patients with early or oligometastatic breast cancer. For all these studies, close
collaborations are maintained with other departments and research divisions. The
Pharmacy has a key role in the clinical pharmacology studies of the high-dose
chemotherapy program and the Molecular Pathology Department is central in the
response prediction studies.
on Breast Cancer Resistance Protein
(ABCG2)-Mediated Transport of
Methotrexate, 7-Hydroxymethotrexate,
Methotrexate Diglutamate, Folic Acid,
Mitoxantrone, Topotecan, and Resveratrol
in In Vitro Drug Transport Models. Mol
Pharmacol 2007; 71:240-249
Intensive chemotherapy Autologous hematopoietic progenitor cell transplantation
continues to be studied, despite the negative attitude towards this treatment modality
that has developed in recent years. In addition to the encouraging results of the
Brouwers EEM, Tibben MM, Rosing H,
Hillebrand MJX, Joerger M,
Schellens JHM, Beijnen JH. Sensitive
inductively coupled plasma mass
spectrometry assay for the determination
of platinum originating from cisplatin,
carboplatin, and oxaliplatin in human
plasma ultrafiltrate. J Mass Spectrom 2006;
41:1186-1194
Figure X.2: Relapse-free survival in 621 patients
with HER-2/neu negative tumors.
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Davies AHG, Larsson G, Ardill J, Friend E,
Jones L, Falconi M, Bettini R, Koller M,
Sezer O, Fleissner C, Taal B, Blazeby JM,
Ramage JK. Development of a diseasespecific quality of life questionnaire module
for patients with gastrointestinal
neuroendocrine tumours. Eur J Cancer
2006; 42:477-484
De Boer S, Dingemans JA, Huijbers EJM,
Zych TR, Beijnen JH. Late cardiotoxicity
after use of anthracycline: The reverse side of
success. Pharm Weekbl 2006; 141:1154-1157
De Bree E, Rosing H, Michalakis J,
Romanos J, Relakis K,
Theodoropoulos PA, Beijnen JH,
Georgoulias V, Tsiftsis DD. Intraperitoneal
chemotherapy with taxanes for ovarian
cancer with peritoneal dissemination.
Eur J Surg Oncol 2006; 32:666-670
Dutch randomized study (Figure X.2) the meta-analysis of the Oxford group
(EBCTCG) has now shown that high-dose chemotherapy has a rather substantial
beneficial effect on both recurrence and breast cancer death, and that this cannot be
explained by a castration effect.
Recent evidence has raised the possibility that alkylating agents in breast cancer are
specifically active in tumors lacking the ability for homologous recombination.
Breast cancers in BRCA1 or BRCA2 carriers have a defect in this DNA-repair pathway
and the same is probably true for 20 to 30% of sporadic breast cancers. The intensive
chemotherapy program will be continued in this subset of breast cancers. A small
clinical study that investigated the feasibility of an intermediate alkylator dose
regimen (with cyclophosphamide, thiotepa and carboplatin) has shown that such a
regimen can be administered in the outpatient-setting twice with a 3-week interval.
A randomized phase II study in locally advanced breast cancer is planned to evaluate
the efficacy of the new tandem intermediate dose alkylator regimen in tumors with a
homologous recombination defect. The pathological complete response rate will be
used as the primary endpoint of this study.
The preoperative chemotherapy program in patients with tumors larger than 3 cm
has continued in 2006. Preoperative chemotherapy has been shown to be particularly
effective in basal-like (triple negative) tumors (50% pathologic complete response
rate). A collaborative study with the Molecular Pathology Department has shown that
a mRNA-expression pattern associated with complete response to dose-dense AC can
be distinguished. This does, however, require prospective validation in the ongoing
study.
De Bree E, Theodoropoulos PA, Rosing H,
Michalakis J, Romanos J, Beijnen JH,
Tsiftsis DD. Treatment of ovarian cancer
using intraperitoneal chemotherapy with
taxanes: From laboratory bench to bedside.
Cancer Treat Rev 2006; 32:471-482
De Jong AE, Hendriks YMC, Kleibeuker JH,
De Boer SY, Cats A, Griffioen G,
Nagengast FM, Nelis FG, Rookus MA,
Vasen HFA. Decrease in mortality in Lynch
syndrome families because of surveillance.
Gastroenterology 2006; 130:665-671
De Jong AE, Nagengast FM, Kleibeuker JH,
Van de Meeberg PC, Van Wijk HJ, Cats A,
Griffioen G, Vasen FA. What is the
appropriate screening protocol in Lynch
syndrome? Fam Cancer 2006; 5:373-378
De Jonge ME, Huitema ADR, Beijnen JH,
Rodenhuis S. High exposures to
bioactivated cyclophosphamide are related
to the occurrence of veno-occlusive disease of
Microarrays, prognosis and response prediction Microarray technology is
gaining credibility as an important diagnostic tool in guiding treatment decisions in
breast cancer. To assess the feasibility of this novel technology in community
hospitals we have conducted a pilot study from 2004 until December 2006 in
seventeen general hospitals that varied in geographical localization, organization and
size (RASTER study). After inclusion of 771 early breast cancer patients we obtained a
prognostic gene expression profile in 80% of pN0 patients within two weeks after
surgery. Fifty percent of the node-negative patients had a poor prognosis signature.
Approximately 22% of patients who had a high risk of distant metastases according
to the microarray test had a low risk according to the Dutch guidelines (CBO 2004),
while 7% had a low risk of distant metastases according to the microarray test, but a
high risk according to the Dutch guidelines.
In 2004 a randomized, multicenter adjuvant study in node-positive primary breast
cancer (MATADOR study; CKTO 2004-04; BOOG 2005-02) was initiated and this
study is currently open in 26 centers in the Netherlands. 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-cyclophophamide regimen
(dose-dense).
Using BAC array comparative genomic hybridization, a predictive profile for a
progression-free survival of over 24 months after high dose chemotherapy with
autologous stem cell support in the metastatic setting has been identified. This
profile will need prospective validation.
the liver following high-dose chemotherapy.
Br J Cancer 2006; 94:1226-1230
THORACIC ONCOLOGY
De Vries NA, Beijnen JH, Boogerd W,
Nico Van Zandwijk, Paul Baas, Sjaak Burgers, JosГ© Belderbos, Houke Klomp, Petra Nederlof,
Van Tellingen O. Blood-brain barrier and
Jan Schellens
chemotherapeutic treatment of brain
tumors. Expert Rev Neurother 2006;
6:1199-1209
De Witte MA, Coccoris M, Wolkers MC,
Van Den Boom MD, Mesman EM,
Non Small Cell Lung Cancer (NSCLC) Prognostic micro-array profiles obtained by
supervised classification of RNA samples from completely resected NSCLC patients1
can be used as a tool to predict disease-free survival and outperform TNM staging.
The confirmation of this result in an independent series of patients has been
established in 2006 and the important question regarding selection of NSCLC
Song JY, Van Der Valk M, Haanen JBAG,
Schumacher TNM. Targeting self-antigens
through allogeneic TCR gene transfer.
Blood 2006; 108:870-877
1
Microarray Consortium: Netherlands Cancer Institute, VU Medical Center, Thoraxklinik Heidelberg,
Medical Academy Gdansk, Medical Academy Bialystok, Agendia
109
MEDICAL ONCOLOGY
Publications (continued)
patients for adjuvant therapy on the basis of micro-array profile is now ready to be
answered in a prospective trial.
Prospective evaluation of the mutation status of the Epidermal Growth Factor
Receptor (EGFR) and the response to small-molecule Tyrosine Kinase Inhibitors
(TKIs) in adenocarcinoma patients has revealed that the majority (90%) of patients
harboring an EGFR mutation will experience significant relief of tumor symptoms
and tumor volume. In the subgroup of patients with exon 19 deletions, all patients
have achieved an objective response and several patients have successfully continued
TKI therapy for more than two years. EGFR mutations can thus be employed as a
selection criterion for TKI treatment. KRAS and EGFR mutations never coincided,
indicating that KRAS mutations can be used as a negative selection criterion for
TKI treatment.
The pharmacogenomic project for patients with advanced NSCLC correlating Single
Nucleotide Polymorphisms (SNP’s) with gemcitabine and cisplatin metabolism and
with clinical outcome is ongoing and has accrued over 40 patients. Regional hospitals
have started to contribute. A similar approach is part of a protocol (NVALT 7), a study
randomizing patients with recurrent NSCLC to either pemetrexed alone or the
combination of pemetrexed and carboplatin.
The final analysis of the EORTC study (08972), which compares sequential versus
concurrent chemoradiotherapy, revealed more adverse events in the concurrent arm.
The efficacy of the two different treatment approaches was comparable.
Den Brok MWJ, Nuijen B, GarcГ­a JL,
Miranda E, Calvo P, Manada C, Beijnen
JH. Compatibility and stability of the novel
anticancer agent ES-285 + HCl formulated
with 2-hydroxypropyl-?-cyclodextrin in
infusion devices. Pharmazie 2006; 61:21-24
Den Brok MWJ, Nuijen B, Harms R,
Buluran JN, Harvey MD, Grieshaber CK,
Beijnen JH. Compatibility and stability of
the novel anti-cancer agent C1311 in
infusion devices and its in vitro
biocompatibility. J Oncol Pharm Pract
2005; 11:13-19
Dorresteijn LD, Stewart FA, Boogerd W.
Stroke As a Late Treatment Effect of
Hodgkin’s Disease. J Clin Oncol 2006;
24:1480
Ekhart C, De Jonge ME, Huitema ADR,
Malignant Pleural Mesothelioma (MPM) The randomized phase III study testing
the value of thalidomide maintenance therapy after pemetrexed and cisplatin
induction chemotherapy (NVALT 5) has now accrued 33% of the projected number of
patients. An interim analysis has confirmed that thalidomide dosing according to our
previous experience (200 mg/day orally) is rather well tolerated. In parallel to this
clinical study, thalidomide is being tested in a conditional knock-out mouse
mesothelioma model (Division of Molecular Genetics).
Eleven patients with very early MPM were included in a feasibility study of induction
chemotherapy followed by surgery and postoperative radiotherapy (EORTC 08031).
The focus is now on further treatment for patients, who are apparently disease-free
after this demanding combined modality approach, and also on improved patient
selection criteria for future trials. Two second-line studies have been activated in
2006: a phase II study of the proteasome inhibitor Bortezomib and in a phase IB/II
study of the alkylating agent S 23906. In both studies, serum and tissue is being
collected to enable translational research.
Schellens JHM, Rodenhuis S, Beijnen JH.
Flat dosing of carboplatin is justified in
adult patients with normal renal function.
Clin Cancer Res 2006; 12:6502-6508
Engwegen JYMN, Gast MCW,
Schellens JHM, Beijnen JH. Clinical
proteomics: searching for better tumour
markers with SELDI-TOF mass
spectrometry. Trends Pharmacol Sci 2006;
27:251-259
Engwegen JYMN, Helgason HH, Cats A,
Harris N, Bonfrer JMG, Schellens JHM,
Beijnen JH. Identification of serum proteins
discriminating colorectal cancer patients
Optical Coherence Tomography (OCT) 2 The OCT apparatus has been investigated
in a mouse model for photodynamic therapy (PDT). Vessel collapse followed by
edema formation was an important feature that could be observed in real time
(Figure X.3).
and healthy controls using surface-enhanced
laser desorption ionisation-time of flight
mass spectrometry. World J Gastroenterol
2006; 12:1536-1544
Figure X.3: Comparison of histology
Geurts TW, Ackerstaff AH,
slides and corresponding OCT image in
Van Zandwijk N, Hart AAM, Hilgers FJM,
a mouse bearing a subcutaneous tumor.
Balm AJM. The psychological impact of
Within three hours after photodynamic
annual chest X-ray follow-up in head and
therapy the borders between the different
neck cancer. Acta Otolaryngol 2006;
layers vanish due to influx of edema and
126:1315-1320
cell swelling. The OCT images were
taken in real time a few minutes before
Haanen JBAG, Baars A, Gomez R,
excision of the tumors.
Weder P, Smits M, De Gruijl TD,
Von Blomberg BME, Bloemena E,
Scheper RJ, Van Ham SM, Pinedo HM,
Van Den Eertwegh AJM. Melanomaspecific tumor-infiltrating lymphocytes but
not circulating melanoma-specific T cells
In three patients with basal cell carcinoma that were treated with PDT the OCT
set-up has been tested and further improvements are being implemented.
may predict survival in resected advancedstage melanoma patients. Cancer Immunol
Immunother 2006; 55:451-458
2
Collaboration with Laser Center Academical Medical Center: Maurice Aalders
110
MEDICAL ONCOLOGY
Publications (continued)
Hannemann J, Kristel P, Van Tinteren H,
Bontenbal M, Van Hoesel QGCM, Smit WM,
Nooij MA, Voest EE, Van Der Wall E,
Hupperets P, De Vries EGE, Rodenhuis S,
Van De Vijver MJ. Molecular subtypes of
Chemoprevention 3 The final analysis of standard H&E and immunohistochemical
stains of bronchial biopsies taken as part of the randomized (phase IIB) fluticasone
intervention study in high risk individuals (volunteers) has not revealed an
appreciable preventive effect of this inhalational corticosteroid. The ability of assays
for HTERT expression to refine the standard criteria for the diagnosis of
premalignant bronchial lesions will be further analyzed.
breast cancer and amplification of
topoisomerase IIa: Predictive role in dose
intensive adjuvant chemotherapy.
GASTROENTEROLOGY
Br J Cancer 2006; 95:1334-1341
Henk Boot, Annemieke Cats, Marianne Smits, Babs Taal, Edwin Jansen, Corrie Marijnen,
Marcel Verheij
Helgason HH, Kruijtzer CMF,
Huitema ADR, Marcus SG,
Ten Bokkel Huinink WW, Schot ME,
Schornagel JH, Beijnen JH,
Schellens JHM. Phase II and
pharmacological study of oral paclitaxel
(Paxoral) plus ciclosporin in anthracyclinepretreated metastatic breast cancer.
Br J Cancer 2006; 95:794-800
Hoebers FJP, Pluim D, Verheij M,
Balm AJM, Bartelink H, Schellens JHM,
Begg AC. Prediction of treatment outcome
by cisplatin-DNA adduct formation in
patients with stage III/IV head and neck
squamous cell carcinoma, treated by
concurrent cisplatin-radiation
(RADPLAT). Int J Cancer 2006;
119:750-756
Hooning MJ, Dorresteijn LD, Aleman BMP,
Kappelle AC, Klijn JG, Boogerd W,
Van Leeuwen FE. Decreased risk of stroke
among 10-year survivors of breast cancer.
Gastric cancer In the USA, adjuvant chemoradiation has become standard
treatment for gastric cancer, whereas in Europe perioperative chemotherapy with
epirubicin, cisplatin and capecitabine has gained wide acceptance. Improvement of
the chemotherapy regimen used in the USA is desired. From December 2002 till
March 2006 we evaluated the feasibility of concurrent chemoradiation with
capecitabine and cisplatin as adjuvant treatment in 38 patients with pT2-4N0-3M0
gastric and esophageal adenocarcinoma. Treatment started with two weeks of
capecitabine (1000 mg/m2 bid). Subsequently, a fixed dose of 45 Gy was given to the
original tumor site and adjacent lymph nodes with concurrent cisplatin and
capecitabine in escalating daily doses. In six patients receiving cisplatin 6 mg/m2
and capecitabine 650 mg/m2 bid, three patients developed dose-limiting toxicity
(neutropenia grade III and IV, thrombocytopenia grade III, and weight loss/nausea
grade III). The MTD therefore was cisplatin 5 mg/m2 i.v. and capecitabine
650 mg/m2 bid orally. This chemoradiation regimen will be the basis for a
multicenter nationwide phase III study comparing perioperative chemotherapy with
postoperative chemoradiation therapy in resectable gastric cancer. Two additional
studies have been performed. Firstly, in the abovementioned chemoradiotherapy
regimen cisplatin was administered on a weekly basis (cisplatin 20-30 mg/m2) in a
phase I fashion with six patients per dose level. In 12 patients, no grade III/IV toxicity
was observed. Secondly, capecitabine monotherapy on radiotherapy days was
escalated from 600-1000 mg/m2 bid. In 66 patients, including those treated at the
highest dose level, no grade III/IV toxicity has been observed. These regimens can be
incorporated in future adjuvant chemoradiotherapy trials.
J Clin Oncol 2006; 24:5388-5394
Jansen EPM, Saunders MP, Boot H,
Oppedijk V, Dubbelman R, Porritt B,
Cats A, Stroom J, ValdГ©s Olmos R,
Bartelink H, Verheij M. Prospective study
on late renal toxicity following postoperative
chemoradiotherapy in gastric cancer. Int J
Radiat Oncol Biol Phys 2007; In Press
Rectal cancer In locally advanced rectal cancer, preoperative chemoradiotherapy
with 5FU is superior in downsizing the tumor and in reducing local recurrences
compared with radiotherapy alone. The question is whether similar efficacy without
increasing toxicity can be achieved by the use of bevacizumab and capecitabine. We
have started a multicenter feasibility study in 60 rectal cancer patients at high risk for
a positive resection margin with chemoradiotherapy with capecitabine (825 mg/m2
bid, day 1-33) to which bevacizumab (5 mg/kg, day –14, 1, 15, 29) is added. The
number of treatment failures is the primary endpoint. Currently, 11 patients have
been included in the study.
Joerger M, Huitema ADR,
Van Den Bongard HJGD, Baas P,
Schornagel JH, Schellens JHM,
Beijnen JH. Determinants of the
elimination of methotrexate and
7-hydroxy-methotrexate following high-dose
infusional therapy to cancer patients.
Br J Clin Pharmacol 2006; 62:71-80
Joerger M, Huitema ADR, Boogerd W,
Van Der Sande JJ, Schellens JHM,
Beijnen JH. Interactions of serum albumin,
valproic acid and carbamazepine with the
Neuroendocrine tumors The clinical features of 244 patients admitted to our
hospital from 1994 through 2004 have been collected in a database (116 men, 128
women; median age 57 years). Liver metastases were present in 65%, skeletal
metastases in 14%. The Octreotide scan was positive in 77% and the MIBG scan in
60% of the patients. An increasing problem is a mesenterial mass, which is present
in 44% of the patients (Figure X.4). In patients with a positive Octreotide scan,
survival is significantly better than in those with a negative scan (67% versus 40%).
A strongly elevated tumor marker, Chromogranin A, is negative predictor of survival.
Evaluation of treatment is mainly based on hormonal activity, which is responsible
for most symptoms. The tumor size is only rarely reduced by drug therapy. To
measure quality of life, an international EORTC questionnaire has been developed
that will be tested in a larger group to evaluate the effect of treatment.
pharmacokinetics of phenytoin in cancer
patients. Basic Clin Pharmacol Toxicol
2006; 99:133-140
3
Chemoprevention Group: Netherlands Cancer Institute, VUmc (Department of Pulmonology):
Egbert Smit
111
MEDICAL ONCOLOGY
Publications (continued)
Figure X.4: Mesenterial mass invading the
superior mesenterial artery, in a patient
Joerger M, Bosch TM, Doodeman VD,
with a neuroendocrine tumor.
Beijnen JH, Smits PHM, Schellens JHM.
Novel deoxycytidine kinase gene
polymorphisms: A population screening
study in Caucasian healthy volunteers. Eur
J Clin Pharmacol 2006; 62:681-684
Joerger M, Huitema ADR,
Van Den Bongard DHJG, Schellens JHM,
Beijnen JH. Quantitative effect of gender,
age, liver function, and body size on the
population pharmacokinetics of paclitaxel
in patients with solid tumors. Clin Cancer
Res 2006; 12:2150-2157
EUS Radial endoscopic ultrasound is important to select patients with esophageal or
gastric tumor for preoperative chemotherapy or chemoradiation. In 2004 the linear
endoscopic EUS became available to puncture enlarged lymph nodes for an optimal
staging. We currently participate in a multicenter trial to evaluate the effect of
mediastinal staging in lung cancer. With linear EUS (n=93) the fine needle aspiration
was representative in 86% and a surgical procedure was prevented in 45%, usually
following the detection of N2 lymph node metastases.
Kartachova MS, ValdГ©s Olmos R,
Haas RLM, Hoebers FJP,
Van Den Brekel MW, Van Zandwijk N,
Herk MV, Verheij M. Mapping of
treatment-induced apoptosis in normal
structures: 99mTc-Hynic-rh-annexin V
SPECT and CT image fusion. Eur J Nucl
Med Mol Imaging 2006; 33:893-899
Kemper EM, Leenders W, sters B,
Lyons S, Buckle T, Heerschap A,
Boogerd W, Beijnen JH, Van Tellingen O.
Development of luciferase tagged brain
tumour models in mice for chemotherapy
intervention studies. Eur J Cancer 2006;
42:3294-3303
Kemper EM, Boogerd W, Beijnen JH,
Van Tellingen O. Paclitaxel alone is not
useful for the treatment of brain tumors.
Pharm Weekbl 2006; 141:380-383
Kramer GWPM, Legrand CL, Van Schil P,
Uitterhoeve L, Smit EF, Schramel F,
Biesma B, Tjan-Heijnen V,
Van Zandwijk N, Splinter T, Giaccone G,
Van Meerbeeck JP. Quality assurance of
thoracic radiotherapy in EORTC 08941:
A randomised trial of surgery versus
thoracic radiotherapy in patients with stage
IIIA non-small-cell lung cancer (NSCLC)
after response to induction chemotherapy.
Eur J Cancer 2006; 42:1391-1398
Kreukels BPC, Schagen SB,
Ridderinkhof KR, Boogerd W,
Hamburger HL, Muller MJ,
Van Dam FSAM, Schagen SB. Effects of
high-dose and conventional-dose adjuvant
chemotherapy on long-term cognitive
sequelae in patients with breast cancer: An
electrophysiologic study. Clin Breast Cancer
2006; 7:67-78
112
MEDICAL ONCOLOGY
Publications (continued)
Kroep JR, Smit EF, Giaccone G,
Overwijk WW, De Visser KE, Tirion FH,
Sparidans RW, Bosch TM, Jörger M,
Van der Born K, Beijnen JH,
De Jong LA, Pols TWH, Van Der Velden YU,
Schellens JHM, Beijnen JH. Liquid
Van Groeningen CJ, Van der Vijgh WJF,
Van Den Boorn JG, Keller AM, Buurman WA,
chromatography-tandem mass spectrometric
Postmus PE, Pinedo HM, Peters GJ.
Theoret MR, Blom B, Restifo NP,
assay for the analysis of uracil, 5,6-dihydrouracil
Pharmacology of the paclitaxelв€’cisplatin,
Kruisbeek AM, Kastelein RA, Haanen JBAG.
and b-ureidopropionic acid in urine for the
gemcitabineв€’cisplatin, and
Immunological and antitumor effects of IL-23 as
measurement of the activities of the pyrimidine
paclitaxelв€’gemcitabine combinations in
a cancer vaccine adjuvant. J Immunol 2006;
catabolic enzymes. J Chromatogr B Analyt
patients with advanced non-small cell lung
176:5213-5222
Technol Biomed Life Sci 2006; 839:45-53
Puri R, Palit V, Loadman PM, Flannigan M,
Sparidans RW, Dost F, Crommentuyn KML,
Shah T, Choudry GA, Basu S, Double JA,
Huitema ADR, Schellens JHM, Beijnen JH.
Kuppens IELM, Dansin E, Boot H,
Lenaz G, Chawla S, Beer M, Van Kalken C,
Liquid chromatographic assay for the non-
Feger C, Assadourian S, Bonneterre ME,
De Boer R, Beijnen JH, Twelves CJ,
peptidic protease inhibitor tipranavir in plasma.
Beijnen JH, Schellens JHM, Bonneterre J.
Phillips RM. Phase I/II Pilot Study of
Biomed Chromatogr 2006; 20:671-673
Dose-finding phase I clinical and
Intravesical Apaziquone (EO9) for Superficial
pharmacokinetic study of orally
Bladder Cancer. [Miscellaneous Article].
Sternberg CN, De Mulder P, Schornagel JH,
administered irinotecan in patients with
J Urol 2006; 176:1344-1348
Theodore C, Fossa SD, Van Oosterom AT,
cancer. Cancer Chemother Pharmacol
2006; 58:509-516
Witjes JA, Spina M, Van Groeningen CJ,
advanced solid tumors. Clin Cancer Res
Rademaker-Lakhai JM, Crul M, Zuur L, Baas P,
Duclos B, Roberts JT, De Balincourt C,
Beijnen JH, Simis YJ, Van Zandwijk N,
Colette L, EORTC Genito-Urinary Cancer
Lagas JS, Vlaming ML, Van Tellingen O,
Schellens JH. Relationship between cisplatin
Group. Seven year update of an EORTC phase
Wagenaar E, Jansen RS, Rosing H,
administration and the development of
III trial of high-dose intensity M-VAC
Beijnen JH, Schinkel AH. Multidrug
ototoxicity. J Clin Oncol 2006; 24:918-924
chemotherapy and G-CSF versus classic M-VAC
2006; 12:3774-3781
resistance protein 2 is an important
in advanced urothelial tract tumours.
determinant of paclitaxel pharmacokinetics.
Rodenhuis S, Rutgers EJT. Adjuvant therapy for
Clin Cancer Res 2006; 12:6125-6132
breast cancer. Br J Surg 2006; 93:1443-1445
Liedert B, Pluim D, Schellens J,
Rodenhuis S, Bontenbal M,
of 5-fluorouracil in the adjuvant setting:
Thomale J. Adduct-specific monoclonal
Van Hoesel QGCM, Smit WM, Nooij MA,
High-dose, low-dose or no-dose leucovorin?
antibodies for the measurement of cisplatin-
Voest EE, Van Der Wall E, Hupperets P,
Commentary. Nat Clin Pract Oncol 2006;
induced DNA lesions in individual cell
Van Tinteren H, Peterse JL, Van De Vijver MJ,
3:300-301
nuclei. Nucleic Acids Res 2006; 34
De Vries EGE. Efficacy of high-dose alkylating
Eur J Cancer 2006; 42:50-54
Taal BG, Cats A, Van Tinteren H. Modulation
Meijerman I, Beijnen JH, Schellens JHM.
chemotherapy in HER2/neu-negative breast
Toebes M, Coccoris M, Bins A, Rodenko B,
cancer. Ann Oncol 2006; 17:588-596
Gomez R, Nieuwkoop NJ, Van De Kasteele W,
Rimmelzwaan GF, Haanen JBAG, Ovaa H,
Herb-drug interactions in oncology: Focus
on mechanisms of induction. Oncologist
Scagliotti GV, Szczesna A, Ramlau R,
Schumacher TNM. Design and use of
2006; 11:742-752
Cardenal F, Mattson K, Van Zandwijk N,
conditional MHC class I ligands. Nat Med
Price A, Lebeau B, Debus J, Manegold C.
2006; 12:246-251
Moser EC, Noordijk EM, Van Leeuwen FE,
Docetaxel-based induction therapy prior to
Le Cessie S, Baars JW, Thomas J, Carde P,
radiotherapy with or without docetaxel for non-
Triesscheijn M, Baas P, Schellens JHM,
Meerwaldt JH, Van Glabbeke M,
small-cell lung cancer. Br J Cancer 2006;
Stewart FA. Photodynamic therapy in oncology.
Kluin-Nelemans HC. Long-term risk of
94:1375-1382
Oncologist 2006; 11:1034-1044
aggressive non-Hodgkin lymphoma. Blood
Siegel-Lakhai WS, Crul M, De Porre P,
Triesscheijn M, Ruevekamp M, Out R,
2006; 107:2912-2919
Zhang S, Chang I, Boot H, Beijnen JH,
Van Berkel TJC, Schellens J, Baas P,
Schellens JH. Clinical and pharmacologic study
Stewart FA. The pharmacokinetic behavior of
Moser EC, Noordijk EM, Van Leeuwen FE,
of the farnesyltransferase inhibitor tipifarnib in
the photosensitizer meso-tetra-hydroxyphenyl-
Baars JW, Thomas J, Carde P, Meerwaldt
cancer patients with normal or mildly or
chlorin in mice and men. Cancer Chemother
JH, Van Glabbeke M, Kluin-Nelemans
moderately impaired hepatic function. J Clin
Pharmacol 2007; In Press
HC. Risk of second cancer after treatment of
Oncol 2006; 24:4558-4564
cardiovascular disease after treatment for
Triesscheijn M, Ruevekamp M, Antonini N,
aggressive non-Hodgkin’s lymphoma; an
EORTC cohort study. Haematologica 2006;
Sparidans RW, Dost F, Crommentuyn KML,
Neering H, Stewart FA, Baas P. Optimizing
91:1481-1488
Huitema ADR, Schellens JHM, Beijnen JH.
Meso-Tetra-Hydroxyphenyl-Chlorin Mediated
Liquid chromatographic assay for the protease
Photodynamic Therapy for Basal Cell
inhibitor atazanavir in plasma. Biomed
Carcinoma. Photochem Photobiol 2006;
Chromatogr 2006; 20:72-76
82:1686-1690
113
MEDICAL ONCOLOGY
Publications (continued)
Vainchtein LD, Thijssen B, Stokvis E,
Van Zandwijk N, Mathy A, Boerrigter L,
Vos EJ, Linn SC, Rodenhuis S. [Effects and
Rosing H, Schellens JHM, Beijnen JH.
Ruijter H, Tielen I, De Jong D, Baas P,
costs of adjuvant chemotherapy for operable
A simple and sensitive assay for the quantitative
Burgers S, Nederlof P. EGFR and KRAS
lymph node positive breast cancer with
analysis of paclitaxel and metabolites in human
mutations as criteria for treatment with tyrosine
HER2/neu overexpression] Effecten en
plasma using liquid chromatography/tandem
kinase inhibitors: retro- and prospective
kosten van adjuvante chemotherapie bij
mass spectrometry. Biomed Chromatogr 2006;
observations in non-small-cell lung cancer.
operabele lymfklierpositieve borstkanker met
20:139-148
Ann Oncol 2007; 18:99-103
HER2/neu-overexpressie. Ned Tijdschr
Vainchtein LD, Rosing H, Mirejovsky D,
Veltkamp SA, Thijssen B, Garrigue JS,
Huynh V, Lenaz L, Hillebrand MJX,
Lambert G, Lallemand F, Binlich F,
Vrieling A, Rookus MA, Kampman E,
Schellens JHM, Beijnen JH. Quantitative
Huitema ADR, Nuijen B, Nol A, Beijnen JH,
Bonfrer JM, Korse CM, Van Doorn J,
analysis of EO9 (apaziquone) and its metabolite
Schellens JHM. A novel self-microemulsifying
Lampe JW, Cats A, Witteman BJ,
EO5a in human plasma by high-performance
formulation of paclitaxel for oral administration
Van Leeuwen FE, Van �t Veer LJ,
liquid chromatography under basic conditions
to patients with advanced cancer. Br J Cancer
Voskuil DW. Isolated Isoflavones do not
coupled to electrospray tandem mass
2006; 95:729-734
Affect the Circulating Insulin-Like Growth
Geneeskd 2006; 150:776-780
spectrometry. J Mass Spectrom 2006;
41:1268-1276
Factor (IGF) System in Men at Increased
Veltkamp SA, Alderden-Los C, Sharma A,
Colorectal Cancer Risk. J Nutr 2007; 137:
Rosing H, Beijnen JH, Schellens JHM.
accepted
Vainchtein LD, Rosing H, Mirejovsky D,
A pharmacokinetic and safety study of a novel
Lenaz L, Schellens JHM, Beijnen JH. Stability
polymeric paclitaxel formulation for oral
Vuylsteke RJCL, Molenkamp BG,
experiments in human urine with EO9
application. Cancer Chemother Pharmacol
Van Leeuwen PAM, Meijer S,
(apaziquone): A novel anticancer agent for the
2007; 59:43-50
Wijnands PGJT, Haanen JBAG,
Scheper RJ, De Gruijl TD. Tumor-specific
intravesical treatment of bladder cancer.
Veltkamp SA, Hillebrand MJX, Rosing H,
CD8+ T cell reactivity in the sentinel lymph
Jansen RS, Wickremsinhe ER, Perkins EJ,
node of GM-CSF - Treated stage I
Van den Berg JH, Beijnen JH, Balm AJM,
Schellens JHM, Beijnen JH. Quantitative
melanoma patients is associated with high
Schellens JHM. Future opportunities in
analysis of gemcitabine triphosphate in human
myeloid dendritic cell content. Clin Cancer
preventing cisplatin induced ototoxicity. Cancer
peripheral blood mononuclear cells using weak
Res 2006; 12:2826-2833
Treat Rev 2006; 32:390-397
anion-exchange liquid chromatography coupled
J Pharm Biomed Anal 2007; 43:285-292
Van den Broek GB, Balm AJM,
with tandem mass spectrometry. J Mass
Vyth-Dreese FA, Kim YH, Dellemijn TAM,
Spectrom 2006; 41:1633-1642
Schrama E, Haanen JBAG, Spierings E,
Van den Brekel MWM, Hauptmann M,
Goulmy E. In situ visualization of antigen-
Schornagel JH, Rasch CRN. Relationship
Vink SR, Schellens JHM, Beijnen JH,
specific T cells in cryopreserved human
between clinical factors and the incidence of
Sindermann H, Engel J, Dubbelman R, Moppi
tissues. J Immunol Methods 2006;
toxicity after intra-arterial chemoradiation for
G, Hillebrand MJX, Bartelink H, Verheij M.
310:78-85
head and neck cancer. Radiother Oncol 2006;
Phase I and pharmacokinetic study of combined
81:143-150
treatment with perifosine and radiation in
Ye H, Gong L, Liu H, Ruskone-
patients with advanced solid tumours. Radiother
Fourmestraux A, De Jong D, Pileri S,
Oncol 2006; 80:207-213
Thiede C, Lavergne A, Boot H, Caletti G,
Van Der Schoot SC, Vainchtein LD,
Beijnen JH, Gore A, Mirejovsky D, Lenaz L,
WГјndisch T, Molina T, Taal BG, Elena S,
Nuijen B. EO-9 bladder instillations:
Vink SR, Lagerwerf S, Mesman E, Schellens
Neubauer A, MacLennan KA, Siebert R,
Formulation selection based on stability
JHM, Begg AC,
Remstein ED, Dogan A, Du MQ. Strong
characteristics and in vitro simulation studies.
Van Blitterswijk WJ, Verheij M.
BCL10 nuclear expression identifies gastric
Int J Pharm 2007; 329:135-141
Radiosensitization of squamous cell carcinoma
MALT lymphomas that do not respond to H
by the alkylphospholipid perifosine in cell culture
pylori eradication. Gut 2006; 55:137-139
Van Der Schoot SC, Nuijen B, Sood P,
and xenografts. Clin Cancer Res 2006; 12:1615-
Thurmond II KB, Stewart DR, Rice JR,
1622
Beijnen JH. Pharmaceutical development,
Zandvliet AS, Schellens JHM, Copalu W,
Beijnen JH, Huitema ADR. A semi-
quality control, stability and compatibility of a
Vlaming MLH, Mohrmann K,
physiological population pharmacokinetic
parenteral lyophilized formulation of the
Wagenaar E, De Waart DR,
model describing the non-linear disposition
investigational polymer-conjugated platinum
Oude Elferink RPJ, Lagas JS,
of indisulam. J Pharmacokinet
antineoplastic agent AP5346. Pharmazie 2006;
Van Tellingen O, Vainchtein LD,
Pharmacodyn 2006; 33:543-570
61:835-844
Rosing H, Beijnen JH, Schellens JHM,
Schinkel AH. Carcinogen and anticancer drug
Zandvliet AS, Copalu W, Schellens JHM,
Van Tuyl SAC, Van Noorden JT, Timmer R,
transport by Mrp2 in vivo: Studies using Mrp2
Beijnen JH, Huitema ADR. Saturable
Stolk MFJ, Kuipers EJ, Taal BG. Detection of
(Abcc2) knockout mice.
binding of indisulam to plasma proteins
small-bowel neuroendocrine tumors by video
J Pharmacol Exp Ther 2006; 318:319-327
and distribution to human erythrocytes.
capsule endoscopy. Gastrointest Endosc 2006;
64:66-72
Drug Metab Dispos 2006; 34:1041-1046
114
SURGICAL ONCOLOGY
X I D IV IS ION OF
SU RG ICA L ON COLOG Y
PLASTIC AND RECONSTRUCTIVE SURGERY
Marieke Van den Berg, J Joris Hage, Arjen Van Turnhout, Leonie Woerdeman
Division head, Group leader Bin Kroon
Board
Bin BR Kroon MD PhD FRCS, Head
Alfons JM Balm MD PhD FRCS FACS
Omgo E Nieweg MD PhD
General Surgical Oncology
Bin BR Kroon MD PhD FRCS Academic staff,
The clinical activities of the Department of Plastic and Reconstructive Surgery are
restricted to reconstructive surgery following oncological resections performed by
general surgeons, urologists, and gynaecologists. This reflects on its research that is
focused on primary and secondary breast reconstruction, and on the vascular
supply and functional anatomy of tissue flaps. Part of the research is conducted in
collaboration with the Departments of Radiology and Radiotherapy of the NKI-AVL,
with Departments of Plastic and Reconstructive Surgery at the Academic Medical
Centre in Amsterdam and the University Medical Centre in Utrecht, and with the
Faculty of Movement Sciences at the Vrije Universiteit in Amsterdam.
Head Department General Surgical Oncology
(till 01-11-2006)
Emiel Rutgers MD PhD Academic staff,
Head Department General Surgical Oncology
(from 01-11-2006)
Frans Zoetmulder MD PhD Academic staff
Frits Van Coevorden MD PhD Academic staff
Omgo Nieweg MD PhD Academic staff
Hester Oldenburg MD PhD Academic staff
Houke Klomp MD Academic staff
Vic Verwaal MD PhD Academic staff
Marie-Jeanne Baas–Vrancken Peeters MD PhD
Academic staff
Reinie Kaas MD PhD Academic staff
Ingrid Kappers MD Academic staff
Philip Meijnen MD Academic staff
Marjan Piek-den Hartog MD Academic staff
Robert Smeenk MD Academic staff
Maartje Van Rijk MD Academic staff
Johanna Van Sandick MD PhD Fellow
Joost Van der Hoeven MD PhD Fellow
Immediate breast reconstruction Hardly any criteria are available to preoperatively
distinguish patients in whom an eventful postoperative course may be expected after
combined skin-sparing mastectomy and immediate prosthetic breast reconstruction.
Therefore, we established which factors increase the risk of surgical complications to
such a level as to adjust our indications for this procedure.
We prospectively studied the clinical relevance of six patient-related and nine
procedure-related potential risk factors for a complicated surgical outcome in 400
combined procedures in 309 patients by univariate and multivariate logistic
regression analysis. Risk factors that proved significantly correlated with loss of
implant by both analyses were accepted as clinical selection criteria.
Mild complications occurred significantly more often in patients who were older than
the mean age of 43 year, and in breasts that were more than averagely sized or were
operated by a fellow in surgical oncology. Implants were significantly more often lost
in patients who were obese or smoked (32% risk of loss vs. 6.3% risk for “optimal”
patients; odds ratio of 5.09), and in breasts that were more than averagely sized
(27% risk of loss vs. 12%; odds ratio 2.32).
We conclude that the clinically relevant increase of risk of implant loss should lead to
reluctance to perform combined skin-sparing mastectomy and immediate prosthetic
breast reconstruction in obese patients who smoke, and in those with more than
average sized breasts.
Nikola Kimmings MD PhD Fellow
Head and Neck Oncology and Surgery
Frans JM Hilgers MD PhD Academic Staff, Head
Annemieke Ackerstaff PhD Academic Staff
Alfons Balm MD PhD FRCS FACS Academic staff
Marcel Copper MD PhD Academic staff
Fenna Ebbens MD Resident
Tom Geurts MD Research physician
Peter Van Vuuren MD Resident
Steven Gonggrijp DDS Academic staff
Petra Jongmans MSc Research fellow
Desiree Kerkdijk DDS Academic staff
Frans Kroon DDS PhD Academic staff
Martin Klop MD Fellow
Peter Lohuis MD PhD Academic staff
Lisette Van der Molen Research fellow
Heike Nyst MD Research physician
Jimmy Pramana MD Research physician
Ludi Smeele MD DDS PhD Academic staff
Bing Tan MD PhD Academic staff
Adriaan Timmers DDS Academic staff
Corina Van As-Brooks PhD Academic staff
Michiel Van den Brekel MD PhD Academic staff
Microsurgical tissue transplantation The possibility to perform a muscle-sparing
procedure and the extra-muscular course of the vascular pedicle may provide suitable
features for free transplantation of a segment of the pectoralis major muscle.
We investigated the length and diameter of this vascular pedicle to determine its
feasibility.
Seventeen anatomical dissections were performed in nine human cadavers.
The length of the pedicle, its arterial diameter, and its entry point into the muscle
were determined.
The mean length of the vascular pedicle up to the medial border of the pectoralis
minor muscle was 6.6 cm. A mean additional length of 1.7 cm was obtained by
proximal dissection of the thoracoacromial trunk to its origin from the axillary artery.
The mean external arterial diameter was 1.8 mm and the diameter of the concomitant
vein was consistently larger. The vascular pedicle consistently entered the muscle
lateral to the midpoint of the clavicular line, a mean of 8.8 cm caudal to this line.
We conclude that the vascular pedicle is sufficient for microvascular anastomosis.
An anatomic landmark for the entry point of the pedicle and, therefore, the cranial
border of the muscle flap could not be defined. The sternocostal part of the pectoralis
major muscle has the potential to be used as a segmental free flap.
115
SURGICAL ONCOLOGY
ANAESTHESIOLOGY AND INTENSIVE CARE
Johannes Huitink, Dirk Buitelaar, Christiaan Keijzer, Tino Stoppa, May Ronday, Julia Ten Cate,
Michael SrГЎmek, Peter Schutte
Awake fibrecapnic intubation in head and neck cancer patients with a difficult
airway: new findings and refinements to the technique Recently we described a
novel intubation technique: awake fibrecapnic intubation. The aim of the present
study was to evaluate the efficacy and risk for complications of this technique in a
consecutive series of head and neck cancer patients with a difficult to intubate airway.
Forty intubations in head and neck cancer patients were prospectively studied.
After topical anaesthesia, a flexible bronchoscope was introduced into the
pharynx. Spontaneous respiration was maintained. A special suction catheter for
carbon dioxide measurements was advanced through the suction channel of
the bronchoscope, into the airway. All adverse events and complications were
documented.
There were no complications of the technique. The median (range) time to
intubation was three minutes (1.5-15 minutes). All patients were intubated
successfully: thirty-nine (98%) with awake fibrecapnic intubation. There was one
patient with severe tumour bleeding and acute airway obstruction caused by
advancement of the tube over the bronchoscope, but this was not caused by the
fibrecapnic intubation technique. It is concluded that awake fibrecapnic intubation is
a safe and valuable technique in head and neck cancer patients with a difficult airway.
Compound A and carbon monoxide production from sevoflurane and seven
different types of carbon dioxide absorbents The degradation of sevoflurane can
lead to the production of compound A, carbon monoxide, and increase of
temperature of the absorbent. Compound A is known to be nephrotoxic in rats.
These reactions depend upon the content of strong bases of the carbon dioxide
absorbent used, and its water content. The purpose of this study, that was done in
collaboration with the department of Anesthesiology of Vrije Universiteit Medisch
Centrum, was to measure the maximum amounts of compound A, carbon monoxide
and temperature increase using seven different carbon dioxide absorbents for
sevoflurane with differing contents of strong bases. Seven absorbents (AmsorbВ®,
Drägersorb 800plus®, Medisorb®, Spherasorb®, Loflosorb®, Superia®) were used
in hydrated and completely desiccated condition in a patient model, using a circle
anaesthesia system connected to an artificial lung. A low flow anaesthesia with an
oxygen/nitrous oxide mixture was maintained using 0.8% sevoflurane.
For quantification of compound A and carbon monoxide, a portable gas
chromatograph was used. Temperature was measured inside the absorbent.
Most absorbents including desiccated AmsorbВ® produced small amounts of
compound A. LoflosorbВ® and SuperiaВ®, which are free of strong bases, did not
produce any compound A or carbon monoxide in hydrated or desiccated condition.
Only desiccated Drägersorb®, Medisorb® and Spherasorb® demonstrated small
amounts of carbon monoxide accompanied by a significant temperature increase.
Guido Van den Broek MD Research fellow
Maya Van Rossum PhD Research fellow
Charlotte Zuur MD Research fellow
Karel Zuur MD Reseach physician
Urologic Oncology
Simon Horenblas MD PhD FEBU
Academic staff, Head
Axel Bex MD PhD Academic staff
Wim Meinhardt MD PhD Academic staff
Henk Van de Poel MD PhD Academic staff
Joost Leijte MD Research fellow
Remco De Vries MD Research fellow
Gynaecology
MatthГ© Burger MD PhD Academic staff, Head
Marc Van Beurden MD PhD Academic staff
Guus Fons MD Academic staff
Lotti Lubsen–Brandsma MD Academic staff
Willemien Van Driel MD PhD Academic staff
Rolien Olivier MD Research fellow
Plastic and Reconstructive Surgery
Joris Hage MD PhD Academic staff, Head
Marieke Van der Berg MD Academic staff
Arjen Van Turnhout MD Academic staff
Leonie Woerdeman MD PhD Academic staff
Brigit Drost MD Academic staff
Diederik Hofstede MD Fellow
DaniГ«lle Derks MD Fellow
Anesthesiology
Peter Schutte MD Academic staff, Head
Dick Buitelaar MD Academic staff
Hans Huitink MD PhD Academic staff
Christiaan Keijzer MD Academic staff
Gene expression effects on breast cancer and brain tumour cells assessed by
microarray analysis The gene expression effects of volatile anaesthetics on breast
cancer cells and brain tumour cells were assessed using microarray techniques in
collaboration with the Central Microarray Facility. Two volatile anaesthetics
(sevoflurane and desflurane) were tested in vitro. All cell lines were tested during one
hour exposure to volatile anaesthetics. Preliminary findings show a profound
difference in gene-expression between the two vapours. This may be of importance
when gene expression profiles are used to predict cancer survival and cancer therapy
effects.
May Ronday MD Academic staff
Michael SrГЎmek MD PhD Academic staff
Tino Stoppa MD Academic staff
Julia Ten Cate MD Academic staff
116
SURGICAL ONCOLOGY
Publications
LYMPHATIC MAPPING
Bex A, Kerst M, Mallo H, Meinhardt W,
Omgo Nieweg, Maartje Van Rijk, Bin K Kroon, Joost Leijte, Pieter Tanis, Renato ValdГ©s Olmos,
Horenblas S, De Gast GC. Interferon
Johannes Peterse, Emiel Rutgers, Hester Oldenburg, Simon Horenblas, Jelle Teertstra,
alpha 2b as medical selection for
Augustinus Hart, Saar Muller, Cornelis Hoefnagel, Bin BR Kroon
nephrectomy in patients with synchronous
metastatic renal cell carcinoma: A
consecutive study. Eur Urol 2006; 49:76-81
Bijker N, Meijnen P, Peterse JL,
Bogaerts J, Van Hoorebeeck I, Julien JP,
Gennaro M, Rouanet P, Avril A,
Fentiman IS, Bartelink H, Rutgers EJ.
Lymphatic mapping Lymphatic mapping with sentinel node biopsy is a staging
technique devised to detect early tumour involvement of the regional lymph nodes.
This technique enables the identification of patients who may benefit from early
regional treatment and from adjuvant systemic therapy. The tumour status of the
sentinel node provides prognostic information. The lymphatic mapping research
program was initiated in 1993 and the technique has evolved into a generally
accepted procedure.
Breast-conserving treatment with or without
radiotherapy in ductal carcinoma-in-situ:
ten-year results of European Organisation
for Research and Treatment of Cancer
randomized phase III trial 10853--a study
by the EORTC Breast Cancer Cooperative
Group and EORTC Radiotherapy Group.
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 tumour control. Studies are done in cooperation with the Medisch Spectrum
Twente, IJsselland Ziekenhuis, Haga Ziekenhuis, Memorial Sloan Kettering Cancer
Center and the John Wayne Cancer Center.
J Clin Oncol 2006; 24:3381-3387
Bogaerts J, Cardoso F, Buyse M, Braga S,
Loi S, Harrison JA, Bines J, Mook S,
Decker N, Ravdin P, Therasse P,
Rutgers E, Van �t Veer LJ, Piccart M. Gene
signature evaluation as a prognostic tool:
Challenges in the design of the MINDACT
trial. Nat Clin Pract Oncol 2006; 3:540-551
Broekhuizen LN, Wijsman JH, Peterse JL,
Rutgers EJT. The incidence and significance
of micrometastases in lymph nodes of
patients with ductal carcinoma in situ and
T1a carcinoma of the breast. Eur J Surg
Oncol 2006; 32:502-506
Breast cancer: Sentinel nodes in unusual locations The preferential lymphatic
drainage pathway from the breast is towards the lower axilla but sentinel nodes are
also found at other sites. Various aspects of sentinel node biopsy outside level I and II
of the axilla and internal mammary chain were investigated. Unusually situated
sentinel nodes were visualized on the lymphoscintigrams in 91 of the 785 breast
cancer patients (12%). These nodes were located in the internal mammary chain,
above and below the clavicle, within the breast and in-between the pectoral muscles
(Rotter’s nodes). Such oddly situated sentinel nodes could be retrieved in 80 of these
91 patients (82%). These were tumour-positive in sixteen patients (20%). In eleven
patients (14%) this was the only involved node and five patients also had a tumourpositive sentinel node in the axilla. Based on these findings, the treatment strategy
was adjusted in twelve individuals with the addition of adjuvant regional or systemic
therapy. Axillary clearance was avoided in two patients with drainage limited to
sentinel nodes elsewhere. The pursuit of unusually situated sentinel nodes provides
additional staging information and this approach results in treatment that is better
tailored to the needs of the individual patient.
Buitelaar DR, Balm AJM, Antonini N,
Van Tinteren H, Huitink JM.
Cardiovascular and respiratory
complications after major head and neck
surgery. Head Neck 2006; 28:595-602
Buyse M, Loi S, Van �t Veer L, Viale G,
Delorenzi M, Glas AM, d’Assignies MS,
Bergh J, Lidereau R, Ellis P, Harris A,
Bogaerts J, Therasse P, Floore A,
Amakrane M, Piette F, Rutgers E,
Sotiriou C, Cardoso F, Piccart MJ,
Decker N, Straehle C. Validation and
clinical utility of a 70-gene prognostic
signature for women with node-negative
breast cancer. J Natl Cancer Inst 2006;
Sentinel node biopsy and neoadjuvant chemotherapy More and more patients
now receive neo-adjuvant chemotherapy. Is sentinel node biopsy reliably possible in
these patients? In eighteen studies published about sentinel node biopsy after
neoadjuvant chemotherapy, the sentinel node was identified in an average 89% of
patients and the false-negative rate was on average 10%. Because of these mediocre
results, none of these investigators dares to omit axillary clearance just yet.
We studied sentinel lymph node biopsy prior to neoadjuvant chemotherapy in
25 T2N0 patients. Ten of them had a tumour-positive axillary sentinel node.
Axillary lymph node dissection was performed in these ten patients after completion
of the chemotherapy regimen. The other fifteen patients (60%) were spared axillary
lymph node dissection. No recurrences have been observed after a median follow-up
of eighteen months. It is concluded that sentinel node biopsy prior to neo-adjuvant
chemotherapy appears successful and reliable. This approach spares patients who
receive neoadjuvant chemotherapy the unnecessary morbidity of axillary lymph node
dissection when the nodes are not involved.
98:1183-1192
Corten EML, Schellekens PPA,
Bleys RLAW, Hage JJ, Kon M. The
segmental pectoralis major free flap:
Anatomical features of its vascular pedicle.
Ann Plast Surg 2006; 56:82-86
Penile cancer At The Netherlands Cancer Institute-Antoni van Leeuwenhoek
Hospital, dynamic sentinel lymph node biopsy is routinely performed in patients
with clinically localized squamous cell carcinoma of the penis. The majority of
patients with penile cancer with a tumour positive sentinel node do not benefit from
complementary lymph node dissection because the remainder of the nodes are
normal. A study was carried out to look for factors that may determine the
involvement of additional nodes. A total of 158 patients with clinically node-negative
penile carcinoma underwent sentinel node biopsy. Factors analyzed for their
117
SURGICAL ONCOLOGY
Publications (continued)
association with additional nodal involvement were T-stage, diameter of the primary
tumour, malignancy grade, vascular invasion of the primary tumour, and size of the
sentinel node metastasis. Tumour-positive sentinel nodes were found in 46 groins
and complementary lymph node dissection was performed. Nine of these 46 groins
(20%) contained additional involved lymph nodes. The size of the sentinel node
metastasis proved to be the only significant prognostic variable for additional lymph
node involvement (p=0.02). None of the fifteen groins with only sentinel metastasis
smaller than 2 mm in the sentinel node contained additional involved nodes.
The conclusion of the study is that additional nodal involvement is related to the size
of the metastasis in the sentinel node. Sentinel node metastases smaller than 2 mm
are not associated with other involved lymph nodes, suggesting that these patients
can be spared complementary lymph node dissection.
Deurloo EE, Klein Zeggelink WFA,
Teertstra HJ, Peterse JL, Rutgers EJT,
Muller SH, Bartelink H, Gilhuijs KGA.
Contrast-enhanced MRI in breast cancer
patients eligible for breast-conserving
therapy: Complementary value for
subgroups of patients. Eur Radiol 2006;
16:692-701
Fons G, Hasibuan SM, Van der Velden J,
Ten Kate FJ. Validation of tissue micro
array technology in endometrioid cancer of
the endometrium. J Clin Pathol 2006;[Epub
MELANOMA
ahead of print]
Omgo Nieweg, Eva Noorda, Bart Vrouenraets, Gooike Van Slooten, Bert Van Geel*,
Alexander Eggermont*, Jolanda Wittenberg**, Bin BR Kroon
Hage JJ, Van Turnhout AAWM. Long-term
outcome of metaidoioplasty in 70 female-to-
Follow-up of patients with clinically localized melanoma appears useless
The purpose was to determine the value of ancillary blood tests and imaging studies
for routine follow-up, and to determine the value of follow-up per se in patients
who have been treated for clinically localized disease.
A systematic literature search in the Medline database and the Cochrane Library was
performed. The keywords and medical subject heading terms were �follow up’ and
�melanoma’, starting in 1984. The selected publications were assessed for patient
population, methodology, intervention and outcome measures. The methodological
quality of every study was appraised and a level of evidence was assigned.
Sixty-six publications form the source of information on which this study is based.
Randomised studies examining a survival benefit from follow up with or without
imaging studies and lab exams are lacking. Few patients with metastases are
identified by these routine tests, and even fewer survive because of these tests. Far
more often the results are false positive, which invariably causes unnecessary concern
and leads to futile additional testing and may even provoke needless operations.
The limited evidence that is available indicates that follow up per se is not beneficial.
There is no literature on quality of life with and without follow up.
The conclusion of the literature study is that there is no convincing evidence that
regional disease control, survival and quality of life improve through surveillance
(level 3). The present findings challenge the current common practice of routine
follow up. As a result, the Dutch national guidelines now recommend follow up only
for patients with a melanoma of more than 1 mm Breslow thickness. Follow up visits
are limited to a history and physical examination. Additional testing is done only for
suspected recurrence.
male transsexuals. Ann Plast Surg 2006;
57:312-316
Hage JJ. Reconstruction of the penis. In:
Grabb WC, Smith JW, Thorne CH, editors.
Philadelphia, Pa.; London: Lippincott
Williams & Wilkins, 2006:
Hage JJ, Karim RB. Risk of breast cancer
among reduction mammaplasty patients
and the strategies used by plastic surgeons to
detect such cancer. Plast Reconstr Surg
2006; 117:727-735
Hage JJ, Van Turnhout AAWM,
Dekker JJML, Karim RB. Saving labium
minus skin to treat possible urethral stenosis
in female-to-male transsexuals. Ann Plast
Surg 2006; 56:456-459
Huitink JM, Burggraaf JP, Rennings JHL,
Slagt C. Air medical transport in the
Netherlands. In: Blumen I, editor.
Principals and Direction of Air Medical
Transport. Salt Lake City: Air Medical
Repeat isolated limb perfusion for recurrent melanoma The treatment of
recurrent limb melanoma after isolated perfusion, occurring in about 70% of the
patients, is a continuing challenge. Major amputation is a deterrent option for local
control and palliation in these patients, who have a rather poor prognosis.
Between 1978 and 2001, 21 patients underwent repeat isolated perfusion with
Tumour Necrosis Factor a (TNFa) and melphalan for recurrent or persistent
melanoma lesions after failure of previous perfusion.
First perfusions had been performed with melphalan alone in 13 patients and with
the addition of TNFa in eight, for a median of nine lesions. Repeat perfusion was
performed for a median of nine lesions. Median follow-up after repeat perfusion was
18 months. Thirteen patients attained a complete response after repeat perfusion.
Nine patients relapsed after complete remission. Median limb recurrence-free
interval was 13 months. The limb salvage rate was 95%. Overall survival was
62 months after complete remission, compared to 13 months for those without
complete remission (p=0.05). Regional toxicity after repeat perfusion was mild.
* Erasmus Medical Center, Daniel den Hoed Cancer Center, Rotterdam, The Netherlands
** Dutch Institute for Healthcare Improvement CBO, Utrecht, The Netherlands
Physician Association Publishing, 2006:
Huitink JM, Buitelaar DR, Schutte PFE.
Awake fibrecapnic intubation: A novel
technique for intubation in head and neck
cancer patients with a difficult airway.
Anaesthesia 2006; 61:449-452
Karim RB, Hage JJ, Van Rozelaar L,
Lange CAH, Raaijmakers J. Complications
of polyalkylimide 4% injections (BioAlcamidTM): a report of 18 cases. J Plastic
Reconstr Aesthet Surg 2006; 59:1409-1414
118
SURGICAL ONCOLOGY
Publications (continued)
Karim RBM, Brito BLR, Dutrieux RPM,
Lassance FPP, Hage JJ. MMP-2
Repeat isolated perfusion with TNF and melphalan is feasible, with a relatively high
complete remission rate, a good limb recurrence-free survival and a high limb
salvage rate.
Assessment as an Indicator of Wound
Healing: A Feasibility Study. Adv Skin
Wound Care 2006; 19:324-327
BREAST CANCER
Emiel Rutgers, Hester Oldenburg, Reinie Kaas, Philip Meijnen, Hans Peterse, Leonie Woerdeman,
Keijzer C, Perez RSGM, De Lange JJ.
Maartje Van Rijk, Marie-Jeanne Vrancken Peeters, Augustinus Hart, Saar Muller, Claudette Loo,
Compound A and carbon monoxide
Omgo Nieweg
production from sevoflurane and seven
different types of carbon dioxide absorbent
in a patient model. Acta Anaesthesiol Scand
2007; 51:31-37
Klein S, Hage JJ. Measurement, calculation,
Stage of tumours detected in the surveillance of women at high risk for breast cancer
The study aimed at analyzing the stage distribution of breast cancers found during
surveillance (1994-2006) with annual mammography and bi-annual palpation in
women at a life time risk of at least 15%. In a total of 152 women a first or
contralateral breast cancer was detected in the period under study.
and normal range of the ankle-arm index:
A bibliometric analysis and
Table XI.1: Stage distribution
recommendation for standardization.
Ann Vasc Surg 2006; 20:282-292
Stage
Goal BOB
Non-BRCA n = 102
Kreulen M, Smeulders MJC, Veeger HEJ,
O+I
Minimal
> 50%
> 30%
> 75%
65/102
38/102
10/50
Stage
Goal BOB
BRCA1 n = 39
Dev Med Child Neurol 2006; 48:436-441
0+I
Minimal
pNo
> 50%
> 30%
> 75%
24/39
9/39
33/39
Kreulen M, Smeulders MJ, Veeger HE,
Minimal = pTisN0 + pT1abN0
Hage JJ. Movement patterns of the upper
BOB: Bevolkingsonderzoek borstkanker
extremity and trunk associated with
*Statistical analysis was done using the chi-square test and when appropriate the Fisher exact test
Hage JJ. Movement patterns of the upper
extremity and trunk before and after
corrective surgery of impaired forearm
rotation in patients with cerebral palsy.
64%
37%
20%
62%
23%
85%
BRCA1/2 n = 50
27/50
76/102
36/50
54%
76%
72%
BRCA2 n = 11
3/11
1/11
3/11
27%
9%
27%
P value
0.25
0.032
0.74
P value
0.044
0.42
0.001*
impaired forearm rotation in patients with
hemiplegic cerebral palsy compared to
healthy controls. Gait Posture 2006;[Epub
ahead of print]
In conclusion, with annual mammography and (self)palpation the detected breast
cancers in non-BRCA carriers comply with the goals for biennial population
screening. The BRCA1 carriers failed in the goal for minimal breast cancer, the
BRCA2 carriers failed all goals, but the numbers are small.
Kriege M, Brekelmans CTM, Boetes C,
Muller SH, Zonderland HM, Obdeijn IM,
Manoliu RA, Kok T, Rutgers EJT,
De Koning HJ, Klijn JGM, Bartels CCM,
Besnard APE, Hoogerbrugge N,
Meijer S, Oosterwijk JC, Seynaeve C,
Tilanus-Linthorst MMA, Tollenaar RAEM.
Differences between first and subsequent
rounds of the MRISC breast cancer
screening program for women with a
familial or genetic predisposition. Cancer
2006; 106:2318-2326
Kriege M, Brekelmans CT, Obdeijn IM,
Boetes C, Zonderland HM, Muller SH,
Kok T, Manoliu RA, Besnard AP,
Tilanus-Linthorst MM, Seynaeve C,
Bartels CC, Kaas R, Meijer S,
Oosterwijk JC, Hoogerbrugge N,
Tollenaar RA, Rutgers EJ, De Koning HJ,
Klijn JG. Factors affecting sensitivity and
specificity of screening mammography and
MRI in women with an inherited risk for
breast cancer. Breast Cancer Res Treat
2006; 100:109-119
Ductal carcinoma in situ diagnosed on core-needle biopsy: risk of invasion
and axillary lymph node metastases The aim of this study was to assess the risk of
invasion and axillary lymph node metastases in patients with DCIS diagnosed on
preoperative core-needle biopsy to select criteria in what patients SN biopsy might be
warranted. One hundred and seventy-two patients diagnosed with DCIS on coreneedle biopsy were analysed. Axillary staging was performed by SN biopsy, basal
node sampling, or level 1-2 axillary lymph node dissection. Invasive breast cancer was
found in the surgical specimens of 45 (26%) patients. Risk factors for invasion were
a palpable lesion (OR: 2.95 (1.20-7.26), p=0.019), presence of a mass on
mammography (OR: 3.06 (1.43-6.56), p=0.004), intermediately (OR: 5.81 (1.18-28.57),
p=0.030), or poorly differentiated tumour grade (OR: 5.46 (1.17-25.64), p=0.031).
Lymph node metastases were found in 10 patients with DCIS and invasion on final
pathology. Factors associated with metastases were age up to 55 years (p=0.030),
invasion larger than 1.0 cm (p<0.001), and presence of vascular invasion (p=0.001).
The presence of invasion in 26% of patients with DCIS diagnosed on core-needle
biopsy poses the problem of identifying patients in whom axillary staging by SN
biopsy is most warranted. While others suggest to perform SN biopsy in all patients
with a core-needle biopsy or at least in all patients who undergo mastectomy, we
think that patients could be selected more carefully to prevent axillary staging in
patients who stand to gain very little from it. SN biopsy should be considered in
patients with an initial diagnosis of DCIS on core-needle biopsy. Patients at risk for
invasion may be identified preoperatively by a palpable lesion, a mass on
mammography, intermediately, or poorly differentiated tumour grade.
119
SURGICAL ONCOLOGY
Publications (continued)
Neomamma Database Neoadjuvant administration of chemotherapy (or primary
systemic treatment) is widely used in breast cancer patients with locally advanced
disease and its use is extending to earlier stages of disease. The goals of this regime
are to treat occult systemic disease, decrease the tumour bulk, and to reduce the
extent of local surgery. Over the past 5 years we have treated over 300 patients with
breast cancer according to different regimes of neoadjuvant chemotherapy.
Most patients were enrolled in the two main randomized trials that were active
during this time frame. This year we have created a data base : Neomamma, to collect
data of these patients to retrospectively evaluate the effect of neoadjuvant
chemotherapy on the outcome of patients with breast cancer. The primary endpoint
of the study is the pathological complete remission rate. Secondary endpoint of the
study is the influence of primary systemic therapy on the extent of local surgery.
Data of 326 patients are ready to be collected from their records. Patient
characteristics are obtained including age and all results of diagnosis and staging
prior the start of chemotherapy. The histological type and grade and receptor status
will be defined. In a subset of patients (NOODCD and NO4POM trial patients)
tumour samples were subjected to micro-array analysis in an attempt to define geneexpression profiles associated with sensitivity or resistance against chemotherapy.
The exact chemotherapy regime is registered. Evaluation of the response by physical
examination and by radiological examination (ultrasound, MRI) will be documented.
Furthermore, the type of surgery is documented and compared with the type of
surgery that was required when no neoadjuvant chemotherapy had been given.
The final pathologic result will be obtained and the pathologic response in the breast
will be measured. In the year 2007 we expect to gather and analyze all data to answer
the primary and secondary end points of the study.
Kroon BK, Nieweg OE, Van Boven H,
Horenblas S. Size of Metastasis in the
Sentinel Node Predicts Additional Nodal
Involvement in Penile Carcinoma. J Urol
2006; 176:105-108
Lakhani SR, Audretsch W,
Cleton-Jensen AM, Cutuli B, Ellis I,
Eusebi V, Greco M, Houslton RS,
Kuhl CK, Kurtz J, Palacios J, Peterse H,
Rochard F, Rutgers E, on behalf of
EUSOMA. The management of lobular
carcinoma in situ (LCIS). Is LCIS the
same as ductal carcinoma in situ (DCIS)?
Eur J Cancer 2006; 42:2205-2211
Leijte JAP, ValdГ©s Olmos R, Mens JWM,
Horenblas S. Use of positron emission
tomography in spindle cell carcinoma of the
penis. Urology 2006; 68
Lont AP, Gallee MPW, Meinhardt W,
Van Tinteren H, Horenblas S. Penis
Conserving Treatment for T1 and T2 Penile
Carcinoma: Clinical Implications of a Local
Recurrence. J Urol 2006; 176:575-580
GYNAECOLOGY
Matthe Burger, Marc Van Beurden, Willemien Van Driel, Guus Fons, Lotti Lubsen-Brandsma,
Lont AP, Kroon BK, Horenblas S,
Roelien Olivier, Manon Van Seters
Gallee MPW, Berkhof J, Meijer CJLM,
Snijders PJF. Presence of high-risk human
Hereditary ovarian carcinoma The predicted value of ovarian cancer screening in
high risk women has been established in 312 women and has been found to be poor
(PPV 40%). The clinical outcome of (PBSO) in BRCA1/2 mutation carriers has been
studied in 90 women. In 8.6% an occult ovarian or fallopian tube carcinoma was
found. A gene expression profile was found in high stage ovarian cancer which is
able to discriminate between poor and good overall-survival with an accuracy of 78%.
A specific proteomic pattern is studied in serum and tissue in patients who are either
at high risk of developing ovarian cancer, or who are diagnosed with ovarian cancer.
Goal is to develop a tumour marker profile in the primary diagnosis of ovarian
cancer, to predict the response to treatment, as well as for screening of high risk
populations.
The psychosocial impact of PBSO has been examined in a nationwide, crosssectional, observational study. Forty-four percent of 846 high-risk women had
undergone PBSO, and 56% had opted for gynaecological screening (GS).
No significant differences were observed in generic QOL in both groups. PBSO was
associated with fewer breast and ovarian cancer worries (P < .001) and more
favourable cancer risk perception (P < .05). However, the PBSO group reported
significantly more endocrine symptoms (P < .001) and worse sexual functioning
(P < .05).(KWF project: NKI 2001-2382)
A multicentered randomized trial to investigate the effect of cognitive behavioural
therapy (CBT) and physical exercise for climacteric symptoms in breast cancer
patients experiencing treatment-induced menopause will be shortly opened.
(KWF project: NKI 2006-3470).
In a multicenter trial the effect of different hormonal replacement regimen on bone
density, breast density and quality of live after prophylactic bilateral salpingo
oophorectomy are examined in a randomised control trial (M05HIR Hirise)
The effect of hormonal replacement therapy on menopausal complaints related to
biochemical changes in surgically and naturally postmenopausal women is currently
being investigated in a prospective observational comparative study.M06HRT
(Novaria).
papillomavirus DNA in penile carcinoma
predicts favorable outcome in survival.
Int J Cancer 2006; 119:1078-1081
Madalinska JB, Van Beurden M,
Bleiker EM, Valdimarsdottir HB,
Lubsen-Brandsma L, Massuger LF,
Mourits MJ, Gaarenstroom KN,
Van Dorst EB, Van der Putten H,
Boonstra H, Aaronson NK. Predictors of
prophylactic bilateral salpingooophorectomy verus hynecological screening
uptake in BRCA1/2 mutation carriers.
J Clin Oncol. In press
Madalinska JB, Van Beurden M,
Bleiker EM, Valdimarsdottir HB,
Hollenstein J, Massuger LF,
Gaarenstroom KN, Mourits MJ,
Verheijen RH, Van Dorst EB,
Van der Putten H, Van der Velden K,
Boonstra H, Aaronson NK. The impact
of hormone replacement therapy on
menopausal symptoms in younger
high-risk women after prophylactic
salpingo-oophorectomy. J Clin Oncol
2006; 24:3576-3582
120
SURGICAL ONCOLOGY
Publications (continued)
Molenaar S, Sprangers M, Oort F,
Rutgers E, Luiten E, Mulder J,
Van Meeteren M, De Haes H. Exploring
the black box of a decision aid: what
information do patients select from an
interactive Cd-Rom on treatment options in
breast cancer? Patient Educ Couns 2007;
65:122-130
Morton DL, Thompson JF, Cochran AJ,
Mozzillo N, Elashoff R, Essner R,
Nieweg OE, Roses DF, Hoekstra HJ,
Karakousis CP, Reintgen DS, Coventry BJ,
Glass EC, Wang HJ. Sentinel-node biopsy
or nodal observation in melanoma [Erratum
in N Engl J Med 2006; 355:1944]. N Engl J
Med 2006; 355:1307-1317
Nieweg OE, Kroon BBR. The conundrum
Ovarian carcinoma A randomised, multicentre, 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. Primary endpoint will be progression
free survival; secondary endpoint will be overall survival, safety and quality of life.
(EORTC 55041).
Preparation are made for a phase III randomised clinical trial for stage III ovarian
carcinoma investigating the effect of secondary debulking surgery with or without
hyperthermic intraperitoneal cisplatinum which will shortly be opened.
Vulva Vulvar cancer and tissue micro array (TMA): COX-2 and Caspase-3 are
identified as potential prognostic markers for vulvar cancer in a test set of
50 vulvectomy specimens using immunohistochemical staining of TMA.
The additional value of this immunohistochemical profile in relation to well known
clinicopathological variables in vulvar cancer is tested in a validation study.
The technique of TMA is validated by comparing immunohistochemical staining
results of triplicate core biopsies on TMA with the results of full section analysis.
Progression of vulvar cancer in waiting time before surgery: Progression in waiting
time is determined in 25 patients by comparing the area of the tumour on two digital
photographs. The first is taken at first visit and the second prior to operation.
of follow-up: should it be abandoned? Surg
Oncol Clin N Am 2006; 15:319-330
Noorda EM, Vrouenraets BC, Nieweg OE,
Kroon BBR. Isolated limb perfusion in
regional melanoma. Surg Oncol Clin N Am
2006; 15:373-384
Noorda EM, Vrouenraets BC, Nieweg OE,
Van Geel AN, Eggermont AMM,
VIN The histology and human papillomavirus (HPV) status in patients with a history
of both LS and VIN without coexistent SCC is studied. VIN related to LS without
coexisting SCC is likely to be undifferentiated, unlike what was previously thought.
HPV DNA was demonstrated in 31% and strongly related to high-grade VIN.
HPV DNA/peptide vaccination. KWF grant is submitted in September 2006
Protec study; Purpose of this study is to assess the benefit of wearing therapeutic
elastic stockings in order to prevent lymphedema for patients treated with inguinal
lymph node dissection. Secondairy endpoints are effect on body image and quality of
life issues. Design of the study is a non-blinded, randomised controlled trial.
Kroon BBR. Repeat isolated limb perfusion
with TNFa and melphalan for recurrent
limb melanoma after failure of previous
HEAD AND NECK ONCOLOGY AND SURGERY
perfusion. Eur J Surg Oncol 2006;
Fons Balm, Jan Paul De Boer, Michiel Van den Brekel, Marcel Copper, Frans Hilgers, Frank Hoebers,
32:318-324
Marina Kartachova, Peter Lohuis, Saar Muller, Coen Rasch, Jan Schornagel, Ludi Smeele, Bing Tan,
Renato Valdes Olmos, Karel Zuur
Olivier RI, Lubsen-Brandsma MAC,
Verhoef S, Van Beurden M. CA125 and
transvaginal ultrasound monitoring in
high-risk women cannot prevent the
diagnosis of advanced ovarian cancer.
Gynecol Oncol 2006; 100:20-26
Olivier RI, Van Beurden M. Role of
BRCA1/BRCA2 in ovarian, fallopian tube,
and peritoneal papillary serous carcinoma.
In: Hayat MA, editor.
Immunohistochemistry and in Situ
Hybridization of Human Carcinomas -molecular genetics, gastrointestinal
carcinoma, and ovarian carcinoma.
Academic Press, 2006: 347-357
Olivier RI, Van Beurden M, Van �t Veer LJ.
The role of gene expression profiling in the
clinical management of ovarian cancer.
Eur J Cancer 2006; 42:2930-2938
Intra-tracheal temperature and humidity assessment in laryngectomized
individuals Airway climate change is an unavoidable consequence of total
laryngectomy. Heat and moisture exchangers (HME) ameliorate pulmonary sequels
of total laryngectomy significantly, but the (patho) physiological mechanisms behind
this beneficial effect are not well understood. Therefore, an intra-tracheal
temperature and humidity analyzer was developed for assessment of airway climate
in laryngectomized individuals and assessment of heat and moisture exchanger
(HME) influence on this climate. Intratracheal positioning of a sensor is not suitable.
Tracheal air has to be sampled with a thin catheter and measured externally and. the
sensor should be able to follow normal breathing frequencies. Primary goals of
engineering were prevention of condensation within the device and achievement of
fast response time characteristics. The airway climate explorer (ACE; see figure XI.1)
developed in close collaboration with the departments of �Medische Technologie
Ontwikkeling’ and ENT of the AMC, consists of a small diameter (5 mm), heated airsampling catheter connected to a heated sensor house incorporating a humidity
sensor. Air is sucked through the catheter by a pump with controlled flow. Validation
was performed in a climate chamber using a calibrated reference sensor and in a twoflow system. The validation revealed that over the clinically relevant range of
humidity values (5–42 mg H2O/L air) sensor output correlated highly to reference
sensor readings (R2 >0.99). The 1–1/e response times were all <0.5 s. In vivo
assessment was successful. In conclusion, this newly developed, validated, and fast
responding temperature and humidity analyzer is suitable for post-laryngectomy
airway climate and HME assessment. Further clinical studies assessing the HME
effect under (climatologically) different circumstances are underway.
121
SURGICAL ONCOLOGY
Publications (continued)
Ortin-Perez J, Van Rijk MC,
Valdes-Olmos RA, Vidal-Sicart S,
Nieweg OE, Vilalta A, Kroon BB, Pons F.
Lymphatic mapping and sentinel node
biopsy in Merkel’s cell carcinoma. Eur J
Figure XI.1: Air climate explorer
Surg Oncol 2006;[Epub ahead of print]
(ACE) for intra-tracheal
temperature and humidity
Rodenhuis S, Rutgers EJT. Adjuvant
assessment in laryngectomized
therapy for breast cancer. Br J Surg 2006;
individuals.
93:1443-1445
Smeenk RM, Bex A, Verwaal VJ,
Evaluation treatment strategies From 2000 to November 2004 236 patients from
5 hospitals with (functionally) inoperable cancer of the oral cavity (19%), oropharynx
(63%) and hypopharynx (18%) were randomly assigned to receive 70 Gy/35
fractions/7 weeks combined with either four courses of intra-arterial (IA) cisplatin
(150 mg/m2) and intravenous Na-thiosulfate on days 2, 9, 16 and 23 (N=118) or
intravenous (IV) cisplatin (100 mg/m2) on days 1, 22 and 43 (N=118). The median
follow-up was 17 months. At two years no significant difference was seen between
IA and IV chemotherapy in loco-regional control (62 vs 68% ) or overall survival
(61% vs 63%) (Figure XI.2). Loco-regional control at 2 years was lower for oral cavity
sites (50%) compared to hypopharynx (76%) and oropharynx (68%, p=0.05). Renal
toxicity > grade II was more frequent in the intravenous arm (1 vs 10%, p=0.02),
neurological toxicity was more frequent in the intra-arterial arm (7 vs 1%, p=0.005).
In conclusion: No tumour control difference is seen between the treatment arms.
Severe renal toxicity is less frequent in the intra-arterial arm, but in this treatment
arm neurological toxicity is more common.
Horenblas S, Zoetmulder FAN.
Pseudomyxoma peritonei and the urinary
tract: Involvement and treatment related
complications. J Surg Oncol 2006; 93:20-23
Van Dalen T, Plooij JM, Van Coevorden F,
Van Geel AN, Hoekstra HJ,
Albus-Lutter C, Slootweg PJ,
Hennipman A, Dutch Soft Tissue Sarcoma
Group. Long-term prognosis of primary
retroperitoneal soft tissue sarcoma.
Eur J Surg Oncol 2006; In Press
Van der Poel HG. Foreseeing cancer
metastases. Eur Urol 2006; 50:648-649
Van der Poel HG. Molecular markers in the
diagnosis of prostate cancer. Crit Rev Oncol
Hematol 2007; 61:104-139
Van der Poel HG, Antonini N,
Hoefnagel CA, Horenblas S,
ValdГ©s Olmos RA. Serum hemoglobin levels
Figure XI.2: Kaplan Meier overall survival
predict response to strontium-89 and
curves comparing randomized phase III intra-
rhenium-186-HEDP radionuclide treatment
arterial and intravenous chemo-irradiation in
for painful osseous metastases in prostate
stage III/IV head and neck cancer patients.
cancer. Urol Int 2006; 77:50-56
In vivo imaging of apoptosis in tumour and normal tissue The purpose of this
study is to evaluate if 99mTc Hynic-rh-Annexin V scintigraphy (TAVS), a technique to
depict apoptosis, is feasible to predict treatment response in patients with head
and neck squamous cell carcinoma (HNSCC) treated with chemoradiation.
Sixteen consecutive patients with stage IV HNSCC underwent TAVS using planar
images and SPECT before and 48-hours after the start of therapy (Figure XI.3).
Treatment-induced changes of tracer uptake (О”U) in the tumour and normal tissues
were calculated. Statistically significant correlation was found between radiation dose
and Annexin uptake in salivary gland and bone marrow. A positive correlation for
maximum U in primary tumour and lymph nodes was observed on an inter-patient
level (r = 0.71; p = 0.006). No correlation was observed between the Annexin-uptake
in tumour or lymph nodes and initial treatment response. Annexin V scintigraphy
showed a radiation-dose-dependent uptake in parotid glands and bone marrow,
indicative of early treatment-induced apoptosis. The fact that Annexin uptake in
primary tumour and nodal metastases are correlated suggests a tumour specific
apoptosis response. Lack of correlation between the tracer tumour uptake and shortterm outcome can be explained by the pre-existing necrosis and lymphocyte
infiltration.
Van der Poel HG, Beetsma DB,
Van Boven H, Horenblas S. Perineal
salvage prostatectomy for radiation resistant
prostate cancer. Eur Urol 2006; In Press
Van der Steen LPE, Hage JJ, Kon M,
Monstrey SJ. Intra-rater repeatability of a
structured method of selecting abstracts for
the annual euraps scientific meeting.
Eur J Plast Surg 2006; 29:111-114
Van Duijnhoven FH, Jansen MC,
Junggeburt JMC, Van Hillegersberg R,
Rijken AM, Van Coevorden F,
Van Der Sijp JR, Van Gulik TM,
Slooter GD, Klaase JM, Putter H,
Tollenaar RAEM. Factors influencing the
local failure rate of radiofrequency ablation
of colorectal liver metastases. Ann Surg
Oncol 2006; 13:651-658
122
SURGICAL ONCOLOGY
Publications (continued)
Van Rijk MC, Peterse JL, Nieweg OE,
Oldenburg HSA, Rutgers EJT, Kroon BBR.
Additional axillary metastases and stage
migration in breast cancer patients with
micrometastases or submicrometastases in
sentinel lymph nodes. Cancer 2006;
107:467-471
Van Rijk MC, Tanis PJ, Nieweg OE,
ValdГ©s Olmos RA, Rutgers EJT,
Hoefnagel CA, Kroon BBR. Clinical
implications of sentinel nodes outside the
axilla and internal mammary chain in
patients with breast cancer. J Surg Oncol
2006; 94:281-286
Van Rijk MC, Nieweg OE, Rutgers EJT,
Oldenburg HSA, Olmos RV,
Hoefnagel CA, Kroon BBR. Sentinel node
Figure XI.3: Pre- (A, C, E) and
biopsy before neoadjuvant chemotherapy
post-radiotherapy (B, D, F) uptake
spares breast cancer patients axillary lymph
of Annexin in a base of tongue
node dissection. Ann Surg Oncol 2006;
carcinoma.
13:475-479
Van Rijk MC, Nieweg OE,
UROLOGY
Oldenburg HSA, Rutgers EJT, Kroon BBR.
Simon Horenblas, Wim Meinhardt, Axel Bex, Henk Van der Poel
The sentinel node biopsy in patients with
breast cancer; many controversies remain.
Eur J Gynaecol Oncol 2006; 27:321-328
Van Rijk MC, Teertstra HJ, Peterse JL,
Clinical and scientific activities of the department of Urology comprise a variety of
topics in the field of oncological urology. Many aspects of bladder, prostate, kidney,
penis, and testicular cancer were studied in both preclinical and clinical evaluations.
The most recent developments are mentioned here:
Nieweg OE, Olmos RAV, Hoefnagel CA,
Kroon BBR. Ultrasonography and fineneedle aspiration cytology in the
preoperative evaluation of melanoma
patients eligible for sentinel node biopsy.
Ann Surg Oncol 2006; 13:1511-1516
Van Rijk MC, Deurloo EE, Nieweg OE,
Gilhuijs KGA, Peterse JL, Rutgers EJT,
Kröger R, Kroon BBR. Ultrasonography
and fine-needle aspiration cytology can
spare breast cancer patients unnecessary
sentinel lymph node biopsy. Ann Surg Oncol
2006; 13:31-35
Van Seters M, Ten Kate FJW,
Van Beurden M, Verheijen RHM,
Meijer CJLM, Burger MPM,
Helmerhorst TJM. In the absence of (early)
invasive carcinoma vulvar intraepithelial
neoplasia associated with lichen sclerosus is
mainly of undifferentiated type: new insights
in histology and aetiology. J Clin Pathol
2006;Published Online
Metastasized renal cancer management In primary metastatic renal cell
carcinoma cytoreductive surgery may prolong survival in selected patients. In 2006
we continued to investigate the timing of nephrectomy and the value of initial
immunotherapy as selection for surgery in a randomized trial. In collaboration with
our department of immunotherapy metastatic patients failing immunotherapy were
included in trials with tyrosine-kinase inhibitors.
Invasive bladder cancer management During the last 10 years we have witnessed
an increase in referrals for the management of muscle infiltrating cancer. This has
led to analyze the changes from a low volume hospital to high volume hospital.
With the aid of the database of the comprehensive cancer center Amsterdam (IKA)
we were able to compare results of the management of bladder cancer. A large
number of parameters were compared. We were especially interested in the local
recurrence rate as a measure of surgical expertise. The analysis did show a trend
towards lower recurrence rates in hospitals with a large patient load of bladder
cancer.
A renewed analysis of a modified cystectomy with complete anatomic reconstruction,
developed at this institute, showed the oncological safety together with the
preservation of sexual functions.
Prostate cancer sentinel node After the development of sentinel node procedures
for penile and bladder cancer, the initiation of a study on the laparoscopic sentinel
node procedure for testicular and urachus cancer we initiated a protocol for the
laparoscopic sentinel node detection of the prostate (Figure X1.4). The current
protocol combines the procedure with an extensive nodal dissection. No falsenegative node sentinel nodes were encountered suggesting that in the future a
sentinel node procedure may be sufficient for the detection of nodal metastases in
prostate cancer and an extensive node dissection will be superfluous for diagnostic
purposes.
123
SURGICAL ONCOLOGY
Publications (continued)
Verbruggen MBM, Verheijen RHM,
Van de Goot FRW, Van Beurden M,
Dorsman JCP, Van Diest PJM. Serous
borderline tumor of the ovary presenting
with cervical lymph node involvement:
a report of 3 cases. [Article]. Am J Surg
Pathol 2006; 30:739-743
Woerdeman LAE, Kortmann JBJ, Hage JJ.
Routine histologic examination of 728
mastectomy scars: Did it benefit our
patients? Plast Reconstr Surg 2006;
118:1288-1292
Woerdeman LAE, Hage JJ,
Figure XI.4: Laparoscopic sentinel node procedure of the prostate: Injection of technetium-nanocolloid
Smeulders MJC, Rutgers EJT,
under transrectal ultrasound (1); scintigraphy and CT-scan matching (2); intraoperative sentinel node
Van der Horst CMAM. Skin-sparing
detection using laparoscopic gamma-probe (3); documentation of removed lymph nodes (4).
mastectomy and immediate breast
reconstruction by use of implants: An
Penile cancer During the last 12 years we developed the world largest experience in
sentinel node biopsy in penile cancer. The results were analyzed and updated leading
to a thesis by Bin Kroon jr. Prognostic significance of HPV presence was analyzed in
collaboration with the department of pathology of the Free University medical center.
assessment of risk factors for complications
and cancer control in 120 patients. Plast
Reconstr Surg 2006; 118:321-330
Wong SL, Morton DL, Thompson JF,
Robot-assisted prostatectomy The introduction of the Da Vinci S surgical robot in
our hospital changed the approach to prostatectomy. Within the first 5 months since
its introduction over 50 robot-assisted laparoscopic prostatectomies (RALPs) were
performed succesfully. Conversion to an open procedure because of intraoperative
problems was never necessary and no complications for which operative intervention
was required occurred. An important advantage of the da Vinci S surgical robot is the
option of image integration (Figure X1.5). Intraoperative transrectal ultrasound
facilitated the dissection of bladder neck and neurovascular bundles and significantly
improved oncological and functional outcome.
Figure XI.5: Intraoperative view of
da Vinci S system presenting
simultaneous transrectal
ultrasound, operative view, and
operative instrumentation.
Gershenwald JE, Leong SPL, Reintgen DS,
Gutman H, Sabel MS, Carlson GW,
McMasters KM, Tyler DS, Goydos JS,
Eggermont AMM, Nieweg OE,
Cosimi AB, Riker AI, Coit DG. Melanoma
patients with positive sentinel nodes who did
not undergo completion lymphadenectomy:
A multi-institutional study. Ann Surg
Oncol 2006; 13:809-816
124
PSYCHOSOCIAL RESEARCH
X II D IV IS ION OF PS YCHOS OCIA L
RES EA R CH A ND EPID EM IOLOG Y
PSYCHOSOCIAL ONCOLOGY
Division head, Group leader Neil Aaronson
Neil Aaronson PhD Group leader
The subdivision of psychosocial oncology is pursuing three major research lines: (1)
quality of life assessment in clinical research and clinical practice (Group Aaronson);
(2) psychosocial issues in cancer clinical genetics (Group Aaronson); and (3)
symptom perception and management (Group Van Dam).
Babs Taal MD PhD Academic staff
Marc Van Beurden MD PhD Academic staff
Senno Verhoef MD Academic staff
Eveline Bleiker PhD Post-doc
Kirsten Douma MSc Graduate student
Karin Gehring MSc Graduate student
Doranne Hilarius MSc Graduate student
Rianne Hoopman MSc Graduate student
Ruud Knols MSc Graduate student
Catharina Korse Msc Graduate student
Chantal Lammens MSc Graduate student
Joanna Madalinska MA MSc Graduate student
Chad Gundy MSc Senior statistical analyst
Miranda Gerritsma Research assistant
Esther Janssen Research assistant
Tanja Nagtegaal Research assistant
Publications
The use of health-related quality-of-life (HRQL) assessments in daily clinical
oncology nursing practice: A community hospital-based intervention study
This sequential cohort study is investigating the efficacy of incorporating
standardized HRQL information as a routine part of the community hospital-based
outpatient adjuvant and palliative chemotherapy treatment in terms of: (1) facilitating
nurse-patient communication; and (2) increasing nurses’ awareness of their patients’
HRQL. Additional outcomes include HRQL-related medical record notations and
actions, patients’ and nurses’ satisfaction, and patients’ self-reported HRQL.
The experimental intervention involves having patients complete a computer-assisted
(touchscreen) version of the EORTC QLQ-C30 and relevant condition-specific
questionnaire modules at 3 points in time. A graphic summary of the HRQL profile
is provided to the responsible nurse and to the patient. In total, 11 nurses and 219
patients have been recruited into the study and data collection has been completed.
Preliminary analyses indicate that the intervention had a significant, salutary effect
on: (1) nurse-patient communication (mean number of HRQL topics discussed per
consultation), with largest increases in the discussion of cognitive functioning,
dyspnea, sleep, and GI complaints; (2) nurses’ awareness of their patients’ level of
activities of daily living, pain, and overall HRQL; (3) HRQL-related medical chart
notations; and (4) HRQL-related advice-giving.
Efficace F, Horneber M, Lejeune S,
Van Dam F, Leering S, Rottmann M,
Aaronson NK. Methodological quality of
patient-reported outcome research was low
in complementary and alternative medicine
in oncology. J Clin Epidemiol 2006; 59
Groenvold M, Petersen MA,
Aaronson NK, Arraras JI, Blazeby JM,
Bottomley A, Fayers PM, de Graeff A,
Hammerlid E, Kaasa S, Sprangers MA,
Bjorner JB. EORTC QLQ-C15-PAL: the
new standard in the assessment of
health-related quality of life in advanced
cancer? Palliat Med 2006; 20:59-61
Hilarius DL, Kloeg PH, Detmar SB,
Muller MJ, Aaronson NK. Level of
agreement between patient self-report and
An empirical study of proxy viewpoint in the evaluation of health-related
quality of life (HRQL) There are situations in which patient self-reported HRQL
must be substituted by proxy assessments. Pickard & Knight (2005) recently
delineated two proxy perspectives: the so-called “proxy-patient” (proxy assessment of
how a patient would assess himself) and the “proxy-proxy” perspective (the proxy’s
own assessment of the patient). The purpose of this study was to evaluate patientproxy concordance based on these two alternative approaches. The EORTC-QLQ-C30
was administered to a sample of 224 cancer patients and their proxies
(i.e., significant others). The proxies were randomly assigned to either the “proxypatient” or the “proxy-proxy” perspective. Contrary to expectations, few differences
between conditions were noted in percentage agreement. The only significant
difference was a higher level of agreement for the “proxy-proxy” condition on the role
functioning scale. The cognitive functioning scale showed a significantly higher
correlation in the “proxy-proxy” condition, with a similar trend for the physical and
role functioning scales. These results suggest that the functional scales of the
QLQ-C30 may show an improvement in proxy-patient agreement if the proxy is
instructed to report his own opinion rather than attempt to estimate how the patient
would respond.
observer ratings of health-related quality of
life communication in oncology. Patient
Educ Couns 2007; 65:95-100
Hoopman R, Muller MJ, Terwee CB,
Aaronson NK. Translation and validation
of the EORTC QLQ-C30 for use among
Turkish and Moroccan ethnic minority
cancer patients in the Netherlands.
Eur J Cancer 2006; 42:1839-1847
Cognitive rehabilitation of glioma patients: A prospective, randomized study
This multicenter, randomized trial is evaluating the effectiveness of a cognitive
rehabilitation program on the neuropsychological test performance, self-reported
cognitive complaints, and HRQL of patients with low-grade glioma.
The rehabilitation program incorporates both a computer-based cognitive retraining
component, and a compensation strategy training component. As of November,
2006, 137 patients have been randomized into the study, 119 patients have completed
the immediate post-training assessment, and 82 the 6 month follow-up. first
follow-up. In 200, 72 patients were recruited into the study. Patient recruitment and
follow-up is ongoing.
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PSYCHOSOCIAL RESEARCH
Publications (continued)
A randomized controlled trial of exercise training in hematological cancer
patients after peripheral blood stem cell transplantation This RCT is being
carried out by a PhD student of ours in Zurich, Switzerland. 134 adult hematologic
cancer patients who have undergone peripheral blood stem cell transplantation
(PBSCT) are randomly assigned to a 12-week ambulatory supervised endurance and
repetitive strength exercise program (minimum 3 hours/week) or a usual care 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,
self-reported and objectively assessed physical walking activity, whole body
composition and hematological values. To date, 65 patients have been randomized
into the study.
Madalinska JB, Van Beurden M,
Bleiker EM, Valdimarsdottir HB,
Hollenstein J, Massuger LF,
Gaarenstroom KN, Mourits MJ,
Verheijen RH, Van Dorst EB,
Van der Putten H, Van der Velden K,
Boonstra H, Aaronson NK.
The impact of hormone replacement therapy
on menopausal symptoms in younger
high-risk women after prophylactic
salpingo-oophorectomy.
J Clin Oncol 2006; 24:3576-3582
Predictors of prophylactic bilateral salpingo-oophorectomy (PBSO) versus
gynecological screening (GS) update in BRCA1/2 mutation carriers As part of a
prospective, multicenter study investigating quality of life, menopausal symptoms
and sexual functioning among high-risk women undergoing PBSO versus GS,
we examined the baseline predictors of PBSO versus GS uptake in a subset of
160 BRCA1/2 carriers. During the 12 month period following the first gynecologic
consultation, 74% of women had undergone PBSO. Women with lower educational
levels, with poorer general health perceptions, those who view ovarian cancer as an
incurable disease, and those who believe more strongly in the benefits of surgery
were found to be significantly more likely to undergo PBSO. These results emphasize
the need to ensure that high-risk women are well-informed about the low predictive
value of gynecologic screening techniques and about the lethal threat posed by
ovarian cancer.
Mols F, Vingerhoets AJJM, Coebergh JW,
Vreugdenhil G, Aaronson NK,
Lybeert MLM, Poll-Franse LV. Better
quality of life among 10-15 year survivors of
Hodgkin’s lymphoma compared to 5-9 year
survivors: A population-based study.
Eur J Cancer 2006; 42:2794-2801
Mols F, Poll-Franse LV, Vingerhoets AJJM,
Hendrikx A, Aaronson NK, Houterman S,
Coebergh JWW, Essink-Bot ML. Long-term
quality of life among Dutch prostate cancer
survivors: Results of a population-based
Long-term psychosocial impact of genetic testing among familial
adenomatous polyposis (FAP) families This 4-year cross-sectional study began in
November 2004, in collaboration with the Netherlands Foundation for the Detection
of Hereditary Tumors (STOET; Hans Vasen). The study is investigating the mid-tolong-term (>1 yr) psychosocial impact of a (clinical or genetic) diagnoses of FAP, the
social and psychosexual impact of such a diagnosis among those diagnosed before
the age of 20 years, the experience with and predictors of compliance with preventive
health options (endoscopic screening), and the knowledge of and attitudes toward
prenatal diagnosis of FAP and pre-implantation techniques. The study will include all
members of families, registered at the STOET, with a high risk for FAP who are
currently 16 years of age or older (estimated n = 219 families; n = 750 family
members). The partners of individuals with positive test results or clinical diagnoses
are also invited to participate (estimated n = 250). Data are collected via self-report
questionnaires and personal interviews. To date, questionnaires have been returned
by 488 family members (response rate = 66%) and 124 partners (response rate =
84%), and 52 semi-structured interviews have been completed.
Psychosocial aspects of genetic testing in families at high risk of multiple
tumors at various sites and ages This multicenter study, which is being conducted
in collaboration with the Family Cancer Clinic (Senno Verhoef), is investigating:
(1) the uptake of genetic testing for Li-Fraumeni Syndrome (LFS) and Von HippelLindau (VHL) and the characteristics of those who do and do not undergo genetic
testing; (2) the psychosocial consequences (e.g., quality of life, cancer worries, family
relationships) of (not) undergoing genetic testing; (3) compliance with recommended
surveillance programs and factors associated significantly with (non) compliance; and
(4) the attitudes of at-risk individuals and their partners toward prenatal diagnosis of
LFS/ VHL and pre-implantation genetic diagnosis. The study will include members
of the 18 families known to have the Li-Fraumeni syndrome (105 adults and
approximately 50 partners) and the 40 families with Von Hippel-Lindau syndrome
(approximately 160 adults and 110 partners). Data are collected via questionnaire and,
for a subset of respondents, via semi-structured interviews. Data collection began in
August 2006 and will continue until September 2007. As of November 2006, 53 of
64 distributed questionnaires have been completed (83% response rate).
study. Cancer 2006; 107:2186-2196
Scott NW, Fayers PM, Aaronson NK,
Bottomley A, De Graeff A, Groenvold M,
Koller M, Petersen MA, Sprangers MAG.
The use of differential item functioning
analyses to identify cultural differences in
responses to the EORTC QLQ-C30. Qual
Life Res 2006; 16:115-129
Van Luijn HEM, Aaronson NK, Keus RB,
Musschenga AW. The evaluation of the
risks and benefits of phase II cancer clinical
trials by institutional review board (IRB)
members: A case study. J Med Ethics 2006;
32:170-176
Figure XII.1: Health-related quality of life
profile scores for use in daily clinical
oncology practice.
126
PSYCHOSOCIAL RESEARCH
SYMPTOM PERCEPTION AND MANAGEMENT
Cognitive impairment of cancer chemotherapy, a multidisciplinary
research line
Group leader Frits Van Dam
Frits Van Dam PhD Group leader
Willem Boogerd MD PhD Academic staff
Sanne Schagen PhD Post-doc
Baudewijntje Kreukels MSc Graduate student
Christien Schilder MSc Graduate student
Chad Gundy MSc Senior statistical analyst
Anky Caspers Research assistant
Tinneke Danhof Research assistant
Elke Eggens Research assistant
Suzanne Leering Research assistant
Annemarie Van Nuland Research assistant
Marion Weevers Research assistant
A prospective randomized controlled study on the effects of chemotherapy for
breast cancer
We continued the analyses of our longitudinal randomized study with a group of
high-risk breast cancer patients that received either adjuvant high-dose CTC
chemotherapy (n=28) or standard-dose FEC (n=39) chemotherapy. The patients were
tested neuropsychologically before the start of chemotherapy and one year after this
first assessment. The results of these patients were compared to the results of a
group of breast cancer patients not treated with chemotherapy (n=57), tested with a
similar time interval, and to the results of a group of matched healthy controls
(n=60), which also underwent repeated neuropsychological testing. We are currently
analyzing a second follow-up assessment for all patients, which took place 6 months
after the first follow-up. We also asked the proxies of all patients to assess the
patients’ cognitive status and compared this with patients’ own assessment of her
cognitive status. 6 months after completion of chemotherapy, there is a high
concordance between the patients’ and the proxies ratings. Currently, we are studying
changes in cognitive complaints reported by patients and their proxies over time,
i.e. from pre-chemotherapy to post treatment.
Animal studies on the effects of cytotoxic agents on cognition In follow-up to
our experiments in which rats were injected with singles dosages of intraveneous
MTX (which showed a significant reduction in cell proliferation in the hippocampus,
with a clear dose response relation, which was also reflected in their cognitive
behavior), we are currently investigating whether other cytotoxic agents cause similar
effects on neurogenesis. 5-FU and cyclophosphamide are the first agents to be
studied.
Effects of endocrine treatment on cognitive functioning A prospective
comparative study into the neuropsychological sequelae of postmenopausal breast
cancer patients receiving exemestane or tamoxifen compared to a healthy group of
women matched for menopausal status and age is conducted since 2003. Patients
are recruited from the TEAM trial; an open label, randomized multicenter study of
either exemestane treatment (25 mg once daily) or 2-3 years adjuvant tamoxifen
(20 mg once daily) followed by 3-2 years exemestane treatment (25 mg once daily).
Patients are being assessed twice: before receiving tamoxifen or exemestane and
after 12 months of treatment. Healthy controls are assessed with the same time
interval. 38 hospitals are participating in this study. At the moment, patient inclusion
is complete: 206 patients and 124 healthy controls are included in the study.
The collection of follow-up data will be complete in March 2007.
Figure XII.2: Spatial learning in the Morris
water maze. Used to study the effects of
cytotoxic agents on the cognitive function of
rats.
Effects of AC-chemotherapy, followed by endocrine treatment on cognitive
functioning
We conducted a cross-sectional study in a group of postmenopausal breast cancer
patients that received endocrine therapy after they completed AC-chemotherapy in
the adjuvant setting. Patients were recruited from the TEAM-trial and received either
exemestane treatment (AC-EXE group; n=51; mean age = 58,5) or tamoxifen
treatment (AC-TAM group, n=30; mean age = 57,9). The patients were tested
neuropsychologically on average 2.2 years after completion of AC-chemotherapy.
The results of these patients were compared to the results of a group of healthy
controls (mean age 60,2; n=48), matched on age and IQ. In both the AC-TAM group
as in the AC-EXE group more women reported memory problems in daily life than in
the group of healthy controls (resp. 27,6% and 25,5 % versus 6,3%, p=.02).
No difference between tamoxifen users and exemestane users with respect to
experienced cognitive problems was found. On the neuropsychological measures,
both the AC-TAM as the AC-EXE group had significantly lower mean scores on tests
of �information processing speed’ than healthy controls (p=.023 and .008).
In addition, tamoxifen users scored significantly lower on a �mental flexibility’ test
127
PSYCHOSOCIAL RESEARCH
Publications
(p=.007) and a �category fluency’ test (p=.000) than healthy controls. Tamoxifen-users
tend to score worse than exemestane users on several tests, particularly attention
tests, a letter fluency test, a verbal memory test and a visual association test.
Furthermore, no correlations were found between reported cognitive problems in
daily life and cognitive test scores, but cognitive problems in daily life were correlated
with fatigue and symptoms of anxiety and depression.
Kreukels BPC, Schagen SB,
Ridderinkhof KR, Boogerd W,
Hamburger HL, Muller MJ,
Van Dam FSAM, Schagen SB.
Effects of high-dose and conventional-dose
adjuvant chemotherapy on long-term
Cognitive functioning and endocrine therapy for prostate cancer Endocrine
therapy plays an increasing role in the treatment of prostate cancer. Since
testosterone seems to influence cognitive functioning, like visuo-spatial functioning
and probably also memory, it has been suggested that endocrine therapy for prostate
cancer may have an effect on cognitive functioning as well.
We performed a cross-sectional study in which the cognitive functioning of patients
with localised prostate cancer (n=25) currently undergoing neo-adjuvant endocrine
treatment with LHRH agonists was examined using standardized neuropsychological
tests. The cognitive performance of these patients was compared with that of prostate
cancer patients who had been treated with brachytherapy (n=32) in the last three
years. All patients were also interviewed about cognitive problems, health-related
quality of life, emotional distress and prostate cancer specific symptoms.
A significant higher percentage of patients undergoing endocrine treatment reported
cognitive problems compared to the patients previously treated with brachytherapy.
Compared with the brachytherapy group, the endocrine therapy group also reported
significantly more feelings of emotional distress, more prostate cancer specific
symptoms, and they indicated more problems regarding several health-related quality
of life aspects. The neuropsychological results showed that 36% of the patients
undergoing endocrine treatment were classified as cognitively impaired, compared
with 6% of the brachytherapy patients (p=.027). For the brachytherapy patients, time
since treatment had no influence on the reported complaints or on the test
performance.
A larger study with a longer follow-up, that would also include a post endocrine
therapy assessment, should point out whether this association between of cognitive
impairment and endocrine treatment still holds.
Complementary and Alternative Medicine (CAM) A substantial number of
patients use one or more alternative treatments in addition to their regular cancer
treatment. From surveys in the Netherlands Cancer Institute over more than twenty
years with more than 3,500 patients, we noticed that the number of patients using
CAM has decreased dramatically over the last years. E.g. the number of patients
using �anti-cancer’ diets has dropped from about 13% in 1999 to less than 2% in
2006. External factors like media attention for patients who have died because of
using solely CAM and the downfall of important alternative therapists seem to have
contributed to this decline in the number of patients using CAM. We plan to
continue our yearly inventory of the number of patients using CAM.
cognitive sequelae in patients with breast
cancer: An electrophysiologic study. Clin
Breast Cancer 2006; 7:67-78
Schagen SB, Muller MJ, Boogerd W,
Mellenbergh GJ, Van Dam FS. Change in
cognitive function after chemotherapy:
a prospective longitudinal study in breast
cancer patients. J Natl Cancer Inst
2006; 98:1742-1745
128
PSYCHOSOCIAL RESEARCH
EPIDEMIOLOGY
The cancer epidemiology group is currently concentrating on two principal research
lines: (1) the etiology of hormone-related cancers; (2) the long-term health
consequences of cancer treatment, particularly in terms of the risk of developing
second malignancies or cardiovascular disease.
Group leader Flora Van Leeuwen
Group leader Matti Rookus
Flora Van Leeuwen PhD Group leader
Matti Rookus PhD Group leader
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
Marieke De Bruin PhD Post-doc
Lisette Hoogendoorn PhD Post-doc
Peggy Manders PhD Post-doc
Ina Mulder PhD Post-doc
Marjanka Schmidt PhD Post-doc
Eric Vermeulen PhD Post-doc
Dorien Voskuil PhD Post-doc
Richard Brohet MSc Graduate student
Mathilde Cardous-Ubbink MSc Senior
Graduate student
Maartje Hooning MD Graduate student
Agnes Olde Damink-Van Rosmalen MSc
Graduate student
Anouk Pijpe MSc Graduate student
Sandra Van den Belt-Dusebout MSc
Graduate student
Janneke Verloop MSc Graduate student
Alina Vrieling MSc Graduate student
Willem Klokman MD MSc Senior Statistical
analyst
Thea Mooij MSc Statistical analyst
Gijs Besseling MSc Research assistant
Ivo Bisschops Research assistant
Steven De Jager Research assistant
Geri De Leeuw-Mantel Research assistant
Simone Dijkhuis MSc Research assistant
Bregje Huisman MSc Research assistant
Esther Janssen Research assistant
Kiki Jeanson MSc Research assistant
Kim Karsenberg MSc Research assistant
Marianne Kuenen Research assistant
Monica Legdeur Research assistant
Gabey Ouwens Research assistant
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 are now expanding the existing cohort and we also started to collect
follow-up information from women who were tested for a BRCA1/2 mutation and
were already included in the study in 1996-2000. Presently, the cohort comprises
616 BRCA1 families and 175 BRCA2 families with 37,912 and 11,001 family
members, respectively. We received risk factor questionnaires from 3,633 family
members and follow-up questionnaires from 457 women who had been tested in
the past.
To obtain sufficient power for the assessment of gene-environment interactions, our
data are incorporated in an international cohort study of BRCA1/2 carriers (IBCCS).
In a retrospective cohort of the first 1,601 BRCA1/2 carriers we examined the impact
of very low dose diagnostic radiation. Any reported exposure to chest X-rays was
associated with an increased risk of breast cancer (HR=1.54). The increased risk was
found among carrier women aged 40 and younger (HR=1.97), and in women born
after 1949 (HR=2.56). Especially, women exposed only before age 20 showed an
increased risk (HR=4.64). Thus, in our series of BRCA carriers we detected a
relatively large effect on breast cancer risk with a level of radiation exposure that is at
least an order of magnitude lower than in previously studied cohorts exposed to
diagnostic radiation. Although part of this increase may be attributable to recall bias,
the observed patterns of risk in terms of age at exposure and attained age are
consistent with those found in previous studies. If confirmed, the results have
important implications for the use of X-ray imaging in young BRCA1/2 carriers.
In another IBCCS analysis we examined endogenous hormonal factors. Breast cancer
risk was not significantly related to age at menarche nor age at menopause.
There was evidence of a protective effect of oophorectomy (HR 0.56) and a significant
trend of decreasing risk with increasing time since oophorectomy, but no apparent
effect of natural menopause.
In 2006, we received funding to expand our cohort of 19,213 women who received
ovarian stimulation for in vitro fertilization (IVF) in the period 1983-1995. We will
include 8800 women who received IVF more recently (1995-1998) to assess the risk
of hormone-related cancers. The control group of 6,017 women will also be expanded
(collaboration with CW Burger, Erasmus MC Rotterdam) and follow-up of the full
cohort (n=38,000) will be extended. In 2006 we continued data collection for our
cohort study on hormone-related cancer risk in women who were exposed to
diethylstilbestrol (DES) in utero (DES daughters). In total, 8,557 DES daughters
returned a risk factor questionnaire. We are validating 4,520 reported health
problems of 3,046 women with clinical medical records.
The Epidemiology group is also closely involved in three intervention studies (using
lycopene and isoflavone supplements) focusing on the insulin-like growth factor
system in relation to breast cancer and colorectal carcinogenesis (see Division
Experimental Therapy).
Overweight and physical activity as determinants of (second) cancer risk and cancer
prognosis are being studied in several projects. Several systematic reviews have been
conducted of the epidemiological literature on physical activity and risk of breast,
colorectal, and endometrial cancer. We concluded that physical activity decreases the
risk of breast cancer, especially in postmenopausal women, and possibly also the risk
of cancer of the colon and endometrium. Currently, several pilot studies are being
conducted to assess the frequency of overweight and (unintentional) weight gain in
breast cancer patients after diagnosis, and to evaluate a combined diet, physical
activity and behavioral counseling intervention on weight loss in overweight breast
cancer patients after primary treatment.
129
EPIDEMIOLOGY
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 radioand chemotherapy (RT and CT) for Hodgkin’s lymphoma (HL) (n=3,400), testicular
cancer (n=2,707) and breast cancer (n=8,000) over a period of up to 30 years after
primary treatment.
It has been shown that CVD causes excess mortality in survivors of HL. However, few
studies have evaluated the long-term incidence of CVD and quantified the effects of
RT and CT. We assessed CVD risk in 1,474 5-year survivors of HL treated before the
age of 41 years in the NKI between 1966 and 1995 (median follow-up time, 18.7
years). In all, 233 patients had developed one or more coronary heart diseases while
only 58 were expected (standardized incidence ratio (SIR)=4.0). SIRs were strongly
elevated for myocardial infarction (MI) (SIR=3.6), angina pectoris (SIR=4.1) and
congestive heart failure (HF) (SIR=4.9). The SIRs remained increased with longer
follow-up; e.g. for MI, the SIR was 4.0 in the 5-25 year follow-up interval and 2.9
among 25-year survivors. Mediastinal radiotherapy significantly increased the risks of
MI, angina pectoris, HF and valvular disorders (2- to 7-fold). Anthracyclines
significantly added to the elevated risks of HF and valvular disorders from
mediastinal RT (Hazard Ratios (HRs): 2.8 and 2.1, respectively).
We also analyzed second malignancy risk in 1155 women treated for HL before age 51.
After a median follow-up of 18.2 years we observed 100 cases of second breast cancer
(SIR 5.4). Breast cancer risk remained increased up to 30 years after treatment (SIR
8.7). Patients treated before age 21 experienced the highest risk (SIR 16.9), resulting
in a cumulative risk of 21% of developing breast cancer before age 45. Patients who
received RT including the breast area had a 5-fold increased risk for breast cancer
compared to those who received no such irradiation. Among these patients, both
chemotherapy and radiotherapy to the ovaries decreased risks for BC (HR 0.5 and
0.3, respectively), probably because these gonadotoxic therapies induce premature
menopause. Gene-expression analysis of breast cancer after HL suggests a distinct
molecular profile of radiation-induced breast cancer (see Division VIII).
Monique Simons MSc Research assistant
Renate Udo Research assistant
Josette Van As MSc Research assistant
Anne Van Dijk Research assistantt
Publications
Aleman BMP, Van den Belt-Dusebout AW,
De Bruin ML, Van �t Veer MB,
Baaijens MH, De Boer JP, Hart AA,
Klokman WJ, Kuenen MA, Ouwens GM,
Bartelink H, Van Leeuwen FE.
Late cardiotoxicity after treatment for
Hodgkin’s lymphoma. Blood 2006;[Epub
ahead of print]
Andrieu N, Easton DF, Chang-Claude J,
Rookus MA, Brohet R, Cardis E,
Antoniou AC, Wagner T, Simard J,
Evans G, Peock S, Fricker JP, Nogues C,
Van �t Veer L, Van Leeuwen FE,
Goldgar DE. Effect of chest X-rays on the
risk of breast cancer among BRCA1/2
mutation carriers in the international
BRCA1/2 carrier cohort study: a report
from the EMBRACE, GENEPSO, GEOHEBON, and IBCCS Collaborators’
Group. J Clin Oncol 2006; 24:3361-3366
Andrieu N, Goldgar DE, Easton DF,
Rookus M, Brohet R, Antoniou AC,
Peock S, Evans G, Eccles D, Douglas F,
Nogues C, Gauthier-Villars M,
Chompret A, Van Leeuwen FE, Kluijt I,
Benitez J, Arver B, Olah E,
Chang-Claude J. Pregnancies, breastfeeding, and breast cancer risk in the
International BRCA1/2 Carrier Cohort
Study (IBCCS). J Natl Cancer Inst 2006;
98:535-544
Hooning MJ, Aleman BMP, Van
Figure XII.3: Risk of breast cancer after Hodgkin’s lymphoma by follow-up time. Based on 1155 female
Rosmalen AJ, Kuenen MA, Klijn JG,
survivors, age at diagnosis <50 yr, 1966-1995 (median follow-up: 18 years)
Van Leeuwen FE. Cause-specific mortality
in long-term survivors of breast cancer:
The effects of treatment on premature menopause were studied separately in
518 female 5-year HL-survivors, younger than 40 years at treatment. Chemotherapy
was associated with a 12-fold increased risk for premature menopause (HR 12.2).
Increased risks were only found for alkylating agents (HR 14.4), especially
procarbazine (HR 12.2) and cyclophosphamide (HR 3.4). The risk associated with
procarbazine was dose-related and independent of the effect of mechlorethamine.
Per additional 1.4 g/m2 of cumulative exposure to procarbazine (which is equal to the
amount given in one cycle of MOPP or two cycles of MOPP/ABV) the risk of
menopause increased with 25%.
A 25-year follow-up study. Int J Radiat
Our study on late effects of testicular cancer treatment focuses on the long-term risk
of second malignancies (SMNs) following radiation and the risk of cardiovascular
disease (CVD) after chemotherapy (CT). The nationwide cohort comprises 2,707
5-year testicular cancer survivors treated between 1965 and 1995.
Monninkhof EM, Elias SG, Vlems FA,
Oncol Biol Phys 2006; 64:1081-1091
Hooning MJ, Dorresteijn LD,
Aleman BMP, Kappelle AC, Klijn JG,
Boogerd W, Van Leeuwen FE. Decreased
risk of stroke among 10-year survivors
of breast cancer. J Clin Oncol 2006;
24:5388-5394
Van der Tweel I, Schuit AJ, Voskuil DW,
Van Leeuwen FE. Physical Activity and
Breast Cancer: A Systematic Review.
Epidemiology 2007; 18:137-157
130
EPIDEMIOLOGY
Publications (continued)
Moser EC, Noordijk EM, Van Leeuwen FE,
Le Cessie S, Baars JW, Thomas J, Carde P,
Meerwaldt JH, Van Glabbeke M,
Kluin-Nelemans HC. Long-term risk of
cardiovascular disease after treatment for
aggressive non-Hodgkin lymphoma. Blood
2006; 107:2912-2919
Schmidt MK, Tollenaar RA, De Kemp SR,
Broeks A, Cornelisse CJ, Smit VT,
Peterse JL, Van Leeuwen FE,
Van �t Veer LJ. Breast Cancer Survival and
Tumor Characteristics in Premenopausal
Women Carrying the CHEK2*1100delC
Germline Mutation. J Clin Oncol 2007;
25:64-69
Van den Belt-Dusebout AW, Nuver J,
De Wit R, Gietema JA,
Ten Bokkel Huinink WW, Rodrigus PT,
Schimmel EC, Aleman BMP,
Van Leeuwen FE. Long-term risk of
cardiovascular disease in 5-year survivors
of testicular cancer. J Clin Oncol 2006;
24:467-475
Figure XII.4: Cumulative risk of cardiovascular
disease after breast cancer by radiotherapy (yes/no)
and period (<1980, ≥1980)
Figure XII.5: Uterine corpus cancer (UCC)-specific
survival according to use of tamoxifen for breast
cancer. Based on 642 women with uterine corpus
cancer after breast cancer.
After a median follow-up of 18 years, we observed 287 SMNs and 357 CVDs of
interest (the most important diagnoses acute myocardial infarction (MI), angina
pectoris (AP) and congestive heart failure (CHF)). The risk of SMN overall was 1.7fold increased with an AER of 32.3 excess cases per 10,000 person-years and the risk
of CVD was 1.2-fold increased compared with the general population. Tumors from
the digestive and genitourinary tract contributed most to the absolute excess risk of
SMNs. The risk of SMN was 2.5-fold increased after subdiaphragmatic radiotherapy
and 1.8-fold increased after chemotherapy, compared with surgery only. We also
analyzed the risk of developing one of multiple adverse outcomes. The risk of
developing SMN or CVD was 1.7-fold increased after subdiaphragmatic radiotherapy
and was significantly increased (1.7-fold) after chemotherapy. A 1.7-fold increased risk
of SMN or CVD was also observed after smoking. We conclude that radiotherapy
strongly increases the risk of SMNs but not of CVD, while chemotherapy slightly
increases the risks of both SMNs and CVDs.
In 2006, we assessed the incidence of CVD in 1601 patients with T1-2N0 breast
cancer treated with postlumpectomy irradiation restricted to tangential fields in 5
different Dutch hospitals between 1980 and 1993. Patients treated with radiation
fields other than breast tangentials and those treated with adjuvant chemotherapy
were excluded. For patients with left-sided breast cancer maximum heart distance
(MHD) was measured by two observers on the original simulator films as a proxy for
irradiated heart volume.
Follow-up was complete for 94% of the patients and median follow-up was 16 years.
The incidence of CVD was 16% in patients with left-sided breast cancer versus 11.6%
in right-sided cases. The hazard ratio (HR) associated with left-sided versus rightsided breast cancer was significantly increased for ischemic heart disease (HR, 1.41),
and other heart disease (HR, 1.53). The risk of CVD did not significantly increase with
increasing MHD.
Possibly, the higher incidence of CVD in patients irradiated with tangential fields for
left-sided breast cancer can be explained by radiation dose-volume aspects or a higher
radiation dose to crucial coronary arteries.
In a previous study on tamoxifen’s late effects, we showed that the risk of poorprognosis uterine corpus cancers increased with duration of tamoxifen treatment for
breast cancer. We are now conducting a new nationwide study to confirm these
findings and to also obtain insight into the mechanism whereby tamoxifen causes
uterine corpus cancer. We are investigating whether the clinicopathologic
characteristics and prognosis of uterine corpus cancers differ between women with
and without long-term tamoxifen treatment. We are also searching for tamoxifenspecific genomic aberrations and differences in gene-expression.
Our existing cohort of 309 patients with uterine corpus cancer following breast
cancer was expanded with 533 of such patients diagnosed since 1996. Information
about breast cancer treatment and follow-up was abstracted from the medical
records. Tissue blocks of 98% of uterine corpus cancers were obtained to review
histology and to examine genomic aberrations. For 200 prospective cases we also
collected fresh-frozen tissue. The 3-year uterine corpus cancer-specific survival was
significantly worse for long-term tamoxifen users than for non-users (82% for ≥2 yr
tamoxifen users vs. 93% for non-users). These results confirm our previous findings.
Genomic and gene-expression analysis are ongoing.
In the Netherlands the law may be changing regarding secondary use of biological
samples obtained for diagnostic or therapeutic purposes. At present, secondary use
for research is allowed when patients have not objected to such use after having been
informed about the possibility of secondary use of samples in a general patient
information leaflet. The new law may include written informed consent, with no
clarity as to whether the permission can apply to a broad research area. Clearly, this
would heavily impact on future research with biological samples. The Cancer
Genomics Center funded a study to evaluate various options for asking informed
consent. The research aims to find out whether patients give consent for research
with their remaining tissue, what information needs of patients are, what kind of
decision procedure patients prefer and which choice options they would want to have.
Data collections begins at the end of 2006.
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PSYCHOSOCIAL RESEARCH
QUALITY OF ONCOLOGIC CARE
Constructive Technology Assessment of the introduction of a 70-gene micro
array prognostic test in breast cancer treatment From 2003 to the end of 2006,
a study was conducted on the feasibility of introducing a 70-gene microarray test as a
prognostic tool in the treatment of node negative breast cancer (the RASTER-study).
As the diffusion of this technology is in a very early stage and the course of
development is difficult to predict, an evaluation approach, constructive technology
assessment (CTA), was chosen that takes the technology dynamics into account. In
total, 789 patients from 16 hospitals were recruited into the study, of whom 417 were
tested. Preliminary results show that approximately 30% of patients receive treatment
advise based on the microarray test that is based on traditional clinical guidelines
(discordant cases). Additionally, it was observed that, on average, it takes 6 months
for a hospital to decide to make use of and to implement this new technology.
Pre- and post introduction measurements concerning logistics, teamwork and patient
centeredness have been performed in 2006 and will continue in 2007. A proposal
has been prepared to continue the CTA as a side study of the MINDACT study, a
large trial in which in one arm the discordant cases are randomised. The proposal
includes quality-related aspects such as timeliness, safety and patient centeredness,
a cost-effectiveness analysis (in cooperation with MGZ-Rotterdam), a study on legalethical aspects of patients rights concerning banked tissues, and scenario-use as a
means to guide implementation processes.
Rehabilitation, Physical Activity and Cancer In recent years it has become evident
that survivorship care and rehabilitation requires greater attention than it has
received to date. In 2006, we have worked on developing a research infrastructure
for future studies studies in this area. In collaboration with the Slotervaart Hospital
and the Jan van Breemen rehabilitation clinic, a multidisciplinary rehabilitation
program was initiated for breast cancer survivors receiving adjuvant treatment.
To date, pre- and post measurements of the pilot groups have indicated that there is
sufficient improvement in quality of life and functional health to embark on a more
formal randomized clinical trial to investigate the program’s clinical effectiveness.
Additionally, a (retrospective) needs assessment survey was performed among
109 breast cancer survivors, indicating that between 20% and 30% of survivors
expressed a desire for rehabilitation activities to improve physical and psychological
well being, and to reduce fatigue.
Finally, a pilot, feasibility study has been initiated on “Active Lifestyle” during
chemotherapy. This approach has been demonstrated to be effective in other patient
populations. This also includes a literature review of theoretical models used in such
interventions with cancer patients.
Group leader Wim van Harten
Wim van Harten MD PhD Group leader
Jolien Bueno De Mesquita MD Research staff
Rutger Dahmen MD Academic staff
Miranda Van Duijn MSc Academic staff
Kirsten Douma MSc Graduate student
Nienke Ballast Research assistant
Inge Haenen Research assistant
Kim Karsenberg MSc Research assistant
Wineke Van Lent MSc Research assistant
Maaike Van Vliet Research assistant
Publications
Van Luenen HGAM, Van Harten WH.
Quality Management and Stimulation of
Technology Transfer in a Research Institute.
Creat Innov Manag 2006; 15:207-217
Van der Ploeg H, Streppel KRM,
Van Der Beek AJ, Van Der Woude LHV,
Vollenbroek-Hutten MMR,
Van Harten WH, Van Mechelen W.
Counselling increases physical activity
behaviour nine weeks after rehabilitation.
Brit J Sports Med 2006; 40:223-229
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DIAGNOSTIC ONCOLOGY
X III D IV IS ION OF
DIA G NOS TIC ONCOLOG Y
DEPARTMENT OF CLINICAL CHEMISTRY
Pharmacological studies in mice
Division head, Group leader Marc Van de Vijver
Nienke Van den Brink-de Vries, Tessa Buckle, Jin Zhao, Roos Oostendorp, Laura Donga,
Olaf Van Tellingen
DEPARTMENT OF CLINICAL CHEMISTRY
Willem Nooijen PhD Academic Staff
Hans Bonfrer PhD Academic Staff
Olaf Van Tellingen PhD Academic Staff
Tessa Buckle Technical staff
Tiny Korse Technical staff
DorothГ© Linders Technical staff
Marian Buning Technical staff
Nienke Van den Brink-de Vries Graduate student
DEPARTMENT OF NUCLEAR MEDICINE
Cornelis A Hoefnagel MD PhD Academic Staff
Philippe Baars MD Academic Staff
Saar Muller PhD Academic Staff
Michiel Sinaasappel PhD Academic Staff
Ferida Sivro-Prndelj MD Academic Staff
Renato ValdГ©s Olmos MD PhD Academic Staff
Remco Knol MD Registrar
Lenka Pereira-Arias MD Registrar
Albert Lafeber MD Registrar
Marina Kartachova MD Clinical Fellow
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
Bert Pool Technical Staff
Lyandra Rooze Technical Staff
Mariska Sonneborn Technical Staff
DEPARTMENT OF PATHOLOGY
Frans Hogervorst PhD Academic Staff
Daphne De Jong MD PhD Academic Staff
Petra Nederlof PhD Academic Staff
RenГ©e Van Pel MD Academic Staff
Hans Peterse MD Academic Staff
Hester Van Boven MD PhD Academic Staff
Marc Van de Vijver MD PhD Academic Staff,
Head Division XIII
Loes Van Velthuysen MD PhD Academic Staff
Laura Van ’t Veer PhD Academic Staff
Jelle Wesseling MD PhD Academic Staff
Limited blood-brain barrier (BBB) penetration of drugs is generally considered to
be a major cause of the overall lack of efficacy of systemic chemotherapy against
intracranial malignancies. We and others have shown the important role of drug
efflux pumps (P-gp and BCRP) at the BBB. We have now shown that these
transporters also limit the BBB penetration of temozolomide (TMZ), the first and
only drug with proven activity against malignant glioma. This finding was not
expected, since there is a general consensus that this activity is based on the good
BBB penetrating properties of this agent.
In vitro experiments clearly demonstrated TMZ vectorial translocation by MDCKBcrp1 cells, but not by LLC-Mdr1a or LLC-MDR1 cells, suggesting that BCRP but not
P-gp is capable of transporting TMZ. Subsequently, we have studied the brain
penetration in wild-type (WT), Bcrp1 KO, Mdr1a/Mdr1b DKO and Mdr1a/Mdr1b/
Bcrp1TKO mice. We found that the accumulation of TMZ in the brain was about
20% higher in KO and DKO mice and about 50% higher in TKO mice compared to
wild-type controls. Thus, both P-gp and BCRP are working together in limiting the
brain penetration of TMZ. Interestingly, the enhanced brain penetration was
achieved without reducing the plasma clearance of this agent. Although the gain in
brain penetration of TMZ was more modest than the 10-fold or higher increases
previously shown for other drugs, we expect that a 50% enhancement of a drug that
is already active against malignant glioma may substantially further improve its
clinical efficacy. We are currently generating TKO nude mice that will be used for
efficacy studies against intracranial xenografts.
More or less similar results have been observed with the tyrosine kinase inhibitors
for EGFR (erlotinib) and the PDGFR (imatinib). Both novel molecularly targeted
agents, primarily developed for other (more common) tumor types, also interfere in
pathways involved in malignant glioma. Therefore, they are being tested in malignant
glioma patients. Our studies show markedly (3 to 4-fold) increased brain levels in
DKO mice, which was further enhanced in TKO mice. Thus both P-gp and BCRP
limit the BBB penetration of these agents. These results highlight the necessity to
inhibit drug efflux pumps at the BBB in order to achieve adequate drug delivery of
agents directed at potentially drugable targets in malignant glioma.
In order to study such novel compounds, better brain tumor models than traditional
xenograft models are essential. We have been successful in the development of
spontaneous models of malignant glioma, based on Cre-Lox conditional mouse
models. We have generated a lentiviral vector containing truncated GFAP promotordriven Cre recombinase that gives rise to grade 3 astrocytomas after stereotactic
intracranial injection in compound conditional kRAS transgenic, Ink4a/Arf knockout
animals. When either PTEN or P53 conditional genes are also deleted the picture
shifts to grade 4 astrocytoma (Glioblastoma multiforme). Concurrent switching of
conditional luciferase allows for non-invasive monitoring, which is an essential
feature for performing intervention studies. We are currently characterizing these
tumors in more detail.
Douwe Atsma Technical Staff
Jolien Bueno de Mesquita Graduate Student
Novel strategies for biomarker discovery using MALDI-MS
based proteomics tools
Stella Mook Graduate Student
DorothГ© Linders, Olaf Van Tellingen, Hans Bonfrer, Wim Nooijen, Theo Luider, Lennard Dekker,
Marie-Christine Hermus Technical Staff
Markus Meyer, Dagmar Niemeyer
Lucie Boerrigter-Barendsen Technical Staff
Aafke Wieringa-Ariaens Technical Staff
Henrique Ruijter-Schippers Technical Staff
Marjanka Schmidt PhD Post-doc
Carla Schippers-Gillissen Technical Staff
In collaboration with investigators from the Erasmus University and Bruker
Daltronics we have undertaken a study to identify potential serum tumor markers
from a group of sarcoma patients using ClinProtВ® beads technology combined with
133
DIAGNOSTIC ONCOLOGY
MALDI-MS detection. We compared 80 controls versus 167 samples from patients
with malignancy using weak cat ion exchange beads to reduce the sample complexity.
We were able to show one differentially expressed peak at m/z of 8938, identified by
MALDI-MS/MS as C3a anaphylotoxin, a fragment of complement C3 precursor.
C3a anaphylotoxin has been identified in other tumor types as well and is a reactive
protein and not a genuine tumor-related product. This repeated finding of fragments
of moderately to high-abundant reactive proteins show one of the major shortcoming
of techniques like SELDI-TOF or MALDI-TOF for biomarker discovery. In serum
about 20 proteins make up about 99% of the proteome. Tumor markers such as
prostate specific antigen (PSA) or human chorionic gonadotropin ( HCG) are present
at much lower quantities. Moreover, the molecular weight of most intact proteins is
too high to allow sensitive detection by MALDI-based mass spectrometry as this
technique is more suited for peptides with sizes ranging up to 10 kDa. We expect that
it will be necessary to invest in sample pretreatment by including comprehensive
fractionation to reduce sample complexity and by digestion of proteins to yield
peptides of shorter lengths. However, such complex sample handling procedures will
destroy quantitative information, making differential profiling by direct sample
comparison impossible. We are now trying to address this quantitative issue by
implementing a technique called Isotope Coded Protein Labeling (ICPL). In ICPL,
protein samples are first derivatised with a primary amine-specific agent (nicotinoyloxy succinimide; Nic) that will covalently attach to the amino acid (AA) of lysines
and to the N-terminal AA of the peptide. This agent is available in two isoforms, with
4 deuterium atoms (heavy; d4-Nic) or 4 hydrogen atoms (light; d0-Nic) in the
nicotinoyl-ring. The reference sample is derivatised with d4-Nic and the unknown
with d0-Nic. Next, the unknown and the reference sample are mixed and can be
subjected to extensive pretreatment without loosing quantitative information because
all labeled proteins in the reference sample will act as internal standard for those in
the unknown sample. They are chemically identical but differ 4 Da in mass per
molecule of Nic. After digestion and further reduction of the sample complexity,
MALDI-MS will visualize labeled peptides as peak pairs differing 4 Da in mass per
substituted Nic moiety, where the peak area ratio of the peaks provide information on
the relative quantity of the peptide in the samples. We are currently trying to show
the potential applicability of this technique for biomarker discovery by searching for
known tumor markers.
DEPARTMENT OF PATHOLOGY
(Continued)
Ivon Tielen Technical Staff
Dennis Veldhuizen Technical Staff
Tin Wu Technical Staff
THE NETHERLANDS CANCER
INSTITUTE FAMILY CANCER CLINIC
Frans Hogervorst PhD Academic Staff
Laura Van ’t Veer PhD Academic Staff
Senno Verhoef MD PhD Academic Staff
Irma Kluijt MD Academic Staff
Priscilla Axwijk Academic Staff
Petra Nederlof Academic staff
Marieke Bronk Genetic associate
Anja Van Rens Genetic associate
Gea Wigbout Genetic associate
Mohamed Achachah Technical staff
Rob Plug Technical staff-Quality staff
Roelof Pruntel Technical staff
Paul Van der Voort Technical staff
Majella Boutmy-de Lange Technical staff
Aafke Wieringa-Arieaens Technical Staff
DEPARTMENT OF RADIOLOGY
Jelle Teertstra MD Academic Staff
Peter Besnard MD Academic Staff
Mesomark, novel marker for mesothelioma
Kenneth Gilhuijs PhD Academic Staff
Hans Bonfrer, Michel Van den Heuvel, Catharina Korse, Paul Baas
Wim Koops MD Academic Staff
Robert Kröger MD Academic Staff
Mesothelin is a 40 kD cell surface glycosylated phosphatidyl-inositol anchored
glycoprotein. Mesothelin is expressed by normal mesothelial cells but highly
overexpressed in tumors such as malignant mesothelioma, ovarian carcinoma and
lung carcinoma. An assay has been developed to measure soluble mesothelin related
peptides (SMRPs) in blood. Increased levels of SMRPs have been found in serum of
patients with malignant mesothelioma and ovarian carcinoma.
We studied retrospectively the concentration of SMRPs by the Mesomark assay
(CisBio, France) in samples from patients with lung cancer. We compared the results
with Carcino Embryonic Antigen (CEA) and Cytokeratin 19 fragments (Cyfra)
concentrations. The latter assays are more established markers for this tumor type.
The median level of SMRP in a healthy group of 50 was 0.56 nmol/l (95%
1.2 nmol/l). In mesotheliomas a median of 1.54 nmol/l was found (p<0.01).
The median Cyfra concentration in the mesothelioma group of 74 patients was
2.93 Вµg/l compared to 0.71 in the healthy population. (The 95% reference level is
1.9 Вµg/l.) We also measured CEA in the mesothelioma patients and as we expected,
in all (but one) the CEA concentration was normal. Drawing ROC curves revealed
that the best discriminating power to diagnose mesothelioma in a group of patients
with NSCLC was a combination of Mesomark and CEA.
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 Academic Staff
Fijs Van Leeuwen PhD Academic Staff
Christian Siedschlag PhD Academic Staff
William Klein Zeggelink Graduate Student
Miret Emanuel Technical Staff
Anita Paape Technical Staff
Kenneth Pengel Technical Staff
Angelique Schlief Technical Staff
Marja Van Vliet Technical Staff
134
DIAGNOSTIC ONCOLOGY
Publications
Technology insight: Tuning into the genetic
Novaria-study
The effect of hormonal replacement therapy on menopausal complaints
related to biochemical changes in surgically and naturally postmenopausal
women
orchestra using microarrays - Limitations
Catharina Korse, Marc Van Beurden, Katja Gaarenstroom, Hans Bonfrer, Matti Rookus,
of DNA microarrays in clinical practice.
Neil Aaronson
Abdullah-Sayani A, Bueno de
Mesquita JM, Van de Vijver MJ.
Nat Clin Pract Oncol 2006; 3:501-516
Adler AS, Lin M, Horlings H,
Nuyten DSA, Van de Vijver MJ, Chang HY.
Genetic regulators of large-scale
transcriptional signatures in cancer.
Nat Genet 2006; 38:421-430
Alderliesten T, Schlief A, Peterse J,
Teertstra H, Muller S, Gilhuijs K.
Validation of Semiautomatic Measurement
of the Extent of Breast Tumors Using
Contrast-Enhanced Magnetic Resonance
Imaging. Invest Radiol 2007; 42:42-49
Aleman BMP, Raemaekers JMM,
Tomisic R, Baaijens MHA, Bortolus R,
Lybeert MLM, Van der Maazen RWM,
Girinsky T, Demeestere G, Lugtenburg P,
Lievens Y, De Jong D, Pinna A,
Henry-Amar M. Involved-field radiotherapy
for patients in partial remission after
chemotherapy for advanced Hodgkin’s
lymphoma. Int J Radiat Oncol Biol Phys
2007; 67:19-30
Baas P, Triesscheijn M, Burgers S,
Van Pel R, Stewart F, Aalders M.
Fluorescence detection of pleural
A recent study found that the effectiveness of Hormonal Replacement Therapy
(HRT) for alleviating endocrine and/or sexual complaints and quality of life was of
limited importance for women who had become postmenopausal as a result of
ovarian ablation. An explanation for this effect may be found in the hypothalamicpituitary-adrenal axis (HPA axis) and the hypothalamic-pituitary-gonadal axis
(HPG axis). The possible differences on the various hormone concentrations of sex
hormones or neurotransmitters between “surgical” and “natural” postmenopausal
women could be an explanation for the results of HRT by these women. We have
started a prospective study to investigate.
1a: Changes in menopausal symptoms and biochemical changes as result of PBSO
in premenopausal women.
1b: Changes in menopausal symptoms and biochemical changes as result of PBSO
in postmenopausal women.
2: Difference in intensity of menopausal symptoms and related biochemical
parameters between surgically and naturally postmenopausal women (control
group 2).
3: The effect of HRT in alleviating menopausal complaints and related biochemical
parameters in surgically postmenopausal women compared to naturally
postmenopausal women (control group 1).
4: Difference in menopausal symptoms and related biochemical parameters in
surgically postmenopausal women between HRT and non-HRT users.
5: The prevalence of serotonin receptor (5-HT2A receptor) and serotonin
polymorphism (5-HTTLPR) in surgically postmenopausal women with respect to
the effect of HRT use.
The study population will be
1: Women who are eligible for PBSO.
2: Women with climacteric complaints visiting a gynecologist.
3: Healthy postmenopausal women >=46 and <56 years.
malignancies using 5-aminolaevulinic
acid. Chest 2006; 129:718-724
DEPARTMENT OF NUCLEAR MEDICINE
Beumer JH, Buckle T, Ouwehand M,
Michiel Van den Brekel, Sjaak Burgers, Axel Bex, Marcel Van Herk, Frank Hoebers, Simon Horenblas,
Franke NEF, Lopez-Lazaro L,
Bin Kroon, Joost Leijte, Wim Meinhardt, Omgo Nieweg, Henk Van der Poel, Maartje Van Rijk,
Schellens JHM, Beijnen JH,
Annette Van der Velden, Marcel Verheij, Babs Taal, Ly Tran, Nico Van Zandwijk, Philippe Baars, Cees
Van Tellingen O. Trabectedin (ET-743,
Hoefnagel, Marina Kartachova, Saar Muller, Ferida Sivro, Michiel Sinaasappel, Renato ValdГ©s Olmos
Yondelisв„ў) is a substrate for P-glycoprotein,
but only high expression of P-glycoprotein
confers the multidrug resistance phenotype.
Invest New Drugs 2007; 25:1-7
Bijker N, Meijnen P, Peterse JL,
Bogaerts J, Van Hoorebeeck I, Julien JP,
Gennaro M, Rouanet P, Avril A,
Fentiman IS, Bartelink H, Rutgers EJ.
Breast-conserving treatment with or without
radiotherapy in ductal carcinoma-in-situ:
ten-year results of European Organisation
for Research and Treatment of Cancer
randomized phase III trial 10853--a study
by the EORTC Breast Cancer Cooperative
Group and EORTC Radiotherapy Group.
J Clin Oncol 2006; 24:3381-3387
Imaging of apoptosis in patients receiving radiotherapy and/or chemotherapy
Changes of 99mTc Hynic-rh-Annexin V tumor uptake were evaluated using
co-registration of SPECT with CT in 16 chemo-naГЇve patients with advanced stage
non-small lung cancer receiving platinum-based chemotherapy. Scintigraphy
performed before and early after the start of treatment showed increase in tumor
uptake varying from 16% to 121% above the baseline in 6 patients with objective
response to chemotherapy. In 10 patients classified as non-responders (stable disease
or progression) tumor uptake varied from –30% to 23%. A significant correlation
(p-value 0.00001) was found between the tumor tracer uptake and treatment
outcome.
In another study involving 16 patients with head and neck squamous cell
carcinoma treated with concurrent cisplatin-based chemoradiotherapy, scintigraphy,
performed before and early after the first course of chemotherapy, showed a
radiation-dose-dependent uptake in parotid glands indicative of early apoptosis
during treatment. The interindividual spread in Annexin-uptake in primary tumors
could not be related to differences in treatment schedule or tumor volume, but the
Annexin-uptake in tumor and lymph nodes was closely correlated.
135
DIAGNOSTIC ONCOLOGY
Sentinel node detection in prostate cancer Lymphoscintigraphy for pelvic
lymphatic mapping and sentinel node identification was evaluated in 26 patients
with prostate cancer of the intermediate prognostic group (T3 or PSA>10ug/l or
Gleason score >6) scheduled for laparoscopic extended pelvic lymphadenectomy.
Tracer administration assisted by transrectal ultrasound enabled injection of
approximately 90% of the planned doses with an effective average dose of 211 MBq
(range 123-266MBq) 99mTc-nanocolloid. Overall sentinel node visualization rate was
96% with rates of 88% at 15 min and 96% at 2 and 4 hours. A total of 128 sentinel
nodes were visualized and at laparoscopy 101 of these nodes were found. In 3 patients
operated more than 24 hours after tracer injection no radioactive nodes could be
intraoperatively detected. Sentinel node metastases were found in 10 patients. At
histology no skip-metastases were seen. SPECT combined with low dose CT was able
to identify sentinel nodes inside the area of pelvic lymphadenectomy in 25 patients,
and outside in 13 patients (10 in aortic-iliac junction and 3 para-aortic). All these
unexpected sentinel nodes mapped by SPECT/CT (Fig. XIII.1) were found using the
laparoscopic gamma probe.
Inguinal lymphatic mapping in melanoma The images of 199 patients with
melanoma draining to the groin area at sentinel node lymphoscintigraphy using
99mTc-nanocolloid were retrospectively evaluated in order to establish the location of
second- and higher-echelon lymph nodes. Secondary lymph nodes were observed in
195 patients (98%). Transmission scanning with a 57Co flood source was used to
outline the body contour and for anatomical orientation lead bands were placed in
the inguinal folds as well as 57Co point sources over the anterior superior iliac spine
and on the pubic tubercle. The groin was divided in the superior and inferior regions
in relation to the saphenofemoral junction as well as the ilio-obturator basin and the
central region connecting the first two regions through the femoral canal to the ilioobturator region. None of the 130 patients with a sentinel node outside the superior
region had secondary nodes in this particular area. None of the 42 patients with a
sentinel node outside the inferior region had drainage to secondary nodes in this
region. This knowledge may allow the surgeon to limit the extent of therapeutic
lymph node dissection in case of an involved sentinel node.
Positron Emission Tomography The results of 19 18F-FDG PET studies
corresponding to 17 patients with penile carcinoma were evaluated and compared to
histopathology. PET was true positive in 14 patients with lymph node metastases and
false positive in 2 patients without metastasis (sensitivity 100%, specificity 71%).
Particularly for the inguinal regions sensitivity of PET was 88% and specificity 100%.
In two patients detection of metastases at distance led to change in treatment.
Following a previous study which demonstrated prominent bowel activity of 18F-FDG
in 57% of Diabetes Mellitus (DM) patients (versus 23% in a control group of patients
without DM) the pattern of bowel activity was evaluated in 32 DM patients scheduled
for PET in the morning and in 38 scanned in the afternoon. Whereas no differences
were observed in blood glucose levels (6.7 mmol/l vs 5.6 mmol/l) in 53% of patients
of the afternoon group intense bowel activity was observed (against 28% in the
morning group). This led to the recommendation that the FDG-PET evaluation of
abdominal malignancies in DM patients should preferentially be planned in the
morning.
Imaging in neuroendocrine tumors The results of scintigraphy with 111Inpentetreotide and 131I-MIBG as well as 99mTc-medronate bone scintigraphy were
evaluated in 52 patients with midgut neuroendocrine tumors. 111In-pentetreotide scan
was slightly better in detecting metastases (71 %) compared to 131I-MIBG (60%).
A combination of the two investigations in metastastatic disease revealed in 5 of 11
patients with a negative 131I-MIBG to have a positive 111In-pentetreotide. By contrast,
in 5 of the 14 patients with the primary tumour negative at the 111In-pentetreotide
scintigraphy proved to be positive at the 131I-MIBG scan. 99mTc-medronate bone
scintigraphy revealed bone metastases in 20% of the patients; in 40% of the patients
with positive bone scan the two other nuclear tests missed the bone abnormalities.
Figure IX.2: Schematic overview of systematic (arrows) and random (ellipses) base-line deviations and their
locations found in a group of 32 lung cancer patients.
Figure XIII.1: A. Lymphatic drainage to the
left para-aortic region (black arrow) in a
patient with a prostate carcinoma. B.
Appearing 15 minutes after transrectal
ultrasound assisted injection of 99mTcnanocolloid into the prostate, the para-aortic
sentinel node is accurately localized by
means of SPECT-CT (white arrow)
136
DIAGNOSTIC ONCOLOGY
Publications (continued)
DEPARTMENT OF PATHOLOGY
Boelens MC, Van den Berg A,
Vogelzang I, Wesseling J, Postma DS,
Timens W, Groen HJM. Differential
expression and distribution of epithelial
adhesion molecules in non-small cell lung
Research in the department of Pathology is aimed at genetic analysis, including gene
expression profiling using microarray analysis, of various malignancies. Part of this
research, carried out by Petra Nederlof, Laura Van �t Veer and Marc Van de Vijver, is
described under Division VIII and XII. A number of more clinically oriented projects
are carried out in the department of Pathology itself and described here.
cancer and normal bronchus. J Clin Pathol
2006;Published online
Booman M, Douwes J, Glas AM,
Riemersma SA, Jordanova ES, Kok K,
Rosenwald A, De Jong D, Schuuring E,
Kluin PM. Mechanisms and effects of loss of
human leukocyte antigen class II expression
in immune-privileged site-associated B-cell
lymphoma. Clin Cancer Res 2006;
12:2698-2705
Booman M, Douwes J, Glas AM,
De Jong D, Schuuring E, Kluin PM.
Primary testicular diffuse large B-cell
lymphomas have activated B-cell-like
subtype characteristics. J Pathol 2006;
EGFR mutation in lung carcinomas and response to Iressa therapy Gefitinib
(Iressa, ZD1839), an inhibitor of epidermal growth factor receptor-tyrosine kinase
(EGFR-TK), has shown potent anti-tumor effects and improved symptom and qualityof-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.
In collaboration with dr N. van Zandwijk, a retrospective series (N=15) of tumors
from NSCLC patients who are/were on Gefinitib therapy as part of a clinical trial was
studied. It was shown that the patients with a mutation in EGFR (N=3) all responded
to IRESSA therapy, whereas the patients without the mutation did not respond.
In a prospective series, Gefinitib eligible patients were screened for mutations.
A total of 12 mutations have been identified in 38 patients (2004 and 2005): one
point mutation in exon 18, eight deletions in exon 19, and three point mutation in
exon 21. In none of the tumors with a EGFR mutations a KRAS mutation could be
identified.
210:163-171
Bosga-Bouwer AG, Van den Berg A,
Haralambieva E, De Jong D, Boonstra R,
Kluin P, Van den Berg E, Poppema S.
Molecular, cytogenetic, and
immunophenotypic characterization of
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 (Figure XIII.2).
follicular lymphoma grade 3B; a separate
entity or part of the spectrum of diffuse large
B-cell lymphoma or follicular lymphoma?
Hum Pathol 2006; 37:528-533
Broekhuizen LN, Wijsman JH, Peterse JL,
Rutgers EJT. The incidence and significance
Pulmonary squamous cell carcinoma following head and neck squamous cell
carcinoma: metastasis or second primary? Of 46 patients with a primary SCC of
the Head and Neck followed by a SCC of the lung, clinical data (stage, tumor status,
radiology, time interval), histology and PCR-based analysis of loss of heterozygosity
(LOH) with 12 markers on 11 chromosome arms were used to distinguish between
metastases and second primary tumors.
of micrometastases in lymph nodes of
patients with ductal carcinoma in situ and
T1a carcinoma of the breast. Eur J Surg
Oncol 2006; 32:502-506
Chi JT, Wang Z, Nuyten DSA,
Rodriguez EH, Schaner ME, Salim A,
Wang Y, Kristensen GB, Helland A,
BГёrresen-Dale A-L, Giaccia A,
Longaker MT, Hastie T, Yang GP,
Van de Vijver MJ, Brown PO. Gene
expression programs in response to hypoxia:
Cell type specificity and prognostic
significance in human cancers.
PLoS Medicine 2006; 3:395-409
Cserni G, Bianchi S, Vezzosi V, Peterse H,
Sapino A, Arisio R, Reiner-Concin A,
Regitnig P, Bellocq JP, Marin C, Bori R,
Penuela JM, Iturriagagoitia AC. The value
of cytokeratin immunohistochemistry in the
evaluation of axillary sentinel lymph nodes
Figure XIII.2: Mutation analysis of the c-KIT gene on paraffin embedded tumor biopsy from a patient
in patients with lobular breast carcinoma.
with a gastrointestinal stromal tumor (GIST), which predicts response to imatinib therapy. A frequent
J Clin Pathol 2006; 59:518-522
c-KIT exon-11 deletion is detected.
137
DIAGNOSTIC ONCOLOGY
Publications (continued)
LOH analysis indicated metastatic disease in 12 cases, probable metastases in
9 cases, second primary SCC in 21 cases and probable second primary disease in
3 cases. In 1 case LOH analysis was inconclusive. Clinical scoring suggested that
39 patients had metastases and 7 had second primaries. Histology gave firm evidence
in 1 case for metastasis and in 1 other for a second primary tumor. For 28 patients
LOH supported the clinical scoring and in 17 cases it did not. These 17 discordant
cases were considered to be second primary tumors by LOH analysis, whereas they
were suggested to be metastasis clinically.
In this study we developed a novel interpretation strategy for LOH-profiling results
to differentiate metastases and second primaries in routine clinical practice.
A considerable number of lung lesions clinically interpreted as metastases, are
suggested to be second primaries by LOH analysis.
In addition, in collaboration with Brakenhof (VUMC) P53 mutation status was
assessed in all tumor-pairs. All P53 results were in concordance with the LOH data.
De Vries NA, Beijnen JH, Boogerd W,
Van Tellingen O. Blood-brain barrier and
chemotherapeutic treatment of brain
tumors. Expert Rev Neurother 2006;
6:1199-1209
Derksen PWB, Liu X, Saridin F,
Van der Gulden H, Zevenhoven J, Evers B,
Van Beijnum JR, Griffioen AW, Vink J,
Krimpenfort P, Peterse JL, Cardiff RD,
Berns A, Jonkers J. Somatic inactivation of
E-cadherin and p53 in mice leads to
metastatic lobular mammary carcinoma
through induction of anoikis resistance and
angiogenesis. Cancer Cell 2006; 10:437-449
RASTER-STUDY
Feasibility of gene expression profiling in community hospitals; preliminary
results of a pilot study in N0 breast cancer patients
Deurloo EE, Klein Zeggelink WFA,
Jolien Bueno de Mesquita, Marc Van de Vijver, Wim Van Harten, Sabine Linn
Muller SH, Bartelink H, Gilhuijs KGA.
Teertstra HJ, Peterse JL, Rutgers EJT,
Contrast-enhanced MRI in breast cancer
Recently we have identified a gene expression profile of 70 genes using microarray
analysis (70-gene MA test), which was a more powerful prognostic factor for freedom
of distant metastases than current clinicopathological features in node negative
breast cancer patients up to 55 years of age (van �t Veer et al., Nature 2002; Van de
Vijver et al., New Engl J Med 2002). To assess whether this 70-gene MA test can be
implemented in daily clinical practice we aimed to answer the following three
questions: I. Is it feasible to collect fresh tumor samples in order to make this test
available in pN0 breast cancer patients in community hospitals? II. What is the
proportion of a “poor prognosis” versus a “good prognosis” in current node negative
patients? III. What is the concordance between the 70-gene MA risk profile and the
metastasis risk as assessed with current Dutch guidelines based on
clinicopathological factors (such as age, pT, tumor grade, hormonal receptor-status)?
In this study, women younger than 55 years presenting with cT1-2N0M0 unifocal
breast cancer were eligible to participate and entered onto this prospective study after
informed consent. Fresh tumor tissue samples were collected within one hour after
surgery and sent in RNAlaterВ® to our hospital. A 70-gene MA test was performed in
node negative patients with a representative tumor tissue sample; pN+ patients were
excluded. According to current Dutch guidelines (used for guiding adjuvant systemic
treatment) for node negative patients, high risk is defined as age < 35 years, pT any
size and grade 2-3 or pT > 1cm and grade 1, or age > 35 years, pT1-2 cm and grade 3,
pT2-3cm and grade 2-3 or pT>3cm and any grade; all others are defined as low risk.
Thus far, 771 patients were included (average age 48 years), 404 (52%) 70-gene
prognostic signatures were obtained and 367 (48%) patients were excluded after
surgery. Of these excluded patients, 24% appeared not to be eligible for the test due
to pN+, 2% due to wrong inclusion, 3% was postoperatively not eligible, 14% due to
sampling failure, 3% due to incorrect procedures, and in 2% the quality of the RNA
was insufficient.
For 200 (50%) patients, a poor prognosis 70-gene MA-risk profile was found; for
200 (50%) a good prognosis profile. In 29% of the patients the 70-gene risk profile
was discordant with risk assessment based on clinicopathological factors according to
the Dutch guidelines.
These first results show that the implementation of the 70-gene MA test in
community hospitals is feasible. Furthermore, the 70-gene MA risk profile is
discordant in a substantial proportion of lymph node-negative breast cancer patients
compared with metastasis risk assessed with conventional clinicopathological factors
according to Dutch guidelines. The accrual will end at December 1st of 2006 after
which an international randomized trial, the MINDACT-trial, will start in which the
70-gene signature will be prospectively validated.
patients eligible for breast-conserving
therapy: Complementary value for
subgroups of patients. Eur Radiol 2006;
16:692-701
Green JA, Berns EMJJ, Coens C,
Van Luijk I, Thompson-Hehir J,
Van Diest P, Verheijen RHM,
Van de Vijver M, Van Dam P, Kenter GG,
Tjalma W, Ewing PC, Teodorovic I,
Vergote I, Van der Burg MEL. Alterations
in the p53 pathway and prognosis in
advanced ovarian cancer: A multi-factorial
analysis of the EORTC Gynaecological
Cancer group (study 55865).
Eur J Cancer 2006; 42:2539-2548
Hannemann J, Velds A, Halfwerk JBG,
Kreike B, Peterse JL, Van de Vijver MJ.
Classification of ductal carcinoma in situ
by gene expression profiling. Breast Cancer
Res 2006; 8
Hannemann J, Kristel P, Van Tinteren H,
Bontenbal M, Van Hoesel QGCM,
Smit WM, Nooij MA, Voest EE,
Van der Wall E, Hupperets P,
De Vries EGE, Rodenhuis S,
Van de Vijver MJ. Molecular subtypes of
breast cancer and amplification of
topoisomerase IIa: Predictive role in dose
intensive adjuvant chemotherapy.
Br J Cancer 2006; 95:1334-1341
Jalving M, Koornstra JJ, Wesseling J,
Boezen HM, De Jong S, Kleibeuker JH.
Increased risk of fundic gland polyps during
long-term proton pump inhibitor therapy.
Aliment Pharmacol Ther 2006;
24:1341-1348
138
DIAGNOSTIC ONCOLOGY
Publications (continued)
Jalving M, De Jong S, Koornstra JJ,
THE NETHERLANDS CANCER INSTITUTE FAMILY CANCER
CLINIC
Boersma-van Ek W, Zwart N, Wesseling J,
Mohamed Achachah, Priscilla Axwijk, Majella Boutmy-de Lange, Marieke Bronk, Daniela Hahn,
De Vries EGE, Kleibeuker JH. TRAIL
Frans Hogervorst, Irma Kluijt, Petra Nederlof, Rob Plug, Roelof Pruntel, Anja Van Rens,
Induces Apoptosis in Human Colorectal
Marielle Ruijs, Laura Van ’t Veer, Senno Verhoef , Paul Van der Voort, Gea Wigbout
Adenoma Cell Lines and Human Colorectal
Adenomas. Clin Cancer Res 2006;
12:4350-4356
Kaas R, Kroger R, Peterse JL, Hart AAM,
Muller SH. The correlation of
mammographic-and histologic patterns of
breast cancers in BRCA1 gene mutation
In 2006 the number of families who have sought genetic advice through our hospital
since the start of the Family Cancer Clinic in 1995 has passed 2200. The steady
increase in number of families seen per year has maintained at about 10% yearly, and
this growth is maximised at this percentage as a consequence of limit imposed by the
insurance companies.
Most families present with a possible genetic predisposition for breast and/or ovarian
cancer.
carriers, compared to age-matched sporadic
controls. Eur Radiol 2006; 16:2842-2848
Kartachova MS, ValdГ©s Olmos R,
Haas RLM, Hoebers FJP,
Van den Brekel MW, Van Zandwijk N,
Herk MV, Verheij M. Mapping of
treatment-induced apoptosis in normal
structures: 99mTc-Hynic-rh-annexin V
SPECT and CT image fusion. Eur J Nucl
The DNA-diagnostic laboratory has screened almost 2000 families for germline
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 153 families a BRCA1 mutation and in
82 families a BRCA2 mutation was identified. This year we noticed again an increase
in the number of predictive, presymptomatic testing for these genes, from 190 to
250. Furthermore, the laboratory is testing new techniques in order to shorten the
through put time of samples. We hope to achieve this using Conformation Sensitive
Capillary Electrophoresis combined with automated pipetting and analysis.
Med Mol Imaging 2006; 33:893-899
Kemper EM, Leenders W, Sters B,
Lyons S, Buckle T, Heerschap A,
Boogerd W, Beijnen JH, Van Tellingen O.
Development of luciferase tagged brain
tumour models in mice for chemotherapy
intervention studies. Eur J Cancer 2006;
42:3294-3303
As good results have been obtained with the research into BRCA1 genetic profiles
using array CGH, a start has been made to implement this technique as an additional
tool for hereditary breast/ovarian cancer screening into clinical practice at the Family
Cancer Clinic (in collaboration with E Van Beers and PM Nederlof, Division VIII and
XIII). In a series of 38 tumors from HBOC families with a negative test result for
BRCA1/2 only 3 cases showed a BRCA1-like aCGH profile, indicating that the routine
diagnostic screening is very efficient. In addition, we have started to apply the aCGH
BRCA1 classifier on unclassified variants (UVs) to gain additional proof of
significance of the variations.
Kreike B, Halfwerk H, Kristel P, Glas A,
Peterse H, Bartelink H, Van de Vijver MJ.
Gene expression profiles of primary breast
carcinomas from patients at high risk for
local recurrence after breast-conserving
therapy. Clin Cancer Res 2006;
12:5705-5712
Kriege M, Brekelmans CTM, Boetes C,
Muller SH, Zonderland HM, Obdeijn IM,
Manoliu RA, Kok T, Rutgers EJT,
The number of requests for microsatellite studies (MSI) and immunohistochemistry
of the MMR genes (performed in collaboration with D De Jong) has stabilized at 80
yearly. In about 20% of the colon tumors microsatellite instability was detected, as an
indication for possible pathogenic mutations in mismatch repair genes. Germline
screening is now offered for hMLH1, hMSH2, hMSH6 and the MutY (MYH) gene.
Furthermore, in case of a MSI high profile in a colon tumor without detectable
mutations in the MMR genes, methylation status of the hMLH1 promotor and the
presence of a specific somatic mutation in the BRAF gene (V600E) have been
assessed. Several tumors were shown to have a methylated promoter which result is
of use for the clinical interpretation of the family history data.
De Koning HJ, Klijn JGM, Bartels CCM,
Besnard APE, Hoogerbrugge N, Meijer S,
Oosterwijk JC, Seynaeve C,
Tilanus-Linthorst MMA, Tollenaar RAEM.
Differences between first and subsequent
rounds of the MRISC breast cancer
screening program for women with a
familial or genetic predisposition. Cancer
2006; 106:2318-2326
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 XII), 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 VIII), 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 P Devilee cs, psychosocial studies, in collaboration with the department of
psychosocial research and epidemiology (Division XII) and clinical and genetic
research in families with gastrointestinal cancer, including stomach cancer
(Dr A Cats, Division X).
139
DIAGNOSTIC ONCOLOGY
Publications (continued)
Progress has been made into the development of guidelines for hereditary breast
cancer management, by the participation of the clinical geneticists of the Family
Cancer Clinic in the WKO (Werkgroep Klinische Oncogenetica) a working party of
the Dutch Clinical Genetics Society, and local guidelines for management of familial
colorectal cancer patients in a multidisciplinary team, whilst a national guideline is in
development through the WKO.
Kriege M, Brekelmans CT, Obdeijn IM,
Boetes C, Zonderland HM, Muller SH,
Kok T, Manoliu RA, Besnard AP,
Tilanus-Linthorst MM, Seynaeve C,
Bartels CC, Kaas R, Meijer S,
Oosterwijk JC, Hoogerbrugge N,
Tollenaar RA, Rutgers EJ, De Koning HJ,
DEPARTMENT OF RADIOLOGY
Klijn JG. Factors affecting sensitivity and
Kenneth Gilhuijs, Tanja Alderliesten, Miret Emanuel, Claudette Loo, Saar Muller, Anita Paape,
specificity of screening mammography and
Kenneth Pengel, Jelle Teertstra, Fijs Van Leeuwen, Angelique Schlief, Christian Siedschlag,
MRI in women with an inherited risk for
Marja Van Vliet
breast cancer. Breast Cancer Res Treat
2006; 100:109-119
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 and pre-operative assessment of tumor extent.
Kroon BK, Nieweg OE, Van Boven H,
Horenblas S. Size of Metastasis in the
Sentinel Node Predicts Additional Nodal
Involvement in Penile Carcinoma. J Urol
Impact of preoperative contrast-enhanced MRI in patients eligible for breastconserving therapy: update on 325 patients The purpose of this study was to
prospectively assess the incidence and the impact of additional findings in preoperative contrast-enhanced (CE) MRI of patients eligible for breast conserving
therapy (BCT).
Between November 2000 and December 2005, 325 patients eligible for BCT on the
basis of conventional imaging and palpation underwent pre-operative CE-MRI with
Gd-DTPA using FLASH 3-D imaging. The images were read by experienced breast
MRI radiologists using the BIRADs lexicon. The incidence of additional findings
(enhancing lesions separate from the known malignancy or larger extent of the
known malignancy than appreciated from conventional imaging) and their impact on
treatment were assessed. The gold standard was histology or follow-up.
Additional findings were detected in 101 patients (30.9%): more extensive disease
was found in 6.4% of patients, additional enhancing lesion(s) in 22.0%, and both in
2.4%. One-hundred additional lesions in 72 patients (72/325=22%) were detected;
47 in the same quadrant (74% of which are malignant), 32 in a different quadrant
(41% of which are malignant) and 21 in the contralateral breast (19 of which are
malignant).
BIRADs scores of additional lesions were benign (n=1), probably benign (n=15),
indeterminate (n=19), suspicious (n=30) and highly suggestive of malignancy (n=35).
A trend was observed towards decreased reporting of benign findings (BIRADs 2 and
3) from 26% to 9%. Additional lesions referred for further workup (n=74) were
visible on ultrasonography in 55.4% of the patients.
Benign lesions (pathology-proven or benign by follow up (median 40 months))
occurred in 48/325 (15%) of the patients. Additional malignant lesions (all pathology
proven) occurred in 36/325 (11%) of the patients. MRI led to a change of treatment in
71 patients (21.8%): mastectomy in 32 (9.8%), wider excision in 27 (8.3%, 1.2% of
which due to benign lesions), contralateral surgery in 3 (0.9%), and neoadjuvant
chemotherapy in 9 (2.8%) patients.
Infiltrating lobular carcinoma (ILC) and ductal carcinoma in situ (DCIS) were found
to be significantly associated with unexpected additional findings at CE MRI
compared with infiltrating ductal carcinoma (IDC) (p=0.02). The density of the
breast parenchyma was borderline significant (p=0.05).
In summary, approximately 50% of the additional lesions detected at preoperative CE
MRI in patients eligible for BCT on the basis of conventional imaging are malignant,
leading to a change of treatment in 22% of the patients, and mastectomy in 10% of
the patients. Treatment changes due to benign findings (wider excision) are
uncommon.
2006; 176:105-108
Lakhani SR, Audretsch W,
Cleton-Jensen AM, Cutuli B, Ellis I,
Eusebi V, Greco M, Houslton RS,
Kuhl CK, Kurtz J, Palacios J, Peterse H,
Rochard F, Rutgers E, on behalf of
EUSOMA. The management of lobular
carcinoma in situ (LCIS). Is LCIS the
same as ductal carcinoma in situ (DCIS)?
Eur J Cancer 2006; 42:2205-2211
Li L, McVety S, Younan R, Liang P,
Du Sart D, Gordon PH, Hutter P,
Hogervorst FBL, Chong G, Foulkes WD.
Distinct patterns of germ-line deletions in
MLH1 and MSH2: the implication of Alu
repetitive element in the genetic etiology of
Lynch syndrome (HNPCC). Hum Mutat
2007; 27:a388
Lopez-Guerrero JA, Riegman PHJ,
Oosterhuis JW, Lam KH, Oomen MHA,
Spatz A, Ratcliffe C, Knox K, Mager R,
Kerr D, Pezzella F, Van Damme B,
Van de Vijver M, Van Boven H,
Morente MM, Alonso S, Kerjaschki D,
Pammer J, Carbone A, Gloghini A,
Teodorovic I, Isabelle M, Passioukov A,
Lejeune S, Therasse P, Van Veen EB,
Dinjens WNM, Llombart-Bosch A.
TuBaFrost 4: Access rules and incentives for
a European tumour bank. Eur J Cancer
2006; 42:2924-2929
140
DIAGNOSTIC ONCOLOGY
Publications (continued)
Morente MM, Mager R, Alonso S,
Pezzella F, Spatz A, Knox K, Kerr D,
Dinjens WNM, Oosterhuis JW, Lam KH,
Oomen MHA, Van Damme B,
Van de Vijver M, Van Boven H,
Kerjaschki D, Pammer J,
Lopez-Guerrero JA, Llombart Bosch A,
Carbone A, Gloghini A, Teodorovic I,
Isabelle M, Passioukov A, Lejeune S,
Therasse P, Van Veen EB, Ratcliffe C,
Riegman PHJ. TuBaFrost 2: Standardising
tissue collection and quality control
procedures for a European virtual frozen
tissue bank network. Eur J Cancer 2006;
42:2684-2691
Niessen RC, Sijmons RH, Ou J,
Olthof SGM, Osinga J, Ligtenberg MJ,
Hogervorst FBL, Weiss MM, Tops CMJ,
Hes FJ, De Bock GH, Buys CHCM,
Kleibeuker JH, Hofstra RMW. MUTYH
and the mismatch repair system: Partners
in crime? Hum Genet 2006; 119:206-211
Nuyten DSA, Kreike B, Hart AAM,
Chi JTA, Sneddon JB, Wessels LFA,
Peterse HJ, Bartelink H, Brown PO,
Chang HY, Van de Vijver MJ. Predicting a
local recurrence after breast-conserving
therapy by gene expression profiling. Breast
Cancer Res 2006; 8:R62
Olivier RI, Lubsen-Brandsma MAC,
Verhoef S, Van Beurden M. CA125 and
Benefit of semi-automatic volumetric measurement of extent of breast tumors
from contrast-enhanced MRI Contrast-enhanced (CE) magnetic resonance
imaging (MRI) is known to accurately visualize invasive breast tumors. The efficacy
of Contrast-enhanced (CE) magnetic resonance imaging (MRI) is currently
investigated to monitor response to neoadjuvant chemotherapy for breast cancer.
Large inter- and intra-observer variations in the assessment of the largest diameter of
the tumor from MRI have, however, been found, leading to reduced ability to
accurately determine changes in tumor extent. The aim of this substudy was to assess
whether semi-automatic volumetric measurement of breast tumors from CE-MRI
decreases measurement variation and increases precision with respect to tumor
extent as measured from histopathology.
25 Patients who underwent breast-conserving therapy for 26 tumors (21 invasive
ductal, 4 invasive lobular and 1 mucinous carcinoma) were included. Detailed
histopathology analysis was performed on the whole excision specimens including
measurement of tumor volume and largest diameter of the tumor. Preoperative
CE-MRI (3D FLASH) was obtained for all breasts. Two experienced breast-MR
radiologists (R1, R2) independently assessed the largest diameter of the tumor from
MRI. In addition, a computerized system developed in our laboratory was used to
automatically segment the breast tumors in 3D and to determine volume as well as
largest diameter of the tumor. Two observers (O1, O2) independently segmented the
26 tumors. Linear regression analysis was used to assess the precision and random
variation in volumetric and largest-diameter measurements of tumor extent.
Semi-automatic volumetric measurement of tumor volume was more accurately
correlated with histopathology than conventional measurement of the largest
diameter of the tumor (O1: slope=0.97, intercept=0.26, adjusted R-square=0.79, O2:
slope=0.92, intercept=0.26, adjusted R-square=0.76 vs. R1: slope=0.68,
intercept=0.72, adjusted R-square=0.52, R2: slope=0.72, intercept=0.47, adjusted
R-square=0.68). The measurement precision relative to pathology increased, on
average, by 30% using automated volumetric measurement. The variation in
measurement dropped from 18% for the manual assessment of the largest diameter
of the tumor to 10% for the semi-automatic volumetric measurement.
Compared to conventional assessment of the largest diameter of the tumor, semiautomatic volumetric measurement of breast tumors from CE-MRI leads to smaller
variations as well as improved precision with respect to tumor extent as measured
from histopathology.
transvaginal ultrasound monitoring in
high-risk women cannot prevent the
diagnosis of advanced ovarian cancer.
Gynecol Oncol 2006; 100:20-26
Ortin-Perez J, Van Rijk MC,
Valdes-Olmos RA, Vidal-Sicart S,
Nieweg OE, Vilalta A, Kroon BB, Pons F.
Lymphatic mapping and sentinel node
biopsy in Merkel’s cell carcinoma. Eur J
Surg Oncol 2006;[Epub ahead of print]
Accurate extent definition of non-small cell lung tumors using pathologyvalidated PET-CT
A project has been initiated aimed at pre-treatment prediction of the distribution of
the tumor cell spread around non-small cell lung cancers (NSCLC) as well as the
biological characteristics of the cancers using pre-treatment respiratory-correlated
PET and CT. To pursue this aim, the pre-treatment PET and CT images of NSCLC
patients is compared in detail with the histopathology of the tumor after surgery.
So far, 8 patients have been included in the study. Non-rigid fusion of the pathology
data and preoperative CT has been applied to account for differences in deformation
of the lung. First results suggest underestimation of the extent of microscopic disease
by a factor of 1.8 when deformations are not taken into account.
Press RR, Buckle T, Beijnen JH,
Van Tellingen O. The effect of
P-glycoprotein and cytochrome P450 3a on
the oral bioavailability of vinorelbine in
mice. Cancer Chemother Pharmacol 2006;
57:819-825
Optimizing functional imaging approaches in a preclinical setting; molecular
imaging in mice
The functional information provided by molecular imaging may reveal the molecular
basis of a tumor in a non-invasive manner-and also enable earlier and more specific
tumor detection, all of which will help to tailor therapy approaches. In a joint effort
between the departments of radiology and nuclear medicine we are currently using
the (advanced) mouse models present at the NKI to develop new clinical molecular
imaging strategies.
For the visualization of both the tumor extent and the biological processes activated
within the tumor, it is very advantageous to register imaging techniques that provide
anatomical features (X-ray, CT, and MRI) with those that enable the visualization of
molecular processes (Gamma camera, PET, and SPECT). To achieve multimodality
imaging in mice, we apply two approaches: 1) imaging immobilized mice in different
141
DIAGNOSTIC ONCOLOGY
Publications (continued)
Riegman PHJ, Dinjens WNM,
Oomen MHA, Spatz A, Ratcliffe C,
Knox K, Mager R, Kerr D, Pezzella F,
Van Damme B, Van de Vijver M,
Van Boven H, Morente MM, Alonso S,
Kerjaschki D, Pammer J,
Lopez-Guerrero JA, Llombart Bosch A,
Carbone A, Gloghini A, Teodorovic I,
Figure XIII.3: Tumor specific monitoring of Pgp-mediated efflux in hereditary breast tumors
Isabelle M, JaminГ© D, Passioukov A,
(in collaboration with Sven Rottenberg).
Lejeune S, Therasse P, Van Veen EB,
Lam KH, Oosterhuis JW. TuBaFrost 1:
modalities using different contrast agents (each modality visualizes a different
process) and 2) imaging immobilized mice using a single contrast agent that is
detectable in different modalities (modalities visualize the same process).
A number of preclinical imaging projects that are currently running: the
development of multimodality imaging strategies and contrast agents for the
visualization of DCIS, evaluation of mouse models for small cell lung cancer
(in collaboration with Tessa Buckle), evaluation of radioactive contrast agents for the
detection of metastatic breast cancer (in collaboration with Hermien Boerhout),
and determination of drug resistance mechanisms in hereditary breast cancer cancer
(in collaboration with Sven Rottenberg). In all these projects we are looking for
a tumor specific and functional readout. This is achieved using a combination of
different imaging modalities such as: X-ray and gamma camera, and PET/CT and
MRI.
Uniting local Frozen Tumour Banks into a
European Network: an overview.
Eur J Cancer 2006; 42:2678-2683
Rodenhuis S, Bontenbal M,
Van Hoesel QGCM, Smit WM, Nooij MA,
Voest EE, Van Der Wall E, Hupperets P,
Van Tinteren H, Peterse JL,
Van de Vijver MJ, De Vries EGE. Efficacy
of high-dose alkylating chemotherapy in
HER2/neu-negative breast cancer. Ann
Oncol 2006; 17:588-596
Rudkin TM, Hamel N, Galvez M,
Hogervorst F, Gille JJP, MГёller P, Apold J,
Foulkes WD. The frequent BRCA1
mutation 1135insA has multiple origins:
A haplotype study in different populations.
BMC Med Genet 2006; 7
Ruijs MWG, Verhoef S, Wigbout G,
Pruntel R, Floore AN, De Jong D,
Van �t Veer LJ, Menko FH. Late-onset
common cancers in a kindred with an
Arg213Gln TP53 germline mutation. Fam
Cancer 2006; 5:169-174
Schmidt MK, Tollenaar RA, De Kemp SR,
Broeks A, Cornelisse CJ, Smit VT,
Peterse JL, Van Leeuwen FE,
Van �t Veer LJ. Breast Cancer Survival and
Tumor Characteristics in Premenopausal
Women Carrying the CHEK2*1100delC
Germline Mutation.
J Clin Oncol 2007; 25:64-69
Sotiriou C, Wirapati P, Loi S, Harris A,
Fox S, Smeds J, Nordgren H, Farmer P,
Praz V, Haibe-Kains B, Desmedt C,
Larsimont D, Cardoso F, Peterse H,
Nuyten D, Buyse M, Van de Vijver MJ,
Bergh J, Piccart M, Delorenzi M. Gene
expression profiling in breast cancer:
Understanding the molecular basis of
histologic grade to improve prognosis.
J Natl Cancer Inst 2006; 98:262-272
142
DIAGNOSTIC ONCOLOGY
Publications (continued)
Van Apeldoorn MJ, Rustemeijer C, Voerman BJ,
Van Rijk MC, Nieweg OE, Rutgers EJT,
Peterse J. Mesothelioma of the Tunica Vaginalis
Oldenburg HSA, Olmos RV, Hoefnagel CA,
Complicated by Chyluria. J Clin Oncol 2006;
Kroon BBR. Sentinel node biopsy before
24:5329-5330
neoadjuvant chemotherapy spares breast cancer
patients axillary lymph node dissection. Ann Surg
Van Beers EH, Joosse SA, Ligtenberg MJ, Fles R,
Oncol 2006; 13:475-479
Hogervorst FBL, Verhoef S, Nederlof PM. A
multiplex PCR predictor for aCGH success of
Van Rijk MC, Teertstra HJ, Peterse JL,
FFPE samples. Br J Cancer 2006; 94:333-337
Nieweg OE, Olmos RAV, Hoefnagel CA,
Kroon BBR. Ultrasonography and fine-needle
Van den Broek GB, Rasch CRN, Pameijer FA,
aspiration cytology in the preoperative evaluation
Peter E, Van den Brekel MWM, Balm AJM.
of melanoma patients eligible for sentinel node
Response measurement after intraarterial
biopsy. Ann Surg Oncol 2006; 13:1511-1516
chemoradiation in advanced head and neck
carcinoma: Magnetic resonance imaging and
Van Rijk MC, Deurloo EE, Nieweg OE,
evaluation under general anesthesia? Cancer
Gilhuijs KGA, Peterse JL, Rutgers EJT, Kröger R,
2006; 106:1722-1729
Kroon BBR. Ultrasonography and fine-needle
aspiration cytology can spare breast cancer
Van der Hout AH, Van den Ouweland AMW,
patients unnecessary sentinel lymph node biopsy.
Van der Luijt RB, Gille HJP, Bodmer D,
Ann Surg Oncol 2006; 13:31-35
BrГјggenwirth H, Mulder IM, Van der Vlies P,
Elfferich P, Huisman MT, Ten Berge AM,
Van Veen EB, Riegman PHJ, Dinjens WNM,
Kromosoeto J, Jansen RPM, Van Zon PHA,
Lam KH, Oomen MHA, Spatz A, Mager R,
Vriesman T, Arts N, Boutmy-de Lange N,
Ratcliffe C, Knox K, Kerr D, Van Damme B,
Oosterwijk JC, Meijers-Heijboer H,
Van de Vijver M, Van Boven H, Morente MM,
Ausems MGEM, Hoogerbrugge N, Verhoef S,
Alonso S, Kerjaschki D, Pammer J,
Halley DJJ, Vos YJ, Hogervorst F, Ligtenberg M,
Lopez-Guerrero JA, Llombart Bosch A,
Hofstra RMW. A DGGE system for comprehensive
Carbone A, Gloghini A, Teodorovic I, Isabelle M,
mutation screening of BRCA1 and BRCA2:
Passioukov A, Lejeune S, Therasse P,
application in a Dutch cancer clinic setting. Hum
Oosterhuis JW. TuBaFrost 3: Regulatory and
Mutat 2006; 27:654-666
ethical issues on the exchange of residual tissue for
research across Europe. Eur J Cancer 2006;
Van der Poel HG, Antonini N, Hoefnagel CA,
42:2914-2923
Horenblas S, ValdГ©s Olmos RA. Serum
hemoglobin levels predict response to strontium-89
Van Zandwijk N, Mathy A, Boerrigter L,
and rhenium-186-HEDP radionuclide treatment
Ruijter H, Tielen I, De Jong D, Baas P,
for painful osseous metastases in prostate cancer.
Burgers S, Nederlof P. EGFR and KRAS
Urol Int 2006; 77:50-56
mutations as criteria for treatment with tyrosine
kinase inhibitors: retro- and prospective
Van der Poel HG, Beetsma DB, Van Boven H,
observations in non-small-cell lung cancer.
Horenblas S. Perineal Salvage Prostatectomy for
Ann Oncol 2007; 18:99-103
Radiation Resistant Prostate Cancer. Eur Urol
2006; In Press
Verdoes M, Florea BI, Menendez-Benito V,
Maynard CJ, Witte MD, Van der Linden WA,
Van Rijk MC, Peterse JL, Nieweg OE,
Van den Nieuwendijk AMCH, Hofmann T,
Oldenburg HSA, Rutgers EJT, Kroon BBR.
Berkers CR, Van Leeuwen FWB, Groothuis TA,
Additional axillary metastases and stage
Leeuwenburgh MA, Ovaa H, Neefjes JJ,
migration in breast cancer patients with
Filippov DV, Van der Marel GA, Dantuma NP,
micrometastases or submicrometastases in sentinel
Overkleeft HS. A Fluorescent Broad-Spectrum
lymph nodes. Cancer 2006; 107:467-471
Proteasome Inhibitor for Labeling Proteasomes In
Vitro and In Vivo. Chem Biol 2006; 13:1217-1226
Van Rijk MC, Tanis PJ, Nieweg OE,
ValdГ©s Olmos RA, Rutgers EJT, Hoefnagel CA,
Zandvoort A, Van der Geld YM, Jonker MR,
Kroon BBR. Clinical implications of sentinel nodes
Noordhoek JA, Vos JTWM, Wesseling J,
outside the axilla and internal mammary chain in
Kauffman HF, Timens W, Postma DS. High
patients with breast cancer. J Surg Oncol 2006;
ICAM-1 gene expression in pulmonary fibroblasts
94:281-286
of COPD patients: a reflection of an enhanced
immunological function. Eur Respir J 2006;
28:113-122
143
BIOMETRICS
BI OME T R I CS DEPARTMEN T
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.
We coordinate the overview of randomized trials of the treatment of prostate cancer.
The statistical analyses are made centrally in collaboration with R Peto and the
Clinical Trials Service Unit at the University of Oxford. This is a continuous effort
that is currently in its 2nd cycle. The initial cycles of this collaboration assessed the
use of maximum androgen blockade (MAB). The present cycle continued its course
in 2006 with the support of the grant by of the Quality of Life program of the
European Commission. The Prostate Cancer Trialists’ Collaborative Group (PCTCG)
was created to bring together these randomized trials and currently counts more than
400 investigators. In the current cycle of the overview trials studying the effect of
immediate endocrine therapy as compared to deferred endocrine treatment in
asymptomatic patients with no recorded metastases are being overviewed and
publication of the effects on prostate cancer mortality and non-prostate cancer
mortality is in discussion.
Collaboration with the Dutch Colorectal Cancer Group (DCCG) has resulted in
several randomized studies in patients with colorectal carcinoma for which we are
the Statistical Center. In the first study in advanced previously untreated patients
sequential versus combination chemotherapy was compared. Seventy institutes
participated randomizing the total accrual planned of 820 patients in 2 years.
The final analysis is in preparation and results will be presented during ASCO 2007.
The second study (CAIRO 2) is a randomized study in the same patients group
studying Cetuximab added to capecitabine, oxaliplatin and bevacizumab.It is expected
that the accrual of 800 patients will be completed in April 2007 and a third study is
already being evaluated by the ethical committee and by the KWF for funding.
An additional study after radical resection of liver metastases of colorectal cancer
comparing bevacizumab in combination with capecitabine and eloxatin vs eloxatin
alone as adjuvant treatment has started in December 2006 and should include in
excess of 600 patients.
The collaboration with the Dutch Chest Physician Association (NVALT) already
resulted in 6 randomized studies in which in excess of 70 hospitals have already
included more than 1200 patients. The first study showed that docetaxel in a weekly
schedule had insufficient activity as compared with docetaxel + carboplatin in a three
weekly schedule in patients with stage IIIb and IV NSCLC. The second study,
performed in collaboration with the British Medical Research Council, is aimed at
evaluating the benefit of neo-adjuvant chemotherapy in operable NSCLC. Accrual to
this study has been completed and final analysis is being finalized. The NVALT3 is a
study in elderly patients with non-small cell lung cancer. In this study 2
chemotherapy regimens are compared in terms of toxicity and quality of life.
A battery of tests and questionnaires is being used and special nurses were trained
in 12 institutes to support the study. Accrual has been completed in 2006 and the
analysis is currently being performed. The NVALT4 study is a randomized placebocontrolled study of docetaxel/carboplatin with celecoxib or placebo in patients with
locally advanced or metastatic non-small cell lung cancer. Accrual to the study began
end of 2003 but was suspended during several months in 2005 waiting for the
evaluation by the FDA of reports of excessive cardiovascular toxicity associated to
Cox2 inhibitors in the treatment of pain. In excess of 400 patients have already been
randomized. We have obtained support from the Dutch Cancer Society for local data
management in the participating sites. We also obtained support for the the
NVALT 5, a phase III trial of the antiangiogenic agent Thalidomide in patients with
malignant pleural mesothelioma after first line chemotherapy. Participation of
Australian institutes is being organized. The NVALT 7 is a study of 2 chemotherapy
regimens in non small cell cancer patients with recurrence after first line
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
Danny Baars Technical staff
Roman Bohoslavsky MSc Technical staff
Monique Carreno Technical staff
Vincent Coppen Technical staff
Suzanne De Boer MSc Technical staff
Marjolijn De Waal MSc Technical staff
Jitske Dijkstra Technical staff
Brigitte Erven-Dufournij Technical staff
Song-Hieng Hau MSc Technical staff
Annelies Hiemstra Technical staff
Edwin Klerkx Technical staff
Jenneke Koorn Technical staff
Bettina Kortekaas Technical staff
Marianne Mahn-Schaefers MSc Technical staff
Ingrid Mandjes MSc Technical staff
Tineke Meindertsma Technical staff
Carla Modder Technical staff
Rick Muussen Technical staff
Cecile Paulus van Pauwvliet Technical staff
Anneke Reinders-Som Technical staff
Jolanda Remmelzwaal Technical staff
Daniel Roberts Technical staff
Marijke Scholten Technical staff
Dea Storm Technical staff
Martha Swart Technical staff
Laura Vader Technical staff
Heleen Vaessen MSc Technical staff
Renato Valdes Olmos Technical staff
Tjalling Valdes Olmos Technical staff
Ludy Valkenet MD Technical staff
Marjolijn Van den Haak MSc Technical staff
Emile Van der Donk Technical staff
Tony Van der 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
Lonny Ziblat Technical staff
144
BIOMETRICS
chemotherapy and a total of 150 patients have already been accrued. Two new studies
in low and high risk patients as determined by preoperative PET SUVmax values are
being developed and should start in 2007.
We collaborate with the radiotherapy department in carrying out several randomized
studies with support from the Dutch Cancer Society. More than 300 patients were
randomized this year into a study of radiotherapy boost in young women with early
breast cancer. In this study tumor material is collected for micro array analysis and
profile determination. Further, accrual has been completed into a study in lung
cancer that will be analyzed and reported in 2007.
The statisticians have collaborated with other departments of the institute, the Free
University and other institutes in the region in a variety of studies, many of which
resulted in co-authorships.
Figure 1: Number of resections in the IKA
region by year
PET prevents futile surgery in NSCLC Positron Emission Tomography (PET)
prevents futile surgery in 1 out of 5 lung cancer (NSCLC) patients. This was the result
of a randomized controlled trial with PET that was performed several years ago in the
IKA region. PET is a relatively new nuclear medicine imaging technique that allows
the visualization of biochemical processes in tissues. Diagnostic accuracy studies had
already shown promising results for PET in NSCLC. However, improved accuracy
does not necessarily imply clinical usefulness, e.g. better patient management and
improved clinical outcome. The assessment of the value of a diagnostic test to patient
management best follows a multi-phase hierarchical process, similar to what we are
familiar with in therapeutic research. The process encompasses the technical aspects
of the test such as image quality and reproducibility, diagnostic accuracy, diagnostic
and therapeutic impact, patient outcomes and finally, the cost-benefit analysis of the
introduction of the technology. Obviously, demonstration of efficacy at each lower
level is logically necessary, but not sufficient, to assure efficacy at a higher level. To
assess the value of PET we developed a framework which basically follows the
postulated hierarchical approach. The starting point of our studies was a notion of
residual inefficiency which might be amenable with the suggested improved accuracy
of FDG-PET (in NSCLC). Retrospective cohort analysis substantiated this notion,
specified the nature of the errors and it proved to be the main incentive for clinicians
to participate in further research. The run-in experiment on the clinical value of PET
was the learning curve of diagnosticians and clinicians. In the end, the coherence of
this approach contributed to a very fruitful environment for collaboration and has
facilitated the successful completion of the two RCTs and the appearance and
implementation of working guidelines in our region. Data from the IKA cancer
registry suggests that the introduction and implementation of the guidelines
effectively reduced the number of resections in the region with an absolute 20%
compared to the average over the five preceding years (Figure 1).
Anno 2006, the PET-situation in the Netherlands has changed substantially. The
PET-scanner capacity has increased dramatically and it is estimated that in about
80% of the patients in the IKA region (2.6 million inhabitants, 1300 NSCLC
annually) with suspected operable NSCLC a PET-scan is integrated in their work-up.
These studies and results are the core of the PhD thesis presented at the Free
University in Amsterdam by Harm van Tinteren.
ICT projects
During 2006 the Department negotiated with the European Commission Services
the Initial Deployment contract of the TENALEA project. A total maximum funding
of 3.7 million was obtained for this 42 months project. In addition of the NKI, which
is the coordinator of the project, the consortium consists of academic and industrial
contractors: the Institut Gustave Roussy, in Paris; the Cochrane Collaboration UK, in
Oxford; the Universitäts Klinikum Köln in Germany; the Medical University of
Gdansk in Poland; the Réseau de Cancérologie d’Aquitaine in Bordeaux; AKZO/
NOBEL NV in The Netherlands and InferMed Ltd in London. TENALEA is an
initiative of the Department that attempts to increase harmonization of the Clinical
Trials Data Handling tools which are being pushed towards the investigators and
study sites by providing best-of-breed solutions at affordable price.
Currently TENALEA provides a service for patient randomization in clinical trials,
based on ALEA software that was developed within the department. The service is
145
BIOMETRICS
Publications
hosted in the Department and a QA system for the operational management of the
service is in place. ALEA is a randomization service which was evaluated by 25 data
centers throughout Europe and was found suitable for the randomization procedure
of all clinical trials operated by these data centers. It was successfully tested for a
clinical trial selected by a pharmaceutical company (ORGANON NV).
In 2006 we have offered randomization services for 33 studies in the Netherlands in
which 2200 patients have been entered. In addition to the studies run by the NKI,
also studies of the IKA, IKO, IKL and Untrecht University have been supported.
Within this new phase of the project a framework to enable controlled extension of
the services to fulfill other requirements of clinical trial data centers, such as Remote
Data Entry will be developed. With Remote Data Entry, the traditional paper Case
Report Forms is replaced by an Electronic system through which the patient data for
a specific trial is collected. InferMed’s MACRO provides a Remote Data Entry service
and is used by several centers in the TENALEA consortium. Therefore, MACRO will
be operated in the TENALEA Shared Services Infrastructure to provide a hosted
Remote Data Entry solution to clinical trial data centers.
TENALEA is not a single service but a placeholder for several clinical trials data
management services. Therefore in addition to the Randomization and Remote Data
Entry other services will be identified and included in the framework as described in
Figure 2. The basis of this framework is a secure, fault tolerant, validated, audited
and compliant Shared Services Infrastructure. This Shared Services Infrastructure
provides a service platform for hosted tools supporting the clinical trials data
management process.
Baas P, Belderbos JSA, Senan S, Kwa HB,
Van Bochove A, Van Tinteren H,
Burgers JA, Van Meerbeeck JP. Concurrent
chemotherapy (carboplatin, paclitaxel,
etoposide) and involved-field radiotherapy
in limited stage small cell lung cancer:
A Dutch multicenter phase II study.
Br J Cancer 2006; 94:625-630
Buitelaar DR, Balm AJM, Antonini N,
Van Tinteren H, Huitink JM.
Cardiovascular and respiratory
complications after major head and neck
surgery. Head Neck 2006; 28:595-602
Den Herder C, Van Tinteren H,
De Vries N. Hyoidothyroidopexy as a
surgical treatment for obstructive sleep
apnoea syndrome. Ned Tijdschr Geneeskd
2006; 150:198-203
Hannemann J, Kristel P, Van Tinteren H,
Bontenbal M, Van Hoesel QGCM,
Smit WM, Nooij MA, Voest EE,
Van der Wall E, Hupperets P,
De Vries EGE, Rodenhuis S,
Van de Vijver MJ. Molecular subtypes
of breast cancer and amplification of
topoisomerase IIa: Predictive role in
dose intensive adjuvant chemotherapy.
Br J Cancer 2006; 95:1334-1341
Figure 2: TENALEA service organization
Koopman M, Antonini NF, Douma J,
Wals J, Honkoop AH, Erdkamp FLG,
The TENALEA Service Management manages the service portfolio of the TENALEA
Service Framework through the introduction of new services, new products for
existing services, periodic auditing of products and services, and the upgrading and
phasing out of existing products. Before a new service is deployed on the TENALEA
Service Framework, a common standard procedure is applied which will be further
detailed in the TENALEA Service Catalogue.
The TENALEA Services Framework will adopt the relevant methodologies and
concepts from the ITIL Service Level Management and ITIL Service Delivery. The
TENALEA Service Catalogue will be an important living document throughout the
initial deployment term. In addition, all services will be provided through Service
Level Agreements.
Not all services from the TENALEA Service Catalogue will become available at the
same point in time (Figure 3). Of the foreseen services in the portfolio, the
Randomization service will be available in the early phase of the project, while the
Remote Data Entry service will start at a later point during the project. Also, the
expected number of trials eventually hosted under the TENALEA Service Framework
will not be the same for all services.
Whereas the TENALEA Service Catalogue includes a Remote Data Entry service
based on MACRO, the eventual goal of TENALEA is to provide Electronic Data
Capture (EDC). The concept of Electronic Data Capture extends on Remote Data
Entry to include the ability to retrieve (part of the) patient data from other electronic
sources within the clinical setting, most prominently the Electronic Patient Record.
Electronic Data Capture best serves the main objective of TENALEA.
Success indicators of the project are: the number of trials which have used one or
more services from the TENALEA portfolio for their operational process and the
number of data centers which have adopted one It is anticipated that by the end of
De Jong RS, Rodenburg CJ,
Vreugdenhil G, Akkermans-Vogelaar JM,
Punt CJA. Randomised study of sequential
versus combination chemotherapy with
capecitabine, irinotecan and oxaliplatin in
advanced colorectal cancer, an interim
safety analysis. A Dutch Colorectal Cancer
Group (DCCG) phase III study. Ann
Oncol 2006; 17:1523-1528
Lont AP, Gallee MPW, Meinhardt W,
Van Tinteren H, Horenblas S. Penis
Conserving Treatment for T1 and T2 Penile
Carcinoma: Clinical Implications of a Local
Recurrence. J Urol 2006; 176:575-580
Rodenhuis S, Bontenbal M,
Van Hoesel QGCM, Smit WM, Nooij MA,
Voest EE, Van der Wall E, Hupperets P,
Van Tinteren H, Peterse JL,
Van de Vijver MJ, De Vries EGE. Efficacy of
high-dose alkylating chemotherapy in
HER2/neu-negative breast cancer.
Ann Oncol 2006; 17:588-596
146
BIOMETRICS ONCOLOGY
DIAGNOSTIC
Publications
Smeenk RM, Verwaal VJ, Antonini N,
Zoetmulder FAN. Progression of
Pseudomyxoma Peritonei after Combined
Modality Treatment: Management and
Initial Deployment 120 clinical trials will operate one or more TENALEA Services,
and between 50 and 70 data centers will be using TENALEA services. Finally, the
main success indicator is whether or not the service can be continued without further
funding. After Initial Deployment, the service must generate sufficient revenues to
cover the operating costs.
Outcome. Ann Surg Oncol
2006;Published online
Triesscheijn M, Ruevekamp M,
Antonini N, Neering H, Stewart FA,
Baas P. Optimizing Meso-TetraHydroxyphenyl-Chlorin Mediated
Photodynamic Therapy for Basal Cell
Carcinoma. Photochem Photobiol 2006;
82:1686-1690
Underberg R, Van Sornsen de Koste J,
Lagerwaard F, Vincent A, Slotman B,
Senan S. A dosimetric analysis of
respiration-gated radiotherapy in patients
with stage III lung cancer. Radiation
Oncology 2006; 1:8
Van der Poel HG, Antonini N, Hoefnagel
CA, Horenblas S, ValdГ©s Olmos RA.
Serum hemoglobin levels predict response to
strontium-89 and rhenium-186-HEDP
radionuclide treatment for painful osseous
metastases in prostate cancer. Urol Int
2006; 77:50-56
Figure 3: TENALEA project plan
Towards Electronic Data Capture (EDC) Technological trends are changing the
current developments in the market in the direction of EDC. Emerging standards for
electronic forms, such as PDF, xFORMS and InfoPath, combined with the emerging
standards in the field of electronic signatures, XML schema’s (ODM, HL7) and
ontology are likely to replace – probably starting this decade - the proprietary frontend of suite vendors to a forms front-end selected by the study site or investigator.
Such forms front-end can be generic, i.e. not for a particular niche segment such as
clinical trials data management and therefore be much more economic. This will
drastically accelerate the take up of electronic online tools for clinical trials data
management.
The electronic Case Record Forms (eCRF) Data Entry module is a generic electronic
form data entry facility which presents the eCRF’s as web forms, PDF forms, xForms
or InfoPath forms on the user’s computer device.
The second system into which the eCRF definition is fed into is an EPR XML Broker.
This is most likely a proprietary system which analyzes the ODM metadata
representation and allows the mapping of data available in the EPR to data required
in the eCRF. This may be a manual procedure performed after retrieving a metadata
eCRF ODM representation or it may be partially, semi or fully automated on the basis
of common ontologies.
The EPR XML Broker and the eCRF data entry module collaborate in a Just In Time
EPR data retrieval mechanism. At the time the user interface of the eCRF Data Entry
tool invokes an electronic Case Report Form, it first invokes the EPR XML broker
which on the basis of the ODM representation of the eCRF, retrieves all known data
from the Electronic Patient Record and inserts it onto the eCRF before it is displayed
to the user. Effectively, the clinician completing the form will only need to provide the
data which could not be located in the EPR.
The introduction of this concept is not restricted to hospitals working with EPR, but
may also be based on an HL7 broker which �listens’ to HL7 messages referring to
patients which are treated in a clinical trial protocol.
147
CLINICAL TRIALS
C LI NI CA L TRIAL S IN THE
N E T HE R LANDS CANCER IN S TITU TE
type of
cancer study
title
study coordinator
in NKI-AVL
phase
activated
(closed)
no pts.
in NKIAVL per
09/01/
2007
M03D24
Dose-finding and pharmacokinetic trial of orally
administered D-24851 to patients with solid tumors
Schellens, JHM
I
02/04/2003
25
M03MEN
A phase I study of intravenous MEN 4901/T-0128
administered once every 6 weeks in patients with solid
tumors
Schellens, JHM
I
10/11/2003
14
M04OAZ
A phase I, open label, multicenter study to assess
the safety, tolerability, pharmacodynamics and
pharmacokinetics of AZD5438 given orally, in 2 dosing
schedules, in patients with advanced solid
malignancies
Schellens, JHM
I
29/06/2004
11
M05AZA
A phase-I open-label, multicenter study to assess
the safety, tolerability and pharmacokinetics of AZD1152
given as as 2 hour intravenous infusion on 2 dose
schedules in patients with advanced solid malignancies
Schellens, JHM
I
23/05/2005
20
M05HOP
An open label phase I dose escalation study of E7080
Schellens, JHM
I
28/06/2005
20
M05PAR
A phase I, pharmacokinetic and biological evaluation of
a small molecule inhibitor of poly ADP-Ribose
Polymerase-1 (PARP-1), KU-0059436, in patients with
advanced tumors
Schellens, JHM
I
15/11/2005
14
M05PFS
A phase I, open-label, dose escalation study to evaluate
safety, pharmacokinetics and pharmacodynamics of
2 dosing schedules of PF 00299804 in patients with
advanced malignant solid tumors
Schellens, JHM
I
27/09/2005
15
M05RTB
A phase III international randomized trial of single
versus multiple fractions for re-irradiation of painful
bone metastases
Oppedijk, V
III
10/01/2006
2
M06BBC
Phase I study to determine the safety, maximum
tolerated dose and pharmacokinetics of BAY57-9352 in
combination with bevacizumab in subjects with
advanced solid tumors
Schellens, JHM
I
19/05/2006
6
M06BCI
Phase I study to determine the safety, maximum
tolerated dose, pharmacokinetics and biomarker status
of BAY 57-9352 in combination with capecitabine and
irinotecan in subjects with advanced solid tumors
Schellens, JHM
I
12/10/2006
3
M06ESO
Effect of esomeprazole on the pharmacokinetics of
BMS-275183 in patients with advanced malignancies
Roelvink, M
I
12/12/2006
1
ALL SITES
148
CLINICAL TRIALS
type of
title
cancer study
study coordinator
in NKI-AVL
phase
activated
(closed)
no pts.
in NKIAVL per
09/01/
2007
M06FRE
Effect of fibrin sealant in reducing REsection surface
related complications - The FRESCO trial
Zoetmulder, FAN
other
06/09/2006
0
M06HYP
A phase II randomised factorial double-blind study to
investigate the management of AZD2171 induced
hypertension and efficacy of AZD2171 at doses of 30
mg and 45 mg in patients with advanced solid tumors
Schellens, JHM
II
21/08/2006
15
M06OGP
Phase I dose escalation study of oral gemcitabine
prodrug (LY2334737) alone and in combination with
erlotinib (Tarceva) in patients with advanced solid
tumors
Schellens, JHM
I
29/08/2006
5
M06PPI
Collection of skin biopsies with hair follicle samples
from patients with advanced solid malignancies
receiving Irinotecan to develop biomarker assays
Schellens, JHM
NA
26/01/2006
2
N03DOR
Oral bioavailability of docetaxel in combination with
ritonavir
Schellens, JHM
I
07/04/2003
22
N03JJP
Relative bioavailability of Paclitaxel in combination with
Cyclosporin A. A pilot investigation
Schellens, JHM
Pilot
26/01/2004
12
N05GEN
Genotyping as tool for dose individualization in highdose chemotherapy with cyclophosphamide, thiotepa
and carboplatin
Huitema, A
NA
14/06/2005
9
N06LTN
Long term neurotoxicity after treatment with the
platinum-containing anti-cancer agents cisplatin and
oxaliplatin
Schellens, JHM
NA
26/04/2006
45
N06PFB
Pleural fluid bank
Heuvel, MM Van den
NA
27/09/2006
*
N98CTO
A phase I study to determine the maximum tolerated
doses of Carboplatin and Topotecan administered
intravenously every 28 days to patients with malignant
tumors
Bokkel Huinink, WW Ten
I
19/04/1999
41
P05VVD
Behoefte-onderzoek naar voedingszorg/voorlichting op
de dagbehandeling
DiГ«tetiek
08/12/2005
*
P06HRM
Psychosocial aspects of genetic testing in families at
high risk of multiple tumors at various sites and ages
Aaronson, N
NA
20/06/2006
0
M04CRG
Cognitive rehabilitation of glioma patients:
A prospective, randomized study
Boogerd, W
NA
28/04/2004
(21/12/2006)
9
N05THB
Totale hersenbestraling met een eenmalige boost bij
patienten met solitaire hersenmetastase van een
epitheliale tumor - een registratiestudie
Dewit, LGH
IV
07/04/2005
5
BRAIN / CNS
* = not registered at trialbureau
type of
cancer study
149
CLINICAL TRIALS
activated
no pts.
(closed)
in NKIAVL per
09/01/
2007
title
study coordinator
in NKI-AVL
phase
E10021
Trial 10021: An IDBBC randomized, double blind,
placebo-controlled, multi-center phase II trial of
anastrozole (Arimidex) in combination with Iressa
(zd1839) or placebo in patients with advanced breast
cancer
Schornagel, JH
II/II
04/11/2003
0
E10031
A phase III trial evaluating the role of ovarian 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 ovarian
function suppression + exemestane
Schornagel, JH
III
20/07/2005
1
E10041
Micro-array in node-negative disease may Avoid
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
Rutgers, EJTh
III
02/01/2007
0
E10981
After mapping of the axilla: Radiotherapy or surgery?
Rutgers, EJTh
III
21/12/2000
331
E16023
Phase I study of lonafarnib (SCH66336) in
combination with herceptin plus paclitaxel in Her 2 neu
overexpressing breast cancer
Schellens, JHM
I
01/08/2003
13
E22051
SUPREMO, an MRC phase III randomised trial to
assess the role of adjuvant chest wall irradiation in
�intermediate risk’ operable breast cancer following
mastectomy
Russell, NS
III
23/11/2006
0
M03ARR
The implementation of microarrays in cancer
diagnosis
Linn, SC
Pilot
21/10/2003
*
M03HTT
Open label, comparative, randomized, multicenter,
study of trastuzumab (Herceptin) given with docetaxel
(Taxotere) versus sequential single agent therapy with
trastuzumab followed by docetaxel as first-line
treatment for metastatic breast cancer (MBC) patients
with HER2-neu overexpression
Schornagel, JH
II
22/08/2003
3
M03RBC
Radiation dose intensity study in breast cancer in
young women: a randomized phase III trial of
additional dose to the tumor bed (“Young Boost”)
Bartelink, GMM
III
29/03/2004
53
M04GTB
A randomized phase II trial of biweekly dose-dense
gemzar plus taxol (GT2) and three weekly gemzar plus
taxol (GT3) in patients with metastatic breast cancer
Schornagel, JH
II
19/08/2004
9
M04MAT
Microarray analysis in breast cancer to tailor adjuvant
drugs or regimens (MATADOR)
Linn, SC
III
26/04/2004
32
M05BRI
Long term risk of breast cancer following treatment of
Hodgkin’s disease
Russell, NS
05/01/2006
3
BREAST
* = not registered at trialbureau
150
CLINICAL TRIALS
type of
title
cancer study
study coordinator
in NKI-AVL
phase
activated
(closed)
no pts.
in NKIAVL per
09/01/
2007
M05HIR
Hormonal substitution after prophylactic adnectomy in
women with an increased risk for breast- and ovarian
cancer due to a genetic predisposition: HIRISE (highrisk women and hormonal substitution exposure)
Beurden, M Van
IV
31/05/2005
*
M06TM2
A randomised, multicenter, prospective, 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 in postmenopausal women
with hormone receptor positive early breast cancer
Linn, SC
III
26/09/2006
0
M99BLU
Lymphatic drainage analysis through preoperative
intradermal injection of blue dye in patients undergoing
a modified radical mastectomy
Nieweg, OE
Pilot
04/05/2000
(18/12/2006)
48
N02ANB
Phase II study of anastrozole as neoadjuvant
treatment in postmenopauzal women with ER+ locally
advanced breast cancer
Oosterkamp, R
II
05/02/2003
1
N03BCC
Proteomic profile and treatment response, in patients,
with advanced breast cancer, treated with Capecitabine
chemotherapy
Helgason, HH
II
07/10/2003
92
N03IGF
The relation between serum and normal breast tissue
Insulin-like Growth Factor (IGF) system components
Voskuil, DW
IV
24/05/2004
40
N04PBC
Proteomic profile in patients with primairy breast
cancer
Helgason, HH
other
28/04/2004
165
N04POM
Tailored preoperative chemotherapy in stage II or III
breast cancer with either a primary tumor over 3 cm in
size or a clinically tumor-positive axilla.
Rodenhuis, S
II
21/03/2005
64
N04RTB
Effecten van bestraling op bloedvaten
Russell, NS
Pilot
05/10/2004
45
N05ECP
Prospectief, dubbelblind, gerandomiseerde placebo
gecontroleerde farmacologisch interventieonderzoek:
evaluatie van de frequentie, duur en intensiteit van
opvliegers na interventie met Venlafaxine (EfexorВ®) en
Clonidine versus placebo bij vrouwen met vervroegd
postmenopauzale klachten tgv adjuvant chemotherapie
en/of hormonale therapie voor een mammacarcinoom
Schellens, JHM
III
10/10/2005
45
N05MIB
99mTc-methoxyisobutylisonitrile (MIBI) for imaging of
apoptosis and prediction of tumor respons to
chemotherapy and/or radiotherapy in cancer patients
Valdes Olmos, RA
other
07/06/2005
6
N05RHN
Feasibility study of intensive alkylating chemotherapy in
residual HER2/neu-negative breast cancer after preoperative chemotherapy
Rodenhuis, S
NA
14/11/2005
13
N05TOM
A clinical trial to compare the efficacy of the lorad
tomosynthesis mammography system to conventional
mammography
Teertstra, HJ
other
07/02/2006
*
* = not registered at trialbureau
151
CLINICAL TRIALS
activated
no pts.
(closed)
in NKIAVL per
09/01/
2007
type of
cancer study
title
study coordinator
in NKI-AVL
phase
N06IAA
Randomized phase II/III study of intensified alkylating
agent chemotherapy with peripheral blood progenitor
cell support in the preoperative chemotherapy of breast
tumors that are deficient for homologous
recombination
Rodenhuis, S
II/II
08/01/2007
0
N06PMK
Meaurement of force of the pectoralis major muscle
after subpectoral implantation of a prosthesis for the
reconstruction of the breast immediately following a
skin sparing mastectomy
Hage, JJ
NA
30/08/2006
*
N06TRZ
The effect of Trastuzumab treatment on B-cell kinase
activities assessed by microarray derived
phosphorylation profiles
Schellens, JHM
NA
11/12/2006
0
N06VNI
Meting van de functie van de arm en de bijdrage
daaraan van de grote borstspieren voor en na de
tweezijdige implantatie van een endoprothese ten
behoeve van een borstreconstructie die direct
aansluitend aan de huidsparende borstoperatie wordt
uitgevoerd
Hage, JJ
other
26/09/2006
N96NKB
The contribution of ATM heterozygosity to the risk of
radiation-induced breast cancer
Russell, NS
Pilot
12/09/1996
(19/12/2006)
255
P00MRI
MRI-scan bij patienten met borstkanker ter bepaling
van de afmetingen van de afwijking
Gilhuijs, KGA
NA
06/10/2000
547
P04PKC
Patienteninformatiebrief en bewustbereidverklaring
micro-array en proteomics
Linn, SC
NA
04/11/2004
*
P05GEO
Onderzoek naar de rol van omgevingsfactoren/
leefgewoonten bij het ontstaan van erfelijke borst- en
eierstokkanker (GEO-onderzoek II)
Rookus, M
NA
23/01/2006
*
P05NAZ
Behoefteonderzoek nazorg/revalidatie na behandeling
voor mammacarcinoom
Hesselink, S
22/03/2006
*
P06VOL
Beslissen over in het verleden opgeslagen
lichaamsmateriaal
Vermeulen, E
23/11/2006
*
P99SHB
Impact van regelmatige controle (screening) bij
vrouwen met een verhoogd risico op borstkanker
vanwege een familiaire predispositie
Rutgers, EJTh
NA
01/05/2000
676
Verwaal, VJ
III
23/06/2003
11
GASTRO INTESTINAL
E40004
CLOCC trial (chemotherapy + local ablation versus
chemotherapy). Randomized phase III study of local
treatment of liver metastases by radiofrequency
combined with chemotherapy versus chemotherapy
alone in patients with F.A.N. unresectable colorectal
liver metastases
* = not registered at trialbureau
152
CLINICAL TRIALS
type of
title
cancer study
study coordinator
in NKI-AVL
phase
activated
(closed)
no pts.
in NKIAVL per
09/01/
2007
M01BAX
POCASTER trial (J-POuch- Colo-Anale anastomose
versus Side-To-End colorectale anastomose na
preoperatieve radiotherapie en Totale Mesorectale
Excisie (TME) bij Rectumcarcinoom; een multicenter,
gerandomiseerd onderzoek
Zoetmulder, FAN
III
06/06/2002
5
M02COL
Een landelijke studie naar de waarde van periodiek
colonoscopisch onderzoek bij personen met een
positieve familieanamnese voor colorectaal carcinoom
Cats, A
Pilot
19/08/2003
1
M02PCR
Postoperative chemo-radiotherapy after surgical
resection of gastric and esophageal cancer.
A multicenter phase II study of a fixed radiotherapy
regimen with concurrent chemotherapy with
capecitabine
Boot, H
II
16/01/2003
44
M03CRE
Chemoradiation for irresectable (T4) esophageal
cancer - Phase II multi-center study
Aleman, BMP
II
26/11/2003
2
M05CRC
Colorectale polipe/maligniteit studie GUT club.
Onderzoek naar de behoefte planning en wachtlijst
problematiek bij dikkedarmkanker
Cats, A
Pilot
15/11/2005
*
M05RAX
Pre-operative chemoradiotherapy regimen with
capecitabine and bevacuzimab in locally advanced
rectal cancer. A feasability study (RAX)
Marijnen, C
II
20/03/2006
4
M06CRI
A multicenter randomized phase III trial of neoadjuvant chemotherapy followed by surgery and
chemotherapy or by surgery and chemoradiotherapy in
resectable gastric cancer (CRITICS-study:
ChemoRadiotherapy after Induction chemo Therapy In
Cancer of the Stomach)
Verheij, M
III
13/11/2006
0
M06SCR
A multicenter phase III randomised trial comparing
total mesorectal excision with pre-operative
radiotherapy with or without post-operative oral
capecitabine in the treatment of operable primary rectal
cancer
Cats, A
III
09/05/2006
1
M06SUS
Surgical gastrojejunostomy or endoscopic duodenal
stent placement for the palliation of malignant gastric
outlet obstruction; a randomized study. Surgery versus
Stent for malignant gastro-duodenal obstruction;
SUSTENT study
Cats, A
III
06/09/2006
0
N02ECC
A single institution phase II study of ECC (Epirubicin,
Cisplatin, Capecitabin) in locally advanced or
metastatic gastric cancer and adenocarcinoma of the
oesophago-gastric junction and distal oesophagus
Boot, H
II
29/08/2002
115
N02NIG
Nutrients and the Insulin-like Growth Factor (IGF)
system in men and women at increased risk of
colorectal cancer
Voskuil, DW
III
28/01/2003
(29/12/2006)
208
* = not registered at trialbureau
153
CLINICAL TRIALS
activated
no pts.
(closed)
in NKIAVL per
09/01/
2007
type of
cancer study
title
study coordinator
in NKI-AVL
phase
N02RCA
Post operative chemo-radiotherapy after resection of
gastric and esophageal cancer
Boot, H
I/II
19/12/2002
50
N03PGE
Proteomics profile and treatment response, in patients,
with advanced gastric or distal esophageal cancer,
treated with first-line Cisplatin, and Capecitabine (ECC)
or 5FU/LV (ECF) chemotherapy
Schellens, JHM
other
23/06/2003
88
N05GEA
Saving the gastro-epiploic artery at omentectomy and
the effect on post-surgical recovering gastric function
Zoetmulder, FAN
NA
21/03/2006
8
N05MCT
A phase II study of treatment with the combination of
unlabelled (“cold”) and radioactive (“hot”) metaiodobenzylguanidin (MIBG) in metastatic carcinoid
tumors
Smits, ME
other
05/12/2005
0
N05STP
Serum and tissue protein profiling and tumor genetic
analysis in patients with potential premalignant
conditions or colorectal cancer
Smits, ME
NA
19/01/2006
49
N06DCM
Feasibility of the analysis of platinum-DNA adducts in
tumor tissue in cisplatinum treated with advanced
gastric cancer
Schellens, JHM
NA
12/12/2006
*
P05FAP
Mid to long-term psychosocial impact of the genetic
testing among familial adenomatous polyposis (FAP)
on families
Aaronson, N
NA
31/08/2005
506
P06GIN
Clinical and psychometric validation of a diseasespecific questionnaire module in assessing the quality
of life of patients with GI-related neuroendocrine
tumors
Taal, BG
NA
02/11/2006
*
P06POR
Decision making in rectal cancer, the costs and benefits
of preoperative radiotherapy
Marijnen, C
NA
05/10/2006
*
GYNAECOLOGICAL
E55041
A randomised, multicentre, phase III study of Erlotinib
vs 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
Driel, WJ Van
III
25/09/2006
1
M01EPO
An open-label phase IIa trial evaluating the safety and
efficacy of EPO906 in patients with advanced ovarian,
primary fallopian, or primary peritoneal cancer
Bokkel Huinink, WW Ten
I/II
22/01/2002
39
M02MCA
Measurement of cisplatinum adduct in patients with
cervical cancer treated with chemoradiotherapy
Verheij, M
Pilot
01/05/2002
(18/12/2006)
4
M05HIR
Hormonal substitution after prophylactic adnectomy in
women with an increased risk for breast- and ovarian
cancer due to a genetic predisposition: HIRISE (HighRisk women and hormonal Substitution Exposure)
Beurden, M Van
IV
31/05/2005
*
* = not registered at trialbureau
154
CLINICAL TRIALS
type of
title
cancer study
study coordinator
in NKI-AVL
phase
activated
(closed)
no pts.
in NKIAVL per
09/01/
2007
M05PPO
Proteomic patterns in blood and tissue of ovarian
cancer patients
Driel, WJ Van
other
12/01/2006
*
M05YDO
An open-label multicenter randomized phase III study
comparing the combination of DOXIL/CAELYX and
YONDELIS with DOXIL/CAELYX alone in subjects with
advanced relapsed ovarian cancer
Bokkel Huinink, WW Ten
III
20/07/2006
1
M06HRT
The effect of hormonal replacement therapy on
menopausal complaints related to biochemical
changes in surgically and naturally postmenopausal
women. A prospective observational comparative study
Korse, CM
NA
25/09/2006
13
M06OVH
Phase III randomised clinical trial for stage III ovarian
carcinoma randomising between secondary debulking
surgery with or without hyperthermic intraperitoneal
chemotherapy (OVHIPEC-1)
Driel, WJ Van
III
04/01/2006
0
N05CGO
Randomized clinical pharmacological dose-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 both administered in
combination with carboplatin as second-line treatment
in patients with advanced ovarian cancer
Schellens, JHM
I
08/09/2005
11
M02STA
The effect of a statin on the progression of the intimamedia thickness induced by radiotherapy
Boogerd, W
III
28/01/2003
43
M05PET
FDG-PET for avoidance of futile direct laryngoscopies
under general anaesthesia with taking of biopsies in
patients with suspicion on recurrent laryngeal
carcinoma after radiotherapy
Brekel, M Van de
NA
11/05/2005
5
N02SNL
Early detection of lymph node metastasis of squamous
cell carcinoma of the larynx with lymphatic mapping
and sentinel node biopsy
Lohuis, PJFM
Pilot
03/04/2002
7
N04LFV
Longfunctie na toediening van inhalatiemedicatie
(salbutamol-ipratropiumbromide dosisaerosol) met en
zonder voorzetkamer bij gelaryngectomeerden
Hilgers, FJM
III
07/02/2005
8
N04RTB
Effecten van bestraling op bloedvaten
Russell, NS
Pilot
05/10/2004
45
N05HME
De kortetermijninvloed van een Heat and Moisture
Exchanger op de endotracheale temperatuur en
luchtvochtigheid bij gelaryngectomeerden
Zuur, JK
Pilot
01/09/2005
*
N05MIB
99mTc-methoxyisobutylisonitrile (MIBI) for imaging of
apoptosis and prediction of tumour respons to
chemotherapy and/or radiotherapy in cancer patients
Valdes Olmos, RA
other
07/06/2005
6
HEAD AND NECK
* = not registered at trialbureau
155
CLINICAL TRIALS
activated
no pts.
(closed)
in NKIAVL per
09/01/
2007
type of
cancer study
title
study coordinator
in NKI-AVL
phase
N05TSP
Prevention of trismus, swallowing and speech
problems in patients treated with chemo-radiation for
advanced head and neck cancer
Hilgers, FJM
NA
14/11/2005
4
P05MDI
Patienteninfobrief en bewustbereidverklaring nader
gebruik lichaamsmateriaal bij hoofd-halskankerpatiГ«nten
Brekel, M Van de
NA
13/04/2005
100
P06AIQ
De AnaesthetIQ Intubator in de praktijk
Hilgers, FJM
NA
18/10/2006
0
LEUKAEMIA / MDS
M02H49
Randomised Phase III study in eldery patients with a
multiple myeloma on the value of Thalidomide added
to Melphalan plus Prednison
Kerst, M
III
31/01/2003
0
M05H65
A randomized phase III study on the effect of
Bortezomib combined with Adriamycin, Dexamethason
(AD) for induction treatment, followed by High Dose
Melphalan and Bortezomib alone during maintenance
in patients with multiple myeloma
Baars, JW
III
03/10/2005
0
M05H68
A randomized phase III study in previously untreated
patients with biological high-risk CLL: fludarabine +
cyclophophamide (FC) versus FC + low dose
alemtuzumab
Kerst, M
III
21/03/2006
0
E08021
A randomized phase III study of follow up with or
without adjuvant Gefitinib (Iressa TM) following
chemotherapy in patients with advanced non-small cell
lung cancer
Burgers, JA
III
11/05/2005
(19/12/2006)
0
E08031
A phase II feasibility trial of induction chemotherapy
followed by extrapleural pneumonectomy and
postoperative radiotherapy in patients with malignant
pleural mesothelioma
Baas, P
II
24/08/2005
10
M03THA
Phase III trial of the antiangiogenic agent Thalidomide
in patients with malignant pleural mesothelioma after
first line chemotherapy (NVALT5)
Baas, P
III
18/02/2004
52
M04ZON
Implementation of Endoscopic Ultrasound guided Fine
Needle Aspiration (EUS-FNA) as a part of the standard
work-up in lung cancer patients (ZonMw-Project)
Burgers, JA
IV
25/01/2005
60
M05ALI
A randomised phase II study of pemetrexed compared
to pemetrexed-carboplatin in pretreated patients with
advanced Non Small Cell lung Cancer
Burgers, JA
II
05/04/2006
21
M05ATD
Accurate target definition of non-small cell lung tumors
using pathology-validated PET and CT imaging for the
optimization of radiation treatment
Stroom, J
NA
05/01/2006
*
LUNG
* = not registered at trialbureau
156
CLINICAL TRIALS
type of
title
cancer study
study coordinator
in NKI-AVL
phase
activated
(closed)
no pts.
in NKIAVL per
09/01/
2007
M05GCP
Pharmacogenomic and pharmacokinetic study in
patients with advanced non small-cell lung cancer
(NSCLC) treated with first-line gemcitabine / platinum
combination chemotherapy
Schellens, JHM
other
13/09/2005
47
M05OPE
Daily oral Perifosine (ZEN-004) in combination with
radiotherapy in non-small cell lung cancer patients
Verheij, M
II
10/01/2006
4
M06BOR
An open label phase II multicentre clinical trial of single
agent bortezomib in patients with malignant pleural
mesothelioma
Baas, P
II
22/11/2006
1
M06NEL
Efficacy of neoadjuvant erlotinib in patients with clinical
stage I/II non-small cell lung cancer (NSCLC)
Klomp, HM
I/II
08/11/2006
1
M06PFN
International, randomized, open-label, phase III trial of
Gemcitabine/Cisplatin plus PF-3512676 versus
Gemcitabine/Cisplatin alone as first-line treatment of
patients with advanced non-small cell lung cancer
Burgers, JA
III
22/05/2006
(03/01/2007)
9
N00ALF
Endoscopic detection op pre-neoplastic lesions and
carcinoma in the bronchial tree with delta-aminolevunic
acid fluorescence
Baas, P
Pilot
30/06/2000
41
N04LSN
Feasibility of lymphoscintigraphy and sentinel node
biopsy in patients with non-small lung cancer
Klomp, HM
Pilot
08/09/2004
5
N05MIB
99mTc-methoxyisobutylisonitrile (MIBI) for imaging of
apoptosis and prediction of tumour respons to
chemotherapy and/or radiotherapy in cancer patients
Valdes Olmos, RA
other
07/06/2005
*
N99HIM
Cytoreduction and hyperthermic intra-thoracic
chemotherapy (HITHOC) in patients with pleural
mestastases opf thymoma or limited mesothelioma
Zoetmulder, FAN
Pilot
26/01/2000
24
P04MMI
Optimization of conformal radiation therapy of NSCLC
by advanced 3-D plannning and multimodality imaging
(NKI projekt 2003-2943)
Belderbos, JSA
I/II
03/06/2004
51
LYMPHOMA - HODGKIN’S DISEASE
E20011
A randomized trial of BEAM plus PBSCT versus single
agent high dose therapy followed by BEAM plus PBSCT
in patients with relapsed Hodgkin’s disease
Baars, JW
III
23/01/2002
(31/12/2006)
0
E20012
BEACOPP (4 cycle’s escalated + 4 cycle’s baseline) vs
ABVD (8 cycle’s) in Stage III & IV Hodgkin’s
Lymphoma.
Baars, JW
III
29/03/2004
0
E20051
The H10 EORTC/GELA randomized Intergroup trial on
early PET-scan guided treatment adaptation versus
standard combined modality treatment in patients with
supradiaphragmatic stage I/II Hodgkin’s lymphoma
Baars, JW
III
23/11/2006
1
* = not registered at trialbureau
type of
cancer study
title
study coordinator
in NKI-AVL
phase
LYMPHOMA - NON-HODGKIN’S
157
CLINICAL TRIALS
activated
no pts.
(closed)
in NKIAVL per
09/01/
2007
E20971
A phase III study on low-dose total body irradiation and
involved field radiotherapy in patients with localized,
stages I and II, low grade non-hodgkin’s lymphoma
Haas, RLM
III
02/02/2004
4
M05H55
Efficacy of maintenance therapy with rituximab after
induction therapy (R-CHOP versus R-FC) for elderly
patients with mantle cell lymphoma not suitable for
autologous stem cell transplantation
Boer, JP De
III
20/07/2005
0
M05H63
Randomized phase III study of Rituximab with
intensified CHOP chemotherapy (R-iCHOP-14) versus
Rituximab with High-Dose Sequential Therapy and
Autologous Stem Cell Transplantation (R-HDT+ASCT)
in adult patients (18-65years) with stage II-IV highintermediate or High Risk Diffuse Large B-cell
Lymphoma
Baars, JW
21/03/2006
0
M05H69
A phase II study of anti-CD52 monoclonal antibody
(alemtuzumab, Mab-Campath) with 2-weekly CHOP
chemotherapy (Camp-CHOP 14) in patients with
mature T-cell non-Hodgkin’s lymphoma
Baars, JW
II
11/01/2005
0
M05H73
Primary rituximab and maintenance (PRIMA) study: a
multicenter phase III open labeled randomized study in
patients with advanced follicular lymphoma evaluating
the benefit of the maintenance therapy with rituximab
after induction of response with chemotherapy plus
rituximab in comparison with no maintenance therapy
Baars, JW
III
21/03/2006
3
M06CMV
Cytomegalovirus (CMV) - specific T cell immunity in B
cell malignancies
Kerst, M
other
28/09/2006
*
M06H77
Efficacy and safety of a single dose of 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
Baars, JW
II
23/05/2006
0
N03OFE
A randomized phase II/III study of eradication therapy
with additional oral fludarabine versus eradication
therapy alone in H.pylori positive gastric MALT
lymphoma
Boer, JP De
II/II
17/06/2003
(18/12/2006)
3
N03OFP
A phase II study of eradication therapy with additional
oral fludarabine in t(11;18) positive gastric MALT
lymphoma
Boer, JP De
II
06/10/2003
4
N03RIM
Radioimmunotherapy (131I-antiCD20 monoclonal
antibody) as consolidation treatment after remission
induction for patients with relapsed or refractory
CD20+ B-cell NHL
Baars, JW
Pilot
25/06/2003
0
N05MIB
99mTc-methoxyisobutylisonitrile (MIBI) for imaging of
apoptosis and prediction of tumor respons to
chemotherapy and/or radiotherapy in cancer patients
Valdes Olmos, RA
other
07/06/2005
*
* = not registered at trialbureau
158
CLINICAL TRIALS
type of
title
cancer study
study coordinator
in NKI-AVL
phase
activated
(closed)
no pts.
in NKIAVL per
09/01/
2007
MELANOMA / SKIN
M05MSL
A phase III multicenter randomized trial of 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
Nieweg, OE
III
11/09/2006
0
M06MDX
A randomized, double-blind, multicenter study
comparing MDX-010 monotherapy, MDX-010 in
combinatio with a melanoma vaccine monotherapy in
HLA-A*0201-positive patients with previously treated
unresectable stage III or IV melanoma
Haanen, JBAG
III
13/09/2006
2
M06TDT
A3671009: a phase 3, open label, randomized,
comparative study of ticilimumab and either
dacarbazine or temozolomide in patients with
advanced melanoma
Haanen, JBAG
III
18/05/2006
16
N03CYL
Pilot study of topical application of salicylate in familial
cylindromas
Oosterkamp, R
Pilot
26/06/2003
5
N03LAM
Longitudinal analysis of melanoma-specific immunity
in stage III and IV melanoma patients
Haanen, JBAG
II
22/08/2003
1
N06TIS
Integrated analyses of melanoma-T cell interactions;
relevance for immunotherapy
Haanen, JBAG
NA
29/08/2006
*
M06PRO
The efficacy of therapeutic compression hoses for
prevention of lymphedema after inguinal lymph node
dissection in cancer patients
Stuiver, M
other
05/09/2006
18
N01RIT
Identifications of molecular mechanisms involved in
radiation-induced telangiectasia
Russell, NS
Pilot
07/05/2002
21
N03THY
Therapeutic management of thymoma and thymic
carcinoma: - a prospective registration study based on
preoperative risk assessment of local failure
Dewit, LGH
Pilot
25/11/2003
11
N05CHO
The efficacy of cordotomies in patients with chest pain
due to primary localized malignancies of the chest
Lukas, A
II
17/01/2006
*
N05SHB
Bestudering van basis van normaal S100B
Gast, GC De
Pilot
21/04/2005
*
N99EOL
Endoscopisch echo-onderzoek van de centrale
luchtwegen onder narcose: feasibility study
Baas, P
Pilot
14/12/1999
*
P05PDE
Prevalentie onderzoek naar het voorkomen van
decubitus
other
14/11/2005
0
III
09/07/2003
7
MISCELLANEOUS
SOFT TISSUE / OSTEOSARCOMA
E62012
Randomised trial of single agent doxorubicin versus
doxorubicin plus ifosfamide in the first line treatment
of advanced or metastatic soft tissue sarcoma
Rodenhuis, S
* = not registered at trialbureau
159
CLINICAL TRIALS
activated
no pts.
(closed)
in NKIAVL per
09/01/
2007
type of
cancer study
title
study coordinator
in NKI-AVL
phase
E62024
Intermediate and high risk localized, completely
resected, gastrointestinal stromal tumors (GIST)
expressing KIT receptor:a controlled randomized trial
on adjuvant Imatinib mesylate (Glivec) versus no
further therapy after complete surgery
Rodenhuis, S
III
25/11/2004
9
E62027
Phase II study of Glivec (imatinib) in locally advanced
and/or metastatic soft tissue sarcoma expressing the
t(17;22)(q22;q13)translocation resulting in a COL1A1/
PDGF fusion protein i.e. DermatoFibroSarcoma
Protuberans (DFSP) and Giant Cell Fibroblastoma
(GSF)
Rodenhuis, S
II
04/11/2004
0
M01EUR
EURO-E.W.I.N.G. 99. EUROpean Ewing tumor Working
Initiative of National Groups
Rodenhuis, S
III
18/09/2001
9
M01ROS
Phase II study of rosiglitazone in advanced
liposarcoma
Rodenhuis, S
II
24/08/2001
5
E30983
Randomized phase II/III study of Taxol-BEP versus BEP
in patients with intermediate prognosis germ cell
cancer
Schornagel, JH
II/II
07/09/1999
3
E30986
Randomized phase II/III study assessing gemcitabine/
carboplatin and methotrexate/carboplatin/Vinblastine
in previously untreated patients with advanced
urothelial cancer ineligible for cisplatinum based
chemotherapy
Schornagel, JH
II/II
07/05/2002
19
E30994
Randomized phase III trial comparing immediate
versus deferred chemotherapy after radical cystectomy
in patients with pT3-pT4, and/or N+ M0 transitional
cell carcinoma (TCC) of the bladder
Kerst, M
III
28/01/2003
17
M00LMT
Identification of occult lymph node metastases in
testicular cancer to select patients for adjuvant
treatment. Feasability of a laparoscopic selective
retroperitoneal lymphadenectomy
Horenblas, S
Pilot
14/11/2000
0
M05AIR
Atrasentran combined with interferon-apha in renal cell
carcinoma: dose escalation phase I study and efficacy
phase II study
Haanen, JBAG
I/II
20/07/2005
6
M06SIL
Phase I dose-escalation trial for the combination of
sorafenib with interleukin-2 treatment in patients with
clear cell renal carcinoma
Haanen, JBAG
I
20/11/2006
0
M94SAL
Salvage regimen incorporating repeated ablative
chemotherapy with autologous PSCT, a phase II study
Rodenhuis, S
II
04/07/1994
24
N02PBI
Feasability of partial bladder irradiation with an
adaptive margin strategy
Pos, F
Pilot
14/03/2003
(15/12/2006)
4
URO-GENITAL
160
CLINICAL TRIALS
type of
title
cancer study
study coordinator
in NKI-AVL
phase
activated
(closed)
no pts.
in NKIAVL per
09/01/
2007
N04ILR
Neoadjuvant plus adjuvant interleukine-2 around
nephrectomy/metastasectomy for patients with a
primary renal cell carcinoma with high-risk of
progression or with a localized metastasis
Gast, GC De
II
06/01/2005
7
N04IRC
Initial immunotherapy as selection for nephrectomy
versus initial nephrectomy followed by immunotherapy
in patients with metastatic renal cell carcinoma and the
primary tumor in situ
Bex, A
II
05/08/2004
16
P04TBB
Verkorte procedure Protocol Toetsingscommissie van
het patient tevredenheidsonderzoek behandeltraject
oppervlakkig blaascarcinoom in het Antoni van
Leeuwenhoek Ziekenhuis
Blok, W De
NA
23/12/2004
(19/12/2006)
*
P06HCP
Het effect van hormonale therapie op het cognitief
functioneren van patiГ«nten met prostaatkanker
Schagen, SSB
09/05/2006
0
* = not registered at trialbureau
161
INVITED SPEAKERS
I N V I T E D S PEAKERS 2006
Reuven Agami, Amsterdam, The Netherlands
Genetic screens identify cancer-related functions of
microRNAs
Peter Cullen, Bristol, UK
Switching off signalling through the Ras proto-oncogene
Anna Akhmanova, Rotterdam, The Netherlands
CLASPing microtubules to the cell cortex
Edwin Cuppen, Utrecht, The Netherlands
Generation and characterization of knockout models in
zebrafish and rat
Patrick Aloy, Barcelona, Spain
Structure-based systems biology: modelling interactions
and complexes
Ilan Davis, Edinburgh, UK
Intracellular mRNA transport and anchoring in
Drosophila
Haico van Attikum, Basel, Switserland
Chromatin dynamics at a chromosomal
DNA double-strand break
Haryana Dhillon, Sydney, Australia
Antipodean Haze - cognitive function research
Fran Balkwill, London, UK
Inflammation and cancer: the cytokine link
Wolfgang Baumeister, Martinsried, Germany
Mapping molecular landscapes inside cells by
cryoelectron tomography
Peter Becker, Munich, Germany
Dosage compensation in flies: the end of generalisations
Derk ten Berge, Stanford, USA
Separate control of cell proliferation and differentiation
by Wnt proteins
Wendy Bickmore, Edinburgh, UK
Higher-order chromatin structure of the mammalian
genome and its relationship to gene expression
Mary-Ann Bjornsti, Memphis, USA
Mechanisms of resistance to DNA topoisomerase I
poisons
Doug Easton, Cambridge, UK
Finding susceptibility genes for breast cancer
Francois Fagotto, Montreal, Canada
How are different cell types sorted? Insights from the
frog embryo
Douglas Fearon, Cambridge, UK
A stem cell-like phase of CD8+ T cell clonal expansion?
Ronald Germain, Bethesda, USA
Building a systems level understanding of adaptive
immunity: from molecules to models to movies
Dan Gottschling, Seattle, USA
Loss of heterozygosity in old cells: A model for ageinduced cancer
Sergio Grinstein, Toronto, Canada
Rapid remodeling of the plasma membrane during
phagocytosis
Jeanine Boesen, Petten, The Netherlands
Molecular imaging
Frank Grosveld, Rotterdam, The Netherlands
Transcription factor networks and long range
interactions
Xandra Breakefield, Boston, USA
Following a twisted path: from dystonia to the
endoplasmic reticulum
Per Hall, Stockholm, Sweden
Genetic and environmental factors influence the
biological behaviour of breast cancer
Harmen Bussemaker, New York, USA
Sequence-based modeling of gene regulatory circuitry
Peter Hordijk, Amsterdam, The Netherlands
Subcellular targeting of Rac1 in cell adhesion and
directional motility
Michael Clarke, Stanford, USA
Normal stem cells and cancer stem cells: two sides of
the same coin
Eran Hornstein, Rehovot, Israel
miRNAs in invertebrate development
John Condeelis, New York, USA
Discovery and testing of an invasion signature for
mammary tumors
Sandra Jacob, Basel, Switserland
Structural biology contributions to the discovery of
nilotinib: overcoming imatinib resistance in CML
Vincent Colot, Paris, France
Studying epigenetic variation and its phenotypic impact
in Arabidopsis using epigenomics
Ritsert Jansen, Groningen, The Netherlands
Genetical genomics - a multifactorial perturbation
strategy
162
INVITED SPEAKERS
Peter-Michael Kloetzel, Berlin, Germany
The UPS and immunoproteasomes: Defining a function
to be non-functional?
Geert Kops, Utrecht, The Netherlands
Mitotic checkpoint kinases in chromosomal instability
and cancer
Rik Korswagen, Utrecht, The Netherlands
Genetic dissection of conserved signal transduction
pathways in C. elegans
Eugene Krissinel, Cambridge, UK
Inference of macromolecular assemblies from crystalline
state
Philippe Lambin, Maastricht, The Netherlands
Individualized radiation oncology: the example of non
small cell lung cancer stage III
Meindert Lamers, Berkeley, USA
Evolution of DNA replication: the crystal structure of E.
coli DNA polymerase III
Thomas Look, Boston, USA
Upstream and downstream modifiers of the BCL-2
survival pathway in cancer
Scott Lowe, Cold Spring Harbor, USA
Dissecting the p53 tumor suppressor network
Anastassis Perrakis, Amsterdam, The Netherlands
Developing methods for structural biology
Jacques PouyssГ©gur, Nice, France
Hypoxia signaling, angiogenesis and cancer
John Quackenbush, Boston, USA
Learning biology from gene expression profiles
Peter Ravdin, San Antonio, USA
Individualizing adjuvant therapy: classical methods and
new possibilities;
adjuvant! online and beyond
Eraz Raz, Göttlingen, Germany
Development and guided migration of primordial germ
cells in zebrafish
Dietrich Rebholz-Schuhmann, Cambridge, UK
Towards integration of automated literature analysis into
bioinformatics services
Gunter Reuter, Halle, Germany
Histone demethylation and the control of gene silencing
in Drosophila
Al Reynolds, Nashville, USA
Novel roles for p120-catenin in Rac-dependent inhibition
of Rho
Nicholas Luscombe, Cambridge, UK
Transcription regulation: a genomic network
Jeffrey Rosen, Houston, USA
Stem/progenitor cells in the etiology and treatment of
breast cancer
Hiten Madhani, San Francisco, USA
Gene regulation in Saccharomyces cerevisiae: MAP
kinases, histone variants and beyond
Gunter Reuter, Halle, Germany
Histone demethylation and the control of gene silencing
in Drosophila
Umar Mahmood, Boston, USA
Optical molecular imaging techniques in cancer
research
Michael Sharpe, Edinburgh, UK
Can cancer nurses manage depression in cancer
outpatients? Results of a randomized efficacy trial of
SMaRT oncology depression care
Jan Paul Medema, Leiden, The Netherlands
Apoptotic cell death in cancer
Kim Nasmyth, Oxford, UK
Genome propagation: the chemistry of sister chromatid
cohesion and its dissolution at anaphase
Laurence Pearl, London, UK
Structural biology of the HSP90 molecular chaperone
system
Petra Peeters, Utrecht, The Netherlands
European prospective investigation into cancer and
nutrition: results for breast cancer
Lucas Pelkmans, ZГјrich, Switserland
Towards systems analysis of endocytosis
Dirk SchГјbeler, Basel, Switserland
Epigenetic gene regulation: Insights from chromosomal
maps
Martin Schwartz, Charlottesville, USA
Integrins in signal transduction and
mechanotransduction
Jerry Shay, Dallas, USA
Aging and cancer: are telomeres and telomerase the
connection?
Holger Stark, Gottingen, Germany
3D structure and dynamics of large macromolecular
machines determined by single particle cryo-EM
Bas van Steensel, Amsterdam, The Netherlands
Chromatin genomics
163
INVITED SPEAKERS
John Strouboulis, Rotterdam, The Netherlands
Characterisation of hematopoietic transcription factor
complexes by in vivo biotinylation tagging
Hugues de ThГ©, Paris, France
Modeling PML/RARA leukemogenesis through therapy
response
David Tuveson, Philadelphia, USA
Modeling and manipulating ductal pancreatic cancer in
mice
Geerten Vuister, Nijmegen, The Netherlands
NMR in structural biology: possibilities, limitations and
prospects
Robert Van Waardenburg, Memphis, USA
Distinct functional domains of Ubc9 dictate cell survival
and resistance to genotoxic stress
164
PROJECTS
P R OJ E C T S SU P PORTED
B Y T HE DUTCH CANCER SOCIETY
Number of
project
Title
Div.
Principal
Investigator
Starting
Date
NKI 99-2035
Quantitative assessment of treatment margins and implementation of
IX/XIII
K Gilhuijs
Sept 1999
multimodality imaging in breast conserving therapy
XI
E Rutgers
IX
H Bartelink
Ended
NKI 99-2042
G protein control of cell proliferation and transformation
III
W Moolenaar
Dec 1999
NKI 99-2055
The role of the nuclear inositide pathway in cell cycle progression
III
N Divecha
Dec 1999
NKI 99-2056
Mechanism of cell cycle regulation and control of cellular lifespan by
VII
M Van Lohuizen
May 1999
VII
A Berns
Aug 1999
VII
M Van Lohuizen
Jan 2000
VII
M Van Lohuizen
March 2000
III
W Moolenaar
Sept 2000
mTHPC mediated photodynamic therapy (PDT): how do drug uptake
VIII
F Stewart
Sept 2000
Apr 2006
and distribution influence tumour and normal tissue response
X
P Baas
X
J Schellens
XII
N Aaronson
Dec 2001
Apr 2006
the Polycomb-group and oncogene Bmi1
NKI 99-2058
Development of a mouse model to study the genetic basis of
mesothelioma
NKI 00-2251
Genetic screens to identify new components of the senescence/
immortalization pathway
NKI 00-2253
Functional analysis of BMI1-RING finger interacting proteins and
application of microarray technology to identify BMI1-responsive genes
NKI 00-2258
Cell-cell communication and connexin signalling: suppression of cell
transformation
NKI 00-2282
NKI 01-2382
The impact of prophylactic oophorectormy in women from hereditary
breast/ovarian cancer families on psychosocial health and symptom
NKI 01-2416
H Boonstra
experience
XI
M Van Beurden
Gamma irradiation and the generation of novel peptides for MHC
VI
J Neefjes
Febr 2001
Analysis of vaccine-induced melanoma specific T cell immunity:
IV/X
J Haanen
Oct 2002
a combined human and mouse study
IV
T Schumacher
X
G De Gast
Cancer susceptibility genes that predispose for radiation induced
VIII
A Broeks
breast cancer
XIII
L Van ’t Veer
XII
F Van Leeuwen
class I molecules
NKI 01-2417
NKI 01-2425
NKI 01-2426
Long-term effects of exposure to DES in utero on the risk of hormone
XII
related cancers
M Rookus
Nov 2001
Nov 2001
F Van Leeuwen
T Helmerhorst
UU 01-2438
Control of cellular proliferation in normal and tumor cells by the
V
B Burgering
Sept 2001
P Borst
March 2002
PI3K/PKB/Forkhead pathway
NKI 01-2473
MRP5 and resistance to purine analogs
V
J Wijnholds
Nov 2006
165
Number of
project
Title
Div.
Principal
Investigator
Starting
Date
NKI 01-2474
Characterization of MRP3, a newly identified transporter of anti-cancer
V
P Borst
Febr 2002
VII
A Berns
July 2001
II
T Sixma
Jan 2001
July 2001
PROJECTS
Ended
drug
NKI 01-2476
Identification of the PIM regulatory network by microarray analysis and
high throughput retroviral tagging in compound mutant mice
NKI 01-2479
Structure/function analysis of Muts homologs in HNPCC
NKI 01-2489
Cloning of novel mammary tumor progression and metastasis genes
VI
J Hilkens
VII
A Berns
NKI 01-2562
T cell tolerance and immunity during spontaneous tumor development
IV
P Krimpenfort
Sept 2001
NKI 01-2570
Improvement of tumor response by combined modality treatment: a
IX
M Verheij
Oct 2001
The Insulin-like Growth Factor (IGF) system in breast and colorectal
VIII
D Voskuil
Oct 2001
carcinogenesis: dietary intervention and molecular studies
XIII/VIII
L Van �t Veer
translational approach
NKI 01-2579
E Kampman
NKI 02-2575
NKI 02-2586
Microarray analysis of breast cancer as a diagnostic tool to guide
XIII
M Van de Vijver
optimal treatment
IX
H Bartelink
XIII/VIII
L Van �t Veer
XII
F Van Leeuwen
Pathological and molecular characteristics of tamoxifen-induced
endometrial tumor
NKI 02-2589
Jan 2002
Jun 2002
H Hollema
XIII
P Nederlof
VIII
A Begg
July 2002
Activated proteolysis, a novel rapid response to DNA damage
VI
R Agami
Febr 2002
A functional genomics approach to identify genes which Influence
II
R Bernards
Jan 2002
I
J Collard
Dec 2002
F Van Leeuwen
Aug 2002
Determinants of radiosensitivity in mammalian cells: elucidating
mechanisms using a putative dominant negative to DNA polymerase
beta
NKI 02-2590
NKI 02-2591
tumor cell behavior in vivo
NKI 02-2592
Function-based screening for genes involved in Invasion of epithelial
tumor cells
NKI 02-2593
Regulation of tumor cell motility and invasion by myosin heavy-chain
III
kinase
NKI 02-2605
NKI 02-2634
W Moolenaar
Functional genomics of gene regulation by heterochromatin proteins
VIII
B Van Steensel
May 2002
The identification of oncogenic events collaborating with loss of
V
H Te Riele
Nov 2002
function of the retinoblastoma gene family in tumor development and
progression
NKI 02-2635
The role of BRCA1 and BRCA2 loss-of-function in breast cancer
V
J Jonkers
Jan 2003
NKI 02-2652
Characterisation of a redox regulated PIP3 synthesis pathway
III
N Divecha
Aug 2002
NKI 02-2654
Inclusion of geometric uncertainties in treatment planning for intensity
IX
M Van Herk
July 2002
modulated radiotherapy
J Lebesque
C Schneider
NKI 02-2764
The role of survivin in cell cycle regulation and lymphomagenesis
V
R Medema
S Lens
March 2002
Oct 2006
166
Title
Div.
Principal
Investigator
Starting
Date
NKI 02-2771
Nov 2002
PROJECTS
Number of
project
UU 03-2783
The incidence, nature and etiology of cognitive problems following
XII
S Schagen
chemotherapy for cancer
XII
F Van Dam
Cognitive rehabilitation of glioma patients: A prospective, randomized
XII
study
M Taphoorn
Oct 2003
M Sitskoorn
N Aaronson
UL 03-2804
Ex vivo in situ analyses of Graft versus Host mechanisms by Minor
IV
F Vyth-Dreese
Dec 2003
Role of regulated exocytosis in migration, invasion and metastasis
I
E Roos
Jan 2003
Role of the chemokine SDF-1 and its receptor CXCR4 in carcinoma and
I
E Roos
March 2003
E Danen
March 2003
Histocompatibility antigen specific T cells; relevance for stem cell
transplantation
NKI 03-2854
NKI 03-2856
melanoma metastasis
NKI 03-2858
Cooperation between the Src oncogene and integrin v 3 in oncogenic
I
transformation of epithelial cells
NKI 03-2859
Improving anti-tumor immunity via TNF receptor family member CD27
A Sonnenberg
IV
J Borst
Aug 2003
and its ligand
NKI 03-2860
Feasibility and limitations of T cell receptor gene transfer
IV
T Schumacher
Nov 2003
NKI 03-2861
Peptidases and improving the MHC class I immune response
VI
J Neefjes
March 2003
Characterization of oncogenic potential and mechanism of gene
VII
M Van Lohuizen
Febr 2003
VII
M Van Lohuizen
April 2003
VII
D Peeper
Febr 2003
Compound Mrp2(Abcc2) and other drug transporter knock-out mice to
VIII
A Schinkel
Nov 2003
study determinants of pharmacokinetics, toxicity risks and possible
X
J Beijnen
NKI 03-2933
repression of TBX2
NKI 03-2935
Efficient in vitro genetic screens to identify new oncogenes involved in
tumor progression
NKI 03-2938
Oncogenic function of human DRIL1, a member of the ARID family of
retinoblastoma protein-interacting factors
NKI 03-2940
J Schellens
therapy optimization for anticancer drugs
NKI 03-2943
Optimization of conformal radiotherapy of non-small cell lung cancer
IX
by advanced 3-D planning and multi-modality imaging
E Damen
April 2003
J Lebesque
L Boersma
NKI 03-2960
Isolation and functional analysis of novel genes that act in the
II
R Bernards
Jan 2003
R Agami
Jan 2003
p16INK4A-cyclin D-pRB-E2F pathway
NKI 03-2962
High throughput functional genetic screens for stable loss-of-function
VI
phenotypes in mammalian cells
NKI 03-2963
NKI 03-2964
Studying tumorigenesis using siRNA-expressing vectors
Role of LPA receptor signaling in cell migration, invasion, proliferation
R Bernards
VI
R Agami
II
R Bernards
March 2003
III
W Moolenaar
May 2003
V
R Medema
Jan 2003
and tumorigenesis
NKI 03-2967
The roles of the human cyclin-dependent kinase subunits HsCks1 and
HsCks2 in mitotic progression
Ended
167
Number of
project
Title
Div.
Principal
Investigator
Starting
Date
NKI 03-2977
Psychological and behavioral issues in cancer genetics
XII
E Bleiker
Nov 2003
N Aaronson
NKI 04-2987
Long-term psychosocial impact of genetic testing among Familial
XII
E Bleiker
Adenomatous Polyposis (FAP) families
XII
N Aaronson
Nov 2004
H Vasen
NKI 04-3062
The role of Tiam1 and Rac in Wnt/beta-catenin-induced tumorigenicity
I
J Collard
Aug 2004
NKI 04-3063
Structure/function analysis of the role of polo-like kinase-1 in cellular
V
R Medema
April 2004
transformation
II
A Perrakis
Function of integrin-CD151 complexes in tumor invasion and
I
A Sonnenberg
Nov 2004
Nov 2004
NKI 04-3067
metastasis
NKI 04-3068
NKI 04-3069
Long-term risk of second cancers and cardiovascular disease following
XII
F Van Leeuwen
treatment of Hodgkin’s disease
IX
B Aleman
Molecular bar code screens to identify genetic interactions with protein
II
R Bernards
Jan 2004
VI
J Neefjes
Jan 2004
IV
J Borst
May 2005
kinases in mammalian cells
NKI 04-3078
On the molecular mechanisms of cross-presentation of primimg for an
efficient anti-tumor immune response
NKI 04-3079
Tumor therapy with ionizing radiation and death ligand Trail: efficacy
and key molecular determinants
NKI 04-3080
The role of RanBP3 in beta-catenin dependent nuclear signalling and
M Verheij
VI
M Fornerod
Sept 2004
Application of Pathology-correlated Imaging to Improve the Accuracy of
IX/XIII
K Gilhuijs
Oct 2004
Surgery and Radiotherapy in Breast-conserving Therapy
XI
E Rutgers
IX
H Bartelink
Subtle gene modification to study the role of the mismatch repair
V
H Te Riele
complex MSH2/MSH6 in mutation avoidance and tumor suppression
II
T Sixma
Dissecting the role of the pp21 INK4a genes in development and tumor
VII
P Krimpenfort
oncogenesis
NKI 04-3082
NKI 04-3084
NKI 04-3086
suppression in mouse models
NKI 04-3087
Manipulation of the CD70 locus to monitor and modulate the anti-
Nov 2004
Dec 2004
A Berns
IV
J Borst
Febr 2004
Sept 2004
tumor immune response and tumor development
NKI 04-3088
NKI 05-3209
NKI 05-3370
NKI 05-3373
Gene-environment interactions in BRCA1/2 breast/ovarian cancer
XII
F Van Leeuwen
families, a project of the Netherlands Collaborative Group on
XII
M Rookus
Hereditary Breast Cancer
XIII
L Van �t Veer
Psychosocial aspects of genetic testing in families at high risk of
XII
E Bleiker
multiple tumors at various sites and ages.
XII
N Aaronson
VII
S Verhoef
The role of TGFb and Notch signaling in the development of radiation-
VIII
F Stewart
induced telangiectasia
IX
N Russell
Radiation-induced atherosclerosis: mechanisms and preventive
VIII
F Stewart
intervention strategies
NKI 05-3375
The role of PRAME in oncogenesis
Nov 2005
April 2005
June 2005
M Daemen
IX
H Bartelink
II
R Bernards
May 2005
PROJECTS
Ended
168
PROJECTS
Number of
project
Title
Div.
Principal
Investigator
Starting
Date
NKI 05-3377
Lipid rafts as novel targets for anti-cancer therapy and induction of
III
W Van Blitterwijk
Jan 2005
apoptosis
IX
M Verheij
High precision radiotherapy in the presence of anatomical changes
IX
J-J Sonke
NKI 05-3378
Nov 2005
M van Herk
C Rasch
NKI 05-3379
A study of drug resistance mechanisms using large scale RNA
II
interference screens
P Borst
June 2005
R Bernards
R Beijersbergen
NKI 05-3387
Identification and characterization of human tumor suppressor genes
VI
in the p53 oathway
NKI 05-3388
Molecular basis of resistance to anti-estrogens in estrogen-receptor
VI
positive breast cancer
NKI 05-3391
TPPII and the role in antigen presentation by MHC Class I molecules
M Voorhoeve
Sept 2005
R Agami
R Michalides
Jan 2005
J Neefjes
VI
J Neefjes
Nov 2005
E Reits
NKI 05-3420
NKI 05-3421
NKI 06-3530
Expression profiling to predict outcome after combined radiotherapy
XI
M Van den Brekel
and chemotherapy in head and neck tumors
VIII
A Begg
IX
C Rasch
X
J Beijnen
XIII
W Bonfrer
X
J Schellens
IV
T Schumache
Clinical proteomics in breast cancer
Generation of human tumor specific T cells with high affinity T cell
March 2005
July 2005
receptors
NKI 06-3545
High Image guided correction strategies to optimize the precision of
IX
radiotherapy
JJ Sonke
Aug 2006
JV Lebesque
M Van Herk
NKI 06-3558
Genetic screens to identify cancer related functions of human miRNAs
VI
NKI 2006-3566
Mechanisms of chemotherapy resistance in spontaneous tumors of
V
genetically modified mice
NKI 2006-3589
Significance of the Fanconi anemia caretaker pathway in development
R Agami
Nov 2006
P Borst
J Jonkers
V
H Te Riele
X
S Linn
and treatment of cancer
NKI 06-3706
Towards patient-tailored systemic therapy in breast cancer: a combined
approach of translation research and mouse model systems
NKI 06-3714
Tumor metastasis: the role of acto-myosin contraction in the
I
downregulation of E-cadherinmediated cell-cell adhesion
J De Rooij
A Sonnenberg
VU 04-3046
Second primary tumors in hereditary retinoblastoma patients
XII
F Van Leeuwen
RUG 04-3157
Cardiovascular morbidity in testicular cancer survivors: case-control
XII
F Van Leeuwen
study of risk factors and assessment of pharmacogenomic
determinants of toxicity
Dec 2006
Ended
169
PROJECTS
M A J OR PR OJECTS
SUPPOR T ED B Y OTHER OR G A N IZ ATION S
Granting agency project
number
Title
Div.
Principal
investigator
AICR
Cytoskeletal regulation of cell-cell adhesion in oncogenic transformation
I
J De Rooij
EU-LSHC-CT-2004-503224
BRECOSM
I
J Collard
NWO/ALW 815-02-002
Analysis of the function of a novel nuclear envelope protein, nesprin-3, in anchoring
I
A Sonnenberg
A Sonnenberg
the nucleus to the intermediate filament system
DEBRA (UK)
Generation of a mouse model for EBS-MD
I
NWO/NW TOP
Molecular mechanism of renal magnesium wasting
I
40-00812-98-06-110
R Bindels
K Jalink
N Knoers
NWO CW 98016
The Process of transposition: three-dimensional structure analysis of Tc1 and Tc3
II
T Sixma
R Bernards
transposase from Caenorhabditis elegans
NWO MW 901-02-191
Genetic screens to identify novel cancer-relevant genes
II
EU QLRT-2001-00988
Structural proteomics in Europe (SPINE)
II
T Sixma
A Perrakis
NWO-CW PIO.01.09-
Structural analysis of the mechanism of ubiquitin conjugation, a generalized cellular
2002/01690
addressing system that controls protein and DNA stability
NWO-CW
Structural and functional analysis of the human L1/Alu retrotransposition machinery
II
T Sixma
II
A Perrakis
700.51.012-2002/03653
O Weichenrieder
NIH
Automatic model building and refinement in crystallography
II
A Perrakis
NWO
Identification of cancer-relevant genes using a functional genetic approach
II
R Bernards
NWO
Identification of cancer-relevant genes using high-hroughput loss-of-function genetic
II
R Bernards
Cancer Genomics
screens in mammalian cells.
Center for Biomedical
Genetics
Consortium
EU-FP6-LSHC-CT-2003-
Identification of novel targets for cancer therapy (INTACT)
506803
EU-FP6-LSCH-2002-503438
Translational and functional onco-genomics: from cancer-oriented genomic
II
R Bernards
VII
M Van Lohuizen
VII
A Berns
II
R Bernards
scrfeenings to new diagnostic tools and improved cancer treatment (TRANSFOG)
N I H-1 P50 CA112970-01
System-based predications of responses to cancer therapy
II
R Bernards
NWO-MW
Spinozapremie
II
R Bernards
EU-LSGH-2005-018683
The Role of Ubiquitin and Ubiquitin-like Modifiers in Cellular Regulation (RUBICON)
II
R Bernards
T Sixma
170
PROJECTS
Granting agency project Title
number
Div.
Principal
investigator
NWO CW VENI
Structural and functional characterization of DNA mismatch repair protein complexes
II
J Lebbink
STW BBC.6035
Exploring new uses of ChhBP as model for ligand-gated ion channel research
II
T Sixma
EU-MRTN-CT-2006-03455
Signal Transduction by Ubiquitination, a Matter of Location (UbiRegulators)
II
T Sixma
CBG
Center for Biomedical Genetics Participation in Top-Research School
II
T Sixma
700.53.407
R Bernards
EU-MTKI-CT-2005-029655
NWO CS VIDI 700.54.427
Breast Cancer Biomarkers and Functional Mediators: Harnessing the New Wealth of
II
R Bernards
-Omic Data (TARGET-BREAST)
VIII
L Van �t Veer
L1 and Alu elements: Structural and functional analysis of human non-LTR
II
O Weichenrieder
II
A Perrakis
II
A Perrakis
retrotransposons
EU-FP6-LSHG-CT-2003-
Biocrystallography (X) on a highly integrated technology platform for European
503420
structural genomics (BIOXHIT)
EU-FW6-LSHG-CT-2004-
A multidisciplinary approach to determine the structures of protein complexes in a
512028
model organism (3D-Repertoire)
MAX-INF EU Network
European Macromolecular Crystallography infrastructure Network, 2004-2008
II
A Perrakis
EMBO Fellowship
The structural and functional basis for coupling of ligand activation to channel opening
II
C Ulens
T Sixma
in the nicotinic acetylcholine receptor
CBG
Lysophospholipid receptor signaling
III
W Moolenaar
EU HPMF-CT2001-01218
Stress mediated regulation of phosphositides
III
N Divecha
The Leukemia and
A novel form of gene regulation that provides specificity by preventing promiscuity
III
F Van Leeuwen
Lymphoma Society
((Special Fellow 3409-04)
NWO-ALW-VIDI
The role of histone methylation in silencing and transcriptional memory
III
F Van Leeuwen
European TMR
(864-04-003)
EU-FP6-LSGH-CT-2004-
NET member of the Epigenome Network of Excellence (EPIGENOME)
503433
III
F Van Leeuwen
VII
B Van Steensel
VII
M Van Lohuizen
NWO
Modulation of autotaxin activity by small molecules
III
H Ovaa
NWO
Chemical biology of ubiquitin and ubiquitin-like modifications
III
H Ovaa
ZON-MW 912-04-32
The role of CD27/CD70-regulated cell survival in shaping effector and memory T cell
IV
J Borst
responses
ZON-MW VIDI 917-056-328
Role of translesion DNA synthesis in immunity and cancer development
IV
H Jacobs
EU-FP6-LSCH-CT-2005-
Adoptive engineered T-cell targeting to activate cancer killing (ATTACK)
IV
T Schumacher
018914
171
Granting agency project
number
Title
Div.
PROJECTS
Principal
investigator
EU-FP6-2005-
Determining the relationships between haematopoietic lineages using genetic
IV
T Schumacher
Mobility-7-039477
barcoding (Genetic Barcoding)
IV
S Naik
Treatment of HPV positive penile cancer by introduction of HPV E7-specific T cell
IV
J Haanen
ZON-MW 432-00-001
immunity thourgh DNA vaccination
T Schumacher
XI
S Horenblas
LSBR
MHC multimer-based adoptive T cell therapy
IV
T Schumacher
TI-Pharma project
TNF ligands in cancer
IV
J Borst
UVA 2006-3606
Integrated analyses of melanoma-T cell interactions in situ; relevance for
IV
F Vyth-Dreese
immunotherapy
NWO-MW Program
Phosphoinositide 3-kinase signaling: Regulation and function
V
901-28-092
NWO MW Vidi 917.36.347
B Burgering
P Coffer
Mammary tumorigenesis in conditional tumor suppressor gene knockout mice:
V
J Jonkers
V
H Te Riele
VI
R Agami
V
L Wessels
disease progression, gene discovery and cellular pathways
EU FP6 -LSCH-CT-2004-
Mutant p53 as target for improved cancer treatment (MUTP53)
502983
NGI-BioRange SP 1.3.2
Developing clinical predictors based on high-dimensional genomics data, pathway
information and directed experimentation
NGI-BioRange SP 1.2.1
A unifying ramework for data-driven pathway discovery
V
L Wessels
NGI-NWO-Horizon
Discovering interactions and functional groups of oncogenic lesions
V
L Wessels
Subtle is the oligo: in vitro and in vivo gene modification by single-stranded DNA
V
Breakthrough Project
050-71-045
Horizon Project 050-71-051
oligonucleotides
ZonMW 912-02-073
The pathofysiological relevance of transporters in the enterohepatic cycle
V
(2002-2006)
R Oude Elferink
P Borst
Susan G. Komen Breast
Identification of collaborating mutations in Brca1 and Brca2 mouse mammary tumors
Cancer Foundation BCTR
by genome-wide screening for chromosomal imbalances
V
J Jonkers
V
P Derksen
V
C Wielders
0403230
NWO-ZonMw Veni
Functional characterisation of E-cadherin signalling in breast cancer progression and
016.056.135
metastasis
Nationaal Regie-Orgaan
Enzymatic production of low-complexity RNAi libraries for high throughput loss-of-
Genomics, Horizon
function screening
Doorbraakproject 050-71-348
UCSF
Prion protein localization
VI
P Peters
NWO 901-02-250
Role of the nuclear pore complex in b type catenin nuclear signalling
VI
M Fornerod
NWO-ALW 811-38-001
Identification of specific transport pathways through the nuclear pore complex using in
VI
M Fornerod
vitro nuclear reconstitution
172
PROJECTS
Granting agency project Title
number
Div.
Principal
investigator
NWO-CW 700.51.013-
Function and structure characterisation of the RILP/RAB7 complex controlling
VI
J Neefjes
2002/02696
lysosomal transport
II
A Perrakis
NWO ZON MW PGS
Manipulation of the phagosomal pathway induced by salmonella and mycobacteria
VI
J Neefjes
912-03-026
and the host immune response
HPMF-CT-2002-02146
Investigating the role of proteins associated with the nuclear pore complex in
Marie Curie Individual
regulating gene expression and DNA double stranded break repair
T Ottenhoff
VI
M Fornerod
CD1 trafficking and loading of Mycobacterium leprae lipids in maturing dendritic cells
VI
P Peters
EU-FP5- QLK2-CT-
Pre-clinical improvement of combined immunotherapy and chemotherapy for new
VI
P Peters
2002-81628
variant Creutzfeld-Jacob disease
EU-FP5- QLK5-CT-
Passage from intestine to brain: assessing the role of dendritic cells in capturing,
VI
P Peters
2002-01044
expanding and disseminating prions
EU–FP6-LSHB-
Development of a pre-clinical blood test for prion diseases (ANTEPRION)
VI
P Peters
Immunological and structural studies of prion diversity (IMMUNOPRION)
VI
P Peters
EU–FP6-FOOD-
Understanding prion strains and species barriers and devising novel diagnostic
VI
P Peters
CT-2006-023183
approaches (STRAINBARRIER)
R01 GM58615 National
Biogenesis of transport vesicles coated by COPI
VI
P Peters
Genome wide search for DNA damage checkpoint functions in human cells
VI
R Agami
VI
R Agami
fellowship
Dutch Leprosy Foundation
702.02.20
CT2006-019090
EU–FP6-FOODCT-2006-023144
Institutes of Health Hsu
ESF NWO EURYI-award
044.016.001
E Zlotorynski
EU-MEIF-CT-2005-514788
Identification and characterization of novel genes involved in DNA damage-response
VI
pathways in humans (DNA damage RNAi)
E Zlotorynski
R Agami
NIH 2 R01 AI028973-17
Pathways of antigen presentation by CD1 a, b and c
VI
P Peters
EU-MRTN-CT-2004-512385
High throughput development of drugs for immunotherapy of autoimmune diseases
VI
J Neefjes
(Drugs for therapy)
EU-MRTN-CT-2003-504227
Microban
VI
J Neefjes
EU-FP6-2005-EIF 041382
The role of the intracellular peptidases in the immune system and in the cellular
VI
J Neefjes
VII
M Van Lohuizen
metabolism (PROTEUS)
NWO Pionier 906-99-001
Mechanism of gene repression by mammalian Polycomb-group proteins: connections
to cell cycle regulation and other silencing processes
EU QLG1-1999 00622
Inducible Melanoma Model
VII
A Berns
EU 5th framework
Molecular mechanisma of senescence and ageing
VII
M Van Lohuizen
QLK6-CT-2001-00616
173
Granting agency project
number
Title
Div.
PROJECTS
Principal
investigator
NWO genomics program
Genome-wide mapping of oncogenic pathways by high-throughput insertional
VII
A Berns
2002, 050-10-008
mutagenesis
NWO/Vidi 864-02-005
Novel functional genomic screens to identify pathways that protect mouse and human
M Van Lohuizen
VII
D Peeper
Identification of novel targets for cancer therapy (MUGEN)
VII
A Berns
Whole-genome analysis of the regulation of chromatin structure and gene expression
VII
B Van Steensel
BSIK program: 03038
Stem cells in development and disease
VII
M Van Lohuizen
Hersenstichting Nederland:
Onderzoek naar de rol van het Bmi1 Polycomb groep gen in het reguleren van neurale
VII
M Van Lohuizen
12F04(2).14
stamcel identiteit
Centre of Biomedical
Participation in Top-Research School together with Universities of Utrecht, Leiden,
VII
A Berns
Genetics
Rotterdam and Hubrecht Laboratory
NOW R.O.
Cancer Genomic Centre
cells against oncogenic transformation by mutant RAS
EU Network of Excellence.
LSHG-CT-2005-005203
European Young
Investigator Award
M Van Lohuizen
VII
A Berns
M Van Lohuizen
EU-LSGH-CT-2006-018739
Platforms for biomedical discovery with human ES cells (ESTOOLS)
VII
M Van Lohuizen
NWO-Genomics Program
Chromatin profiling of human gene silencing complexes
VIII
B Van Steensel
DIRF: An in vivo screen to identify proteins that bind to a genomic region of interest
VIII
B Van Steensel
Multi-gene chromatin domains
VIII
B Van Steensel
Human Frontier Science
Whole-genome analysis of the interplay between chromatin context and gene
VIII
Program RGP56/2003
expression control
050-10-002
Netherlands Genomic
Initiative 050-71-026
Netherlands Genomic
Initiative 050-71-048
B Van Steensel
H Bussemaker
K White
STW 2004-2008
Generation and validation of cytochrome P450 3A knockout and transgenic mice as
VIII
A Schinkel
(BFA.6165)
tools for improved drug development and research
X
J Schellens
J Beijnen
J Smit
EU-LSCH-CT-2005-018911
Detections of minimal tumor cell admixture in patients with breast cancer (DISMAL)
VIII
L Van �t Veer
EU-LHC-CT-2004-503426
Translating molecular knowledge into early breast cancer management: building on the
VII
L Van ’t Veer
Breast International Group network BIG for improved treatment tailoring (TRANSBIG)
XI
E Rutgers
EU-LSGH-CT-2004-505785
BioCare
IX
H Bartelink
EU-015997
EUROCAN + PLUS
EU DGXII
IX
H Bartelink
VII
A Berns
A code of practice for dosimetry of boron neutron capture therapy (BNCT) in Europe
IX
B Mijnheer
Application of an electronic portal imaging device for dosimetry in radiotherapy
IX
B Mijnheer
SMT4-CT98-2145
STW BGN 33-3240
174
PROJECTS
Granting agency project
number
Title
Div.
Principal
investigator
STW BGN 33-3240
Portal image analysis
IX
M Van Herk
Pfizer and Roche
TEAM II: A randomized, multicentre, prospective phase III trial investigating: a.
X
Neoadjuvant hormonal therapy with exemestane for three versus six months and/or b.
S Linn
C Van de Velde
The efficacy and safety of the addition of ibandronate to adjuvant hormonal therapy in
postmenopauzal women with hormone receptor positive early breast cancer
NWO 612-066-201
Learning and using understandable prognostic models in medicine
X
P Lucas
B Taal
NCI PO1 CA29605-12
Multicenter selective lymphadenectomy trial
XI
O Nieweg
Atos Medical
Development and evaluation of a third generation ProvoxTM voice prosthesis
XI
F Hilgers
I Tan
A Ackerstaff
Erasmus MC, 3M company
Clinical, virological and immunological studies of imiquimod in VIN
XI
M Van Beurde
T Helmerhorst
Michel Keijzer Foundation
Airway pathophysiology after total laryngectomy and the influence of heat and
XI
F Hilgers
XI
F Hilgers
moisture exchangers
Breuning-ten Cate
Intelligibility of tracheoesophageal speech
Foundation
L Pols
International Health
IVF treatment, unexplained subfertility and number of retrieved oocytes in relation to
Foundation
age at menopause
IARC
International BRCA1/2 Carrier Cohort Study
XII
F Van Leeuwen
Nefkens Foundation
Effects of chemotherapy on cognitive skills and evoked potentials in women receiving
XII
F Van Dam
adjuvant treatment for breast cancer
X
W Boogerd
An international field study of the reliability and validity of the EORTC QLQ-C30 and a
XII
N Aaronson
EORTC 15011-30011
XII
F Van Leeuwen
C Burger
disease-specific questionnaire module (QLQ-PR25) for assessing the quality of life of
patients with prostate cancer
Lance Armstrong
Long term risk of second cancers and cardiovascular disease following treatment of
XII
F Van Leeuwen
Foundation
testicular cancer
IX
B Aleman
Pharmacia BV MO2TEA
Assessment of neuropsychological sequelae of tamoxifen and exemestane in
XII
postmenopausal women with early breast cancer
Medical Centre Alkmaar
The use of health related quality of life assessments in daily clinical oncology nursing
S Schagen
F Van Dam
XII
N Aaronson
XII
N Aaronson
XII
N Aaronson
XII
F Van Leeuwen
practice: A community hospital based intervention study
Organon
Hormonal substitution after prophylactic adnectomy in women with an increased risk
of breast and ovarian cancer due to a genetic predisposition: HIRISHE (High-risk
women and hormonal substitution exposure)
University Hospital Zurich
Physical exercise to improve health related quality of life in peripheral stem cell and
(UHZ)
bone marrow transplantation receipts
Cancer Genomics Center
The benefits and risks of cancer genomics for society
N Aaronson
Granting agency project
number
Title
Clinical Research Foundation Effects of chemotherapy on neurogenesis and learning abilities in experimental animal
/ University of Groningen
research
EU-2004-012926
Radiation exposure at an early age: Impact of genotype on breast cancer risk (GENE
Div.
XII
175
PROJECTS
Principal
investigator
S Schagen
F Van Dam
XII
F Van Leeuwen
RAD RISK)
EU QLG1-CT-2002-30242
PCTCG overview
Biom
O Dalesio
EU eTEN program- C510736
Trans European Network for patient randomization in clinical trials
Biom
O Dalesio
E Van der Donk
CKTO 2006-02
NVALT-Oncology
A multicenter randomized phase III trial of neo-adjuvant chemotherapy followed by
M Verheij
surgery and chemotherapy or by surgery and chemoradiotherapy in respectable gastric
C Van de Velde
cance
A Cats
Statistical Center NVALT Clinical Trials in oncology
Biom
O Dalesio
176
PERSONNEL INDEX
PE R S O NNEL INDEX
Aaronson, Neil 124
Aarts, Marieke 49
Achachah, Mohamed 133
Achanta, Geetha 62
Ackerstaff, Annemieke 114
Adriaansz, Sandra 103
Agami, Reuven 62
Albers, Harald 38
Alderden, Carolien 103
Alderliesten, Tanja 133
Aleman, Berthe 88, 128
Alendar, Andrej 71
Antonini, Ninja 143
Arens, Ramon 43
Armstrong, Nicola 58
Atsma, Douwe 132
Axwijk, Priscilla 133
Baank, Saskia 132
Baars, Danny 143
Baars, Joke 102
Baars, Philippe 132
Baas, Paul 80, 102
Baas–Vrancken Peeters, Marie-Jeanne 114
Bais, Evert 102
Bakker, Arne 43
Bakker, Martine 132
Bakker, Sietske 49
Ballast, Nienke 131
Balm, Alfons 114
Bardina, Elena 49
Bartelink, Harry 86, 88
Beaudry, Jean Bernard 69
Beers-Bauhuis, Carolien 132
Begg, Adrian 78
Beijersbergen, Roderick 26
Beijnen, Jos 82, 102
Belderbos, Jose 88
Ben Jelloul, Marouane 30
Bergman, AndrГ© 71
Berkers, Celia 38
Bernad-Fernandez, Rafael 64
Bernards, RenГ© 24
Berns, Anton 71
Berns, Katrien 24
Besnard, Peter 133
Besseling, Gijs 128
Betgen, Anja 88
Bex, Axel 115
Bijker, Nina 88
Bin Ali, Rahmen 71
Bins, Adriaan 45
Bisschops, Ivo 128
Bleiker, Eveline 124
Bloemers, Monique 88
Blom, Marleen 69
Boekhout, Annelies 103
Boer, Mandy 75
Boerhout, Hermien 53
Boerrigter-Barendsen, Lucie 132
Bohoslavsky, Roman 143
Bolijn, Marije 82
Bonfrer, Hans 132
Boogerd, Willem 102, 126
Boot, Henk 102
Boren, Joan 71
Borlado, Luis 24
Borst, Gerben 88
Borst, Jannie 41
Borst, Piet 51
Bos, Erik 67
Bosma, Astrid 84
Boss, David 102
Boutmy-de Lange, Majella 133
Bouwman, Peter 53
Braaf, Linde 84
Braat, Koen 69
Brand, Bob 88
Braunschweig, Ulrich 56
Broeks, Annegien 84
Brohet, Richard 128
Bronk, Marieke 133
Brouwers, Elke 102
Bruggeman, Sophia 69
Bruin, Natascha 132
Bruin, Sjoerd 84
Bruinvis, Iain 88
Buchwald, Gretel 28
Buckle, Tessa 132
Bueno de Mesquita, Jolien 55, 131, 132
Buikhuisen, Wieneke 102
Buitelaar, Dick 115
Bultsma, Yvette 36
Buning, Marian 132
Burg, Danny 38
Burger, MatthГ© 115
Burgers, Sjaak 102
Calbo, Joaquim 71
Calogero, Anna 43
Cardous-Ubbink, Mathilde 128
Carreno, Monique 143
Caspers, Anky 126
Cats, Annemieke 102
Cavalli, Silvia 38
Celie, Patrick 30
Chang, Qing 62
Christodoulou, Evangelos 30
Citterio, Elisabetta 69
Coccoris, Miriam 43
Cohen, Serge 30
Collard, John 19
Coppen, Vincent 143
Copper, Marcel 114
Dahmen, Rutger 131
Dalesio, Otilia 143
Damen, Carola 102
Damen, EugenГЁ 88
Danhof, Tinneke 126
Dannenberg, Jan Hermen 71
De Boer, Jan Paul 102
De Boer, Roel 88
De Boer, Suzanne 143
De Bois, Josine 88
De Bruin, Marieke 128
De Bruin, Michiel 55
De Gast, Bert 102
De Groot, Renate 84
De Haan, Patricia 88
De Haas, Marcel 51
De Jager, Steven 128
De Jong, Daphne 132
De Leeuw-Mantel, Geri 128
De Marco, Valeria 30
De Ridder, Jeroen 58
De Rooij, Johan 17
De Visser, Karin 53
De Vries, Diederick 30
De Vries, Evert 41
De Vries, Hilda 71
De Vries, Remco 115
De Vries, Sandra 49
De Waal, Marjolijn 143
De Widt, John 34
De Wit, Elzo 56
De Witte, Moniek 43
De Wolf, Cocky 51
Deenen, Maarten 82, 102
Dekker, Marleen 49
Dellemijn, Trees 45
Delzenne-Goette, Elly 49
Derks, DaniГ«lle 115
Derksen, Patrick 53
Desmet, Christophe 73
Dewit, Luc 88
Dijkhuis, Simone 128
Dijkstra, Jitske 143
Dirac, Annette 24
Divecha, Nullin 36
Doodeman, Petra 132
Doodeman, Valerie 103
Douma, Kirsten 124, 131
Douma, Sirith 73
Doumont, Gilles 53
Droogendijk, Helga 102
Drost, Brigit 115
Drost, Jarno 62
Drost, Rinske 53
Dubbelman, Ria 103
Duppen, Joop 88
Duursma, Anja 62
Ebbens, Fenna 114
Egan, David 26
Eggens, Elke 126
Eichhorn, Pieter 24
Ekhart, Corine 102
177
PERSONNEL INDEX
Ellenbroek, Saskia 19
Elouarrat, Dalila 36
Emanuel, Miret 133
Engelsma, Dieuwke 64
Engwegen, Judith 102
Epping, Mirjam 24
Erven-Dufournij, Brigitte 143
Evers, Bastiaan 53
Faber, Alex 40
Fabius, Armida 26
Faesen, Alex 28
Feddema, Wouter 51
Feenstra, Christel 132
Fish, Alex 28
Fles, Renske 84
Floot, Ben 78, 80
Foekema, Joke 103
Foijer, Floris 49
Fons, Guus 115
Fornerod, Maarten 64
Frankfort, Suzanne 102
Frederiks, Floor 40
Frenay, Michel 88
Gaasbeek, Ruben 71
Gadiot, Jules 71
Gast, Marie-Christine 102
Gebretensae, Abadi 103
Gehring, Karin 124
Geiger, Thomas 73
Genest, Paul-AndrГ© 51
Gerard, Audrey 19
Gerlach, Carmen 43
Gerritsma, Miranda 124
Geurts, Tom 114
Geutjes, Ernst-Jan 24
Gilhuijs, Kenneth 133
Godsave, Sue 67
Gomez, Raquel 45
Gonggrijp, Steven 114
Greil, Frauke 56
Griekspoor, Alexander 60
Groothuis, Tom 60
Guelen, Lars 56
Gundy, Chad 124, 126
Gunnarsdottir, Sjofn 51
Haanen, John 45, 102
Haas, Rick 88
Haenen, Inge 131
Hage, Joris 115
Hajdo-Milasinovic, Amra 19
Halfwerk, Hans 86
Halstead, Jonathon 36
Hannemann, Juliane 86
Haramis, Anna Pavlina 69
Hart, Guus 88
Hau, Song-Hieng 143
Hauptmann, Michael 58
Heemsbergen, Wilma 88
Heessen, Stijn 64
Heideman, Marinus 47
Heijens, Claudia 102
Helgason, Helgi 102
Hendriksen, Jolita 64
Hengeveld, Trudi 32
Hermus, Marie-Christine 132
Hibbert, Rick 28
Hiemstra, Annelies 143
Hijmans, Marielle 24
Hilarius, Doranne 124
Hilge, Mark 30
Hilgers, Frans 114
Hilkens, John 75
Hilkmann, Henk 38
Hillebrand, Michel 103
Hoebers, Frank 78, 88
Hoefnagel, Cornelis 132
Hofland, Ingrid 78
Hofstede, Diederik 115
Hogan, Greg 56
Hogervorst, Frans 84, 132, 133
Holstege, Henne 53
Holtkamp, Marjo 103
Hömig, Cornelia 73
Hoogendoorn, Lisette 128
Hooning, Maartje 128
Hoopman, Rianne 124
Hoppes, Rieuwert 38
Horenblas, Simon 115
Horlings, Hugo 86
Houben, Anna 32
Houben, Diane 67
Hoving, Saske 80
Huang, Sidong 24
Huisman, Bregje 128
Huitema, Alwin 102
Huitink, Hans 115
Hulsman, Danielle 69
Hundscheid, Stephanie 86
Huseinovic, Angelina 30
IJpenberg, Annemieke 71
Jacobs, Heinz 47
Jacobs, Jacqueline 71
Jager, Agnes 102
Jalink, Kees 23
Jansen, Edwin 88
Jansen, Hans 67
Jansen, Robert 102
Janssen, Esther 124, 128
Janssen, Lennert 60
Jeanson, Kiki 128
Joling, Jeanine 43
Jones, David 36
Jongmans, Petra 114
Jongsma, Johan 71
Jonkers, Jos 53
Joosse, Simon 84
Joosten, Krista 30
Jörger, Markus 102
Jorritsma, Annelies 45
Kaas, Reinie 114
Kalverda, Bernike 64
Kappers, Ingrid 114
Karsenberg, Kim 128, 131
Kartachova, Marina 132
Kassiem, Mobien 30
Kedde, Martijn 62
Keessen, Marianne 103
Keijzer, Christiaan 115
Keizer, Ron 102
Keller, Anna 41
Kerkdijk, Desiree 114
Kersbergen, Ariena 51
Kerst, Martijn 102
Ketema, Mirjam 17
Keune, Willem-Jan 36
Kim, Yeung-Hyen 45
Kimmings, Nikola 114
Klarenbeek, Jeffrey 34
Klein Zeggelink, William 133
Klerkx, Edwin 143
Klokman, Willem 128
Klomp, Houke 114
Klop, Martin 114
Klous, Marjolein 102
Kluijt, Irma 133
Knegjens, Joost 88
Knipscheer, Puck 28
Knol, Remco 132
Knols, Ruud 124
Kok, Marleen 55, 84
Kool, Jaap 69
Koolen, Stijn 102
Koopman-Kroon, Ciska 103
Koops, Wim 133
Koorn, Jenneke 143
Korse, Tiny 124, 132
Kortekaas, Bettina 143
Kortlever, Roderik 24
Kreft, Maaike 17
Kregel, Eva 53
Kreike, Bas 86, 88
Krenn, Bea 67
Kreukels, Baudewijntje 126
Krijger, Peter 47
Krimpenfort, Paul 71
Kristel, Petra 86
Kröger, Robert 133
Kroon, Bin BR 114
Kroon, Frans 114
Kruse, Jacqueline 80
Kuenen, Marianne 128
Kuijl, Coen 60
Kuiken, Johan 26
Kuikman, Ingrid 17
Kuilman, Thomas 73
Kuiper, Maria 103
Kujala, Pekka 67
Lafeber, Albert 132
Lagas, Jurjen 76
Lai, Carmen 58
178
PERSONNEL INDEX
Lambooij, Jan Paul 71
Lammens, Chantal 124
Langerak, Petra 47
Langeslag, Michiel 23
Le Sage, Carlos 62
Lebbink, Joyce 28
Lebesque, Joos 88
Leering, Suzanne 126
Legdeur, Monica 128
Leijte, Joost 115
Linders, DorothГ© 132
Linn, Sabine 55, 84, 102
Lippuner, Christoph 60
Littler, Dene 30
Liu, Xiaoling 53
Lohuis, Peter 114
Loo, Claudette 133
Los, Alrik 34, 36
Lotz, Carina 45
Lubsen–Brandsma, Lotti 115
Lukas, Anne 102
Maas, Chiel 41
Madalinska, Joanna 124
Madiredjo, Mandy 24
Mahn-Schaefers, Marianne 143
Mallo, Henk 103
Manders, Peggy 128
Mandjes, Ingrid 143
Mandjes, Miranda 58
Marchetti, Serena 82
Margadant, Coert 17
Marijnen, Corrie 88
Marsman, Marije 60
Masselink, Harry 88
Masztalerz, Agnieszka 21
Mattiroli, Francesca 28
McDermott, Leah 88
Meijer, Joost 21
Meijerman, Irma 82
Meijnen, Philip 114
Meindertsma, Tineke 143
Meinhardt, Wim 115
Menendez, Victoria 60
Mertens, Sander 19
Meuleman, Wouter 58
Michalides, Rob 66
Michaloglou, Chrysiis 73
Middendorp, Sabine 41
Mijnheer, Ben 88
Minderhoud, Tom 89
Modder, Carla 143
Molloy, Tim 84
Monserrat, Veronica 60
Mooij, Thea 128
Mook, Stella 84, 132
Moolenaar, Wouter 32
Moonen, Luc 88
Moorman, Celine 56
Mulder, Ina 128
Mulder, Lennart 86
Mullenders, Jasper 24
Muller, Saar 132, 133
Muussen, Rick 143
Nacerdine, Karim 69
Nagel, Remco 62
Nagtegaal, Tanja 124
Nair, Suresh 62
Nan-Offeringa, Lianda 103
Nawijn, Martijn 71
Nederlof, Petra 84, 128, 132, 133
Neefjes, Jacques 60
Neering, Herman 102
Neijenhuis, Sari 78
Neijssen, Joost 60
Nieweg, Omgo 114
Nijkamp, Wouter 26
Nijwening, Jeroen 26
Noback, Sonja 69
Nol, Annemarie 103
Nooijen, Anke 84
Nooijen, Willem 132
Notenboom, Valerie 28
Nuyen, Bastiaan 102
Nuyten, Dimitry 86, 88
Nyst, Heike 114
Olde Damink-Van Rosmalen, Agnes 128
Oldenburg, Hester 114
Olijve, Albert 103
Olivier, Rolien 115
Olszewska, Agnieszka 88
Ong, Nico 67
Oostendorp, Roos 82, 102
Oosterhuis, Koen 45
Oosterkamp, Rianne 24
Opperdijk, Vera 88
Oude Vrielink, Joachim 62
Ouwehand, MariГ«t 103
Ouwens, Gabey 128
Ovaa, Huib 38
Paape, Anita 133
Pagie, Ludo 56
Pajic, Marina 51
Pala, Zeliha 76
Pameijer, Frank 133
Pang, Baoxu 60
Paul, Petra 60
Paulus van Pauwvliet, Cecile 143
Peeper, Daniel 73
Pegtel, Michiel 19
Pengel, Kenneth 89, 133
Peperzak, Victor 41
Pereira-Arias, Lenka 132
Peric-Hupkes, Daniel 56
Perrakis, Anastassis 30
Peters, Peter 67
Peterse, Hans 86, 132
Pickersgill, Helen 56, 64
Piek-den Hartog, Marjan 114
Pierson, Jason 67
Pietersen, Alexandra 69
Pijpe, Anouk 128
Plug, Rob 133
Pluim, Dick 82
Ponsioen, Bas 23, 32
Pool, Bert 132
Pos, Floris 88
Pramana, Jimmy 78, 114
Prasad, Asheeta 69
Prevoo, Warner 133
Prieur, Alexandre 73
Pritchard, Colin 71
Proost, Natalie 71
Pruntel, Roelof 133
Quaak, Susanne 103
Rasch, Coen 88
Reinders-Som, Anneke 143
Remeijer, Peter 88
Remmelzwaal, Jolanda 143
Repanas, Kostas 30
Res, Pieter 67
Reumer, Annet 28
Roberts, Daniel 143
Rocha, Nuno 60
Rodenhuis, Sjoerd 86, 102
Rodenko, Boris 38
Roelvink, Marja 103
Romeijn, Martijn 67
Rondaij, Mariska 66
Ronday, May 115
Rookus, Matti 128
Roos, Ed 21
Rooze, Lyandra 132
Rosing, Hilde 102
Rossi, Maddalena 89
Rottenberg, Sven 51
Rowland, Benjamin 24
Rubio, Miguel 24
Ruevekamp, Marjan 80
Ruijs, Marielle 84
Ruijter-Schippers, Henrique 132
Russell, Nicola 80, 88, 128
Russo, Francesco 56
Rutgers, Emiel 86, 114, 128
Rygiel, Tomasz 19
Rzadkowski, Adrian 32
Sachs, Norman 17
Salomon, Izhar 60
Salverda, Govert 88
Savaskan, Nicolai 32
Schagen, Sanne 126
Schellens, Jan 82, 102
Schepers, Koen 43
Schilder, Christien 126
Schinkel, Alfred 76
Schippers-Gillissen, Carla 132
Schlief, Angelique 133
Schmidt, Marjanka 84, 128, 132
Schneider, Christoph 88
Schnell, Silke 47
Scholten, Marijke 143
179
PERSONNEL INDEX
Schornagel, Jan 102
Schot, Margaret 103
Schrier, Mariette 62
Schumacher, Ton 43
Schutte, Peter 115
Schwarz, Marco 88
Sein, Johan 45
Siedschlag, Christian 133
Simons, Monique 129
Sinaasappel, Michiel 132, 133
Sivro-Prndelj, Ferida 132
Sixma, Titia 28
Smeele, Ludi 114
Smeenk, Robert 114
Smit, Linda 24
Smit, Marjon 73
Smits, Marianne 102
Smitsmans, Monique 88
Snel, Mireille 36
Snoek, Margriet 71
Sonke, Gabe 102
Sonke, Jan-Jakob 88
Sonneborn, Mariska 132
Sonnenberg, Arnoud 17
Sparmann, Anke 69
Sprong, Debbie 80
SrГЎmek, Michael 115
Steenbakkers, Roel 88
Stewart, Fiona 80
Stoppa, Tino 115
Storm, Dea 143
Stortelers, Catelijne 32
Stroom, Joep 88
Strumane, Kristin 19
Sutherland, Kate 71
Swart, Erwin 43
Swart, Martha 143
Taal, Babs 102, 124
Taghavi, Panthea 69
Talhout, Wendy 56
Tan, Bing 114
Tanger, Ellen 69
Te Poele, Johannes 80
Te Riele, Hein 49
Teertstra, Jelle 133
Ten Bokkel Huinink, Wim 102
Ten Cate, Julia 115
Ter Heine, Rob 103
Ter Riet, Bas 51
Teunissen, Hans 69
Theodorou, Vassiliki 75
Theuws, Jacqueline 88
Tibben, Mathijs 103
Tielen, Ivon 133
Tielenburg, RenГ© 89
Timmers, Adriaan 114
Tjin, Esther 45
Toaldo, Cristiane 66
Toebes, Mireille 43
Tolhuis, Bas 69
Tran, Ly 103
Triesscheijn, Martijn 80
Udo, Renate 84, 129
Ulens, Chris 28
Urbanus, Jos 43
Uren, Anthony 71
Vader, Laura 143
Vaessen, Heleen 143
Vainchtein, Liia 103
Vainio, Saara 51
ValdГ©s Olmos, Renato 132, 143
ValdГ©s Olmos, Tjalling 143
Valkenet, Ludy 143
Valle, Noelia 64
Van Amerongen, RenГ©e 71
Van As, Josette 129
Van As-Brooks, Corina 114
Van Baal, Jurgen 34, 36
Van Beers, Erik 84
Van Beurden, Marc 115, 124
Van Blitterswijk, Wim 34
Van Boven, Hester 132
Van Bunningen, Bart 88
Van Coevorden, Frits 114
Van Dam, Frits 126
Van de AhГ©, Fina 71
Van de Kasteele, Willeke 45
Van de Poel, Henk 115
Van de Steeg, Evita 76
Van de Ven, Peter 89
Van de Vijver, Marc 86, 132
Van de Wetering, Koen 51
Van Deemter, Liesbeth 51
Van den Belt-Dusebout, Sandra 128
Van den Berg, Joost 103
Van den Berk, Paul 47
Van den Bongard, Desiree 88
Van den Boom, Marly 43
Van den Bout, Iman 17
Van den Brekel, Michiel 115
Van den Brink-de Vries, Nienke 132
Van den Broek, Guido 115
Van den Haak, Marjolijn 143
Van den Hoorn, Tineke 60
Van den Hoven, Jolanda 103
Van der Berg, Marieke 115
Van der Burg, Eline 53
Van der Donk, Emile 143
Van der Gulden, Hanneke 53
Van der Heijden, Ingrid 55
Van der Hoeven, Joost 114
Van der Horst, Ferdi 84
Van der Horst, Gerda 41
Van der Kammen, Rob 19
Van der Krogt, Gerard 23
Van der Kruijssen, Corina 76
Van der Molen, Lisette 114
Van der Plas, Arnout 84
Van der Sande, Jaap 102
Van der Sar, Jana 103
Van der Schoot, Sabien 103
Van der Stoop, Petra 69
Van der Torre, Jaco 71
Van der Velde, Hella 64
Van der Velde, Tony 143
Van der Velden, Annette 102
Van der Velden, Yme 69
Van der Voort, Paul 133
Van der Wal, Anja 49
Van der Weide, Marchien 102
Van der Wel, Wel 67
Van Dijk, Anne 129
Van Dijk, Pim 28
Van Dongen, Miranda 24
Van Doorn, Remco 73
Van Driel, Willemien 115
Van Duijn, Miranda 131
Van Duijse, Josyanne 62
Van Eijndhoven, Monique 82
Van Esch, Anita 76
Van Gerwen, Suzan 30
Van Gijn, Roel 102
Van Harten, Wim 131
Van Heijst, Jeroen 43
Van Herk, Marcel 88
Van Herwaarden, Antonius 76
Van Hien, Richard 84
Van Leeuwen, Fijs 38, 133
Van Leeuwen, Flora 84, 128
Van Leeuwen, Fred 40
Van Lent, Wineke 131
Van Lohuizen, Maarten 69
Van Luenen, Henri 51
Van Lummel, Menno 34
Van Maanen, Rianne 102
Van Meeteren, Laurens 32
Van Montfort, Erwin 71
Van Netten, Gabry 143
Van Nuland, Annemarie 126
Van Oosterwijk, Els 143
Van Pel, RenГ©e 132
Van Rens, Anja 133
Van Rijk, Maartje 114
Van Rossum, Maya 115
Van Rossum, Sari 28
Van Sandick, Johanna 114
Van Steensel, Bas 56
Van �t Veer, Laura 84, 128, 132, 133
Van Tellingen, Olaf 132
Van Tilburg, Erica 38
Van Tinteren, Harm 143
Van Turnhout, Arjen 115
Van Velthuysen, Loes 132
Van Vliet, Maaike 131
Van Vliet, Marja 133
Van Vliet, Martin 58
Van Vliet-Vroegindeweij, Corine 88
Van Vuuren, Peter 114
Van Waardenberg, Wil 143
Van Waterschoot, Robert 76
180
PERSONNEL INDEX
Van Welsem, Tibor 40
Van Winden, Annemieke 103
Van Zandwijk, Nico 102
Van Zeijl, Leonie 32
Van Zon, Maaike 67
Van Zon, Wouter 49
Vargas, Mark 28
Veldhuizen, Dennis 133
Veltkamp, Sander 82
Vendrig, Tineke 45
Vens, Conchita 78
Veraar, Elise 41
Verbrugge, Inge 41
Verheij, Marcel 34, 88
Verhoef, Senno 84, 124, 128, 133
Verhoeven, Els 69
Verloop, Janneke 128
Vermeulen, Christie 78
Vermeulen, Eric 128
Verwaal, Vic 114
Verwijs, Manon 78
Verwoerd, Desiree 66
Verzijlbergen, Kitty 40
Vincent, Andrew 143
Vink, Stefan 82
Vissers, Joep 69
Vlaming, Marijn 76
Vlaskamp, Marcel 143
Vogel, Maartje 56
Voogel-Fuchs, Marja 103
Voorhoeve, Mathijs 62
Vormer, Tinke 49
Voskuil, Dorien 84, 128
Vredeveld, Liesbeth 73
Vrieling, Alina 84, 128
Vrieling, Conny 88
Vyth-Dreese, Florry 45
Wagenaar, Els 76
Wals, Anneke 143
Weevers, Marion 126
Weichenrieder, Oliver 30
Weigelt, Britta 84
Wesseling, Jelle 132
Wessels, Lodewyk 58
Westerman, Bart 69
Wever, Lidwina 143
Wielders, Camiel 49
Wielders, Eva 49
Wientjens, Ellen 53
Wieringa-Ariaens, Aafke 132, 133
Wigbout, Gea 133
Wijnands, Yvonne 21
Wijnholds, Jan 51
Wilhelmsen, Kevin 17
Willemse, Els 143
Winterwerp, Herrie 28
Wissink, Esther 41
Wittkämper, Fritts 88
Woerdeman, Leonie 115
Wolthuis, Rob 49
Wu, Tin 133
Zandvliet, Anthe 103
Zerp, Shuraila 34
Zevenhoven, John 71
Ziblat, Lonny 143
Zijp, Lambert 89
Zimmermann, Christian 76
Zlotorynski, Eitan 62
Zoetmulder, Frans 114
Zuur, Charlotte 115
Zuur, Karel 115
Zwaagstra, Annabel 75
Zwart, Wilbert 66
181
COLOFON
COLOFON
Coordinators
Suzanne Bakker
Suzanne Corsetto
Henri Van Luenen
Photograph HM The Queen Beatrix
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Photograph AJM Berns
Loek Zuijderduin
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