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THE<br />
NETHERLANDS<br />
CANCER<br />
INSTITUTE<br />
SCIENTIFIC<br />
ANNUAL<br />
REPORT <strong>2008</strong><br />
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SCIENTIFIC ANNUAL REPORT <strong>2008</strong><br />
3
4<br />
Patron HM The Queen Beatrix
SCIENTIFIC ANNUAL REPORT <strong>2008</strong><br />
THE NETHERLANDS CANCER INSTITUTE<br />
CANCER RESEARCH LABORATORY AND CANCER HOSPITAL<br />
www.nki.nl<br />
5
6 COPYRIGHT<br />
Scientific Annual Report <strong>2008</strong><br />
Illustrations and unpublished data in these reports may not be<br />
used without permission of the author.<br />
Copyright ©:<br />
The Netherlands Cancer Institute<br />
Antoni van Leeuwenhoek Hospital<br />
Plesmanlaan 12 1<br />
1066 CX Amsterdam<br />
The Netherlands<br />
Phone +3 1 20 5 12 9 1 1 1<br />
Fax +3 1 20 6 17 2625<br />
ISSN 1387-86 1 1<br />
COLOFON<br />
Coordinators<br />
Suzanne Corsetto<br />
Henri Van Luenen<br />
Photograph HM The Queen Beatrix<br />
Enquiry’s/permission:<br />
Rijksvoorlichtingdienst, afd. Pers en Publiciteit/FOTO<br />
Postbus 20009<br />
2500 EA Den Haag<br />
Photo Ruud Taal/Capital Press<br />
Copyright RVD<br />
Photograph AJM Berns<br />
Loek Zuijderduin<br />
Other Photographs and Illustrations<br />
Audiovisual Services<br />
The Netherlands Cancer Institute<br />
Antoni van Leeuwenhoek Hospital<br />
Plesmanlaan 12 1<br />
1066 CX Amsterdam<br />
The Netherlands<br />
Printed by Thieme Amsterdam
CONTENTS<br />
Board Members 8<br />
Research Divisions 9<br />
Introduction 11<br />
Education in Oncology 16<br />
Division of Biochemistry 18<br />
Division of Cell Biology I 22<br />
Division of Cell Biology II 31<br />
Division of Experimental Therapy 38<br />
Division of Gene Regulation 46<br />
Division of Division of Immunology 53<br />
Division of Molecular Biology 62<br />
Division of Molecular Carcinogenesis 75<br />
Division of Molecular Genetics 79<br />
Division of Psychosocial Research and Epidemiology 89<br />
Division of Diagnostic Oncology 100<br />
Division of Medical Oncology 116<br />
Division of Radiotherapy 126<br />
Division of Surgical Oncology 143<br />
Biometrics Department 156<br />
Clinical Trials 161<br />
Invited Speakers 177<br />
Projects 180<br />
Personnel Index 193<br />
7
President of Board of Governors W Kok<br />
BOARD MEMBERS<br />
INTERNATIONAL SCIENTIFIC ADVISORY BOARD<br />
T De Lange, Leon Hess Professor, The Rockefeller<br />
University, New York, USA<br />
RA Flavell, Professor of Immunobiology, Yale<br />
University School of Medicine, New Haven, USA<br />
WGJ Hol, Professor of Biochemistry and Biological<br />
Structure, University of Washington, Seattle, USA<br />
J Mendelsohn, President MD Anderson Cancer Center,<br />
University of Texas, Houston, USA<br />
P Nurse, Professor of Microbiology, President of<br />
The Rockefeller University, New York, USA<br />
R Nusse, Professor of Developmental Biology, Stanford<br />
University, Stanford, USA<br />
HL Ploegh, Professor of Biology, Whitehead Institute<br />
for Biomedical Research, Cambridge, USA<br />
K Vousden, Director, Beatson Institute for Cancer<br />
Research, Glasgow, UK<br />
RA Weinberg, Professor of Biology, Massachusetts<br />
Institute of Technology, Whitehead Institute for<br />
Biomedical Research, Cambridge, USA<br />
NATIONAL SCIENTIFIC ADVISORY BOARD<br />
DD Breimer, Professor of Pharmacology, Leiden<br />
University<br />
JC Clevers, Professor of Clinical Immunology,<br />
Hubrecht Institute, Utrecht<br />
EGE de Vries, Professor of Medical Oncology,<br />
University of Groningen<br />
JHF Falkenburg, Professor of Experimental<br />
Hematology, Leiden University<br />
CG Figdor, Professor of Experimental Immunology,<br />
Radboud University Nijmegen<br />
JHJ Hoeijmakers, Professor of Molecular Genetics,<br />
Erasmus University Rotterdam<br />
P Lambin, Professor of Radiation Oncology, Maastricht<br />
University<br />
RH Medema, Professor of Experimental Oncology,<br />
Utrecht University<br />
CJH van de Velde, Professor of Surgical Oncology,<br />
Leiden University<br />
9<br />
BOARD MEMBERS<br />
BOARD OF DIRECTORS<br />
AJM Berns, chairman and director of research<br />
S Rodenhuis, director clinical research and development<br />
WH Van Harten, director organization and management<br />
BOARD OF GOVERNORS<br />
W Kok, president<br />
HCJ Van der Wielen, vice-president<br />
T De Swaan, treasurer<br />
FH Schröder<br />
D Sinninghe Damsté<br />
MC Smeets<br />
PC Van der Vliet<br />
GP Vooijs<br />
SCIENTIFIC ADVISORY COUNCIL<br />
AJM Berns, chairman<br />
JJ Neefjes, secretary<br />
S Rodenhuis<br />
T Sixma<br />
M Verheij<br />
J Borst
10 RESEARCH DIVISIONS<br />
RESEARCH DIVISIONS<br />
Biochemistry<br />
Titia Sixma (head)<br />
Anastassis Perrakis<br />
Franciska Manuputty (office manager)<br />
Cell Biology I<br />
Arnoud Sonnenberg (head)<br />
Wim van Blitterswijk<br />
John Collard<br />
Kees Jalink<br />
Wouter Moolenaar<br />
Ed Roos<br />
Cell Biology II<br />
Jacques Neefjes (head)<br />
Rob Michalides<br />
Huib Ovaa<br />
Peter Peters<br />
Marieke van der Velde (office manager)<br />
Experimental Therapy<br />
Laura van ’t Veer<br />
Adrian Begg<br />
Jan Schellens<br />
Fiona Stewart<br />
Thea Eggenhuizen (office manager)<br />
Gene Regulation<br />
Reuven Agami<br />
Maarten Fornerod<br />
Fred van Leeuwen<br />
Bas van Steensel<br />
Suzanne Corsetto (office manager)<br />
Immunology<br />
Jannie Borst (head)<br />
Christian Blank<br />
John Haanen<br />
Heinz Jacobs<br />
Ton Schumacher<br />
Florry Vyth-Dreese<br />
José Overwater (office manager)<br />
Molecular Biology<br />
Hein Te Riele (head)<br />
Piet Borst (honorary staff member)<br />
Jos Jonkers<br />
Sabine Linn<br />
Alfred Schinkel<br />
Lodewyk Wessels<br />
Rob Wolthuis<br />
Tom de Knegt (office manager)<br />
Linda Römer (secretary)<br />
Molecular Carcinogenesis<br />
René Bernards (head)<br />
Roderick Beijersbergen<br />
Franciska Manuputty (office manager)<br />
Molecular Genetics<br />
Maarten van Lohuizen (head)<br />
Anton Berns<br />
Jan-Hermen Dannenberg<br />
Jacqueline Jacobs<br />
Anna Haramis<br />
John Hilkens<br />
Paul Krimpenfort<br />
Daniel Peeper<br />
Margriet Snoek<br />
Erica Delwel (office manager)<br />
Marie Anne van Halem (secretary)<br />
Psychosocial Research and Epidemiology<br />
Neil Aaronson (head)<br />
Eveline Bleiker<br />
Wim van Harten<br />
Flora van Leeuwen<br />
Sanne Schagen<br />
Yvonne Driessen-Ruwaard (office manager)<br />
Diagnostic Oncology<br />
Laura van ’t Veer (head)<br />
Tanja Alderliesten<br />
Priscilla Axwijk<br />
Philippe Baars<br />
Olga Balague Ponz<br />
Peter Besnard<br />
Hans Bonfrer<br />
Daphne de Jong<br />
Kenneth Gilhuijs<br />
Cees Hoefnagel<br />
Frans Hogervorst<br />
Irma Kluijt<br />
Wim Koops<br />
Robert Kröger<br />
Charlotte Lange<br />
Claudette Loo<br />
Saar Muller<br />
Petra Nederlof<br />
Willem Nooijen<br />
Frank Pameijer<br />
Renée van Pel<br />
Warner Prevoo<br />
Efraim Rosenberg<br />
Michiel Sinaasappel<br />
Ferida Sivro<br />
Jelle Teertstra<br />
Renato Valdes Olmos<br />
Hester van Boven<br />
Fijs van Leeuwen<br />
Olaf van Tellingen<br />
Loes van Velthuysen<br />
Mark van de Vijver<br />
Senno Verhoef<br />
Wouter Vogel<br />
Jelle Wesseling<br />
Christine Arkes (secretary)<br />
Carla van Tiggelen (secretary)
Medical Oncology<br />
Sjoerd Rodenhuis (head)<br />
Joke Baars<br />
Paul Baas<br />
Jos Beijnen<br />
André Bergman<br />
Christian Blank<br />
Willem Boogerd<br />
Henk Boot<br />
Dieta Brandsma<br />
Sjaak Burgers<br />
Annemieke Cats<br />
Jan Paul de Boer<br />
Leo Gualtherie van Weezel<br />
John Haanen<br />
Alwin Huitema<br />
Martijn Kerst<br />
Sabine Linn<br />
Anne Lukas<br />
Jan Schellens<br />
Jan Schornagel<br />
Marianne Smits<br />
Gabe Sonke<br />
Babs Taal<br />
Wim ten Bokkel Huinink<br />
Margot Tesselaar<br />
Michiel van den Heuvel<br />
Marchien van der Weide<br />
Nico van Zandwijk<br />
Mariëlle de Kwant (secretary)<br />
Radiotherapy<br />
Marcel Verheij (head)<br />
Berthe Aleman<br />
Harry Bartelink<br />
José Belderbos<br />
Eugene Damen<br />
Roel de Boer<br />
Luc Dewit<br />
Paula Elkhuizen<br />
Rick Haas<br />
Olga Hamming-Vrieze<br />
Guus Hart<br />
Wilma Heemsbergen<br />
Frank Hoebers<br />
Edwin Jansen<br />
Han Krewinkel<br />
Joos Lebesque<br />
Corrie Marijnen<br />
Harry Masselink<br />
Ben Mijnheer<br />
Luc Moonen<br />
Floris Pos<br />
Coen Rasch<br />
Babs Reichgelt<br />
Peter Remeijer<br />
Nicola Russell<br />
Govert Salverda<br />
Christoph Schneider<br />
Joep Stroom<br />
Bart van Bunningen<br />
Marcel van Herk<br />
Baukelien van Triest<br />
Corine van Vliet<br />
Marieke van Zwienen<br />
Frits Wittkämper<br />
Patricia Haye-Fewer (management assistant)<br />
Surgical Oncology<br />
Theo Ruers (head)<br />
Marc van Beurden<br />
Michiel van den Brekel<br />
Alfons Balm<br />
Annemieke Ackerstaff<br />
Axel Bex<br />
Biljana Zupan-Kajcovski<br />
Christiaan Keijzer<br />
Dirk Buitelaar<br />
Emiel Rutgers<br />
Frans Hilgers<br />
Frits van Coevorden<br />
Henk van de Poel<br />
Hester Oldenburg<br />
Houke Klomp<br />
Bing Tan<br />
Ingeborg Vergouwe<br />
Inka Nieuweboer-Krobotova<br />
Joris Hage<br />
Johanna van Sandick<br />
Julia ten Cate<br />
Katina Efthymiou<br />
Leonie Woerdeman<br />
Lotti Lubsen–Brandsma<br />
Ludi Smeele<br />
Marc van Beurden<br />
Marianne Piek-den Hartog<br />
Marie-Jeanne Baas–Vrancken Peeters<br />
Marieke van der Berg<br />
Martine van Huizum<br />
May Ronday<br />
Michael Srámek,<br />
Michel Wouters<br />
Michiel van den Brekel<br />
Omgo Nieweg<br />
Peter Schutte<br />
Peter Lohuis<br />
Sanne Vos<br />
Simon Horenblas<br />
Tino Stoppa<br />
Vic Verwaal<br />
Wietze van der Veen<br />
Willemien van Driel<br />
Wim Meinhardt<br />
Annemieke Hoogland (office manager)<br />
Biometrics Department<br />
Otilia Dalesio<br />
Financial Administration<br />
Didier Dohmen<br />
General Facilities, ICT and Personnel Department<br />
Eric De Wilde<br />
General Research Coordination<br />
Jacques Neefjes, deputy scientific director<br />
Henri van Luenen, director of operations<br />
11<br />
RESEARCH DIVISIONS
12<br />
INTRODUCTION<br />
Director of Research Ton Berns<br />
INTRODUCTION<br />
I am pleased to present our Scientific Annual Report<br />
<strong>2008</strong>. It provides an overview of the scientific activities<br />
at The Netherlands Cancer Institute - Antoni van<br />
Leeuwenhoek Hospital (<strong>NKI</strong>-AVL). Additional<br />
information can be found at www.nki.nl. A lucid<br />
overview of our activities with illustrations can be found<br />
in our brochure (available on our website)<br />
The <strong>NKI</strong>-AVL is a Comprehensive Cancer Center,<br />
combining hospital and research laboratories under one<br />
roof in a single independent organization. The hospital<br />
comprises 180 beds, an outpatient clinic and a large<br />
radiotherapy department. Facilities for clinical research<br />
include a large patient database, clinical data<br />
management, extensive diagnostic facilities, a pharmacy<br />
with a production unit for experimental drugs, and<br />
active research groups in pharmacy, epidemiology and<br />
psychosocial oncology. The laboratory covers all major<br />
areas of cancer research, with special emphasis on cellbased<br />
screens, mouse tumor models, cell biology,<br />
structural biology, and immunology. Translational<br />
research is an integral activity of many research groups<br />
and is fostered by collaboration between clinical and<br />
basic scientists. This scientific report deals mainly with<br />
the basic and clinical science in the <strong>NKI</strong>-AVL.<br />
Information on patient-care is described in our General<br />
Annual Report.<br />
In <strong>2008</strong> most of our research groups relocated to the<br />
renovated research labs that were constructed in the<br />
former hospital building. We have taken this opportunity<br />
to restructure the divisions in order to enhance<br />
interactions between the research groups. As a result,<br />
two new divisions were formed while two existing<br />
divisions were combined. The transition to the new labs<br />
went very smoothly thanks to the excellent preparation<br />
and organization of the whole process.<br />
Our clinical activities again exhibited growth, although<br />
not as much as expected due to the space limitations in<br />
the outpatient clinics and the operating rooms. In 2009,<br />
we will add an extra operating room specially equipped<br />
for minimally invasive surgery. We also foresee<br />
additional, temporary facilities to relieve the pressure on<br />
our outpatient facility. Filling current vacancies for<br />
nursing staff, however, is at present our most serious<br />
concern. Expansion of our radiotherapy department is<br />
planned to take place through the establishment of two<br />
off-site satellites at clinical centers elsewhere. In<br />
addition, we are planning, together with the Erasmus<br />
University, the University Medical Center Leiden and the<br />
Delft Technical University, a proton/heavy ion radiation<br />
center for treating specific groups of patients that will<br />
benefit from the more targeted delivery of radiation.<br />
The transition to a new reimbursement system in the<br />
health care system is progressing, and this enables us to<br />
further expand our clinical activities. However, we foresee<br />
harsher financial challenges due to the current economic<br />
crisis. This is a more general problem we are facing,<br />
although we do not expect major setbacks in the coming<br />
year. We concluded <strong>2008</strong> with a small profit for the<br />
hospital. Also our research expenditure remained within<br />
budget in <strong>2008</strong>. We expect to balance the budget for<br />
both the hospital and the research in 2009 as well,<br />
despite steep increases in some expenses beyond our<br />
control.<br />
In <strong>2008</strong> our PhD students organized, together with<br />
Cancer Research UK, an international scientific meeting<br />
for PhD students only. The meeting was held at the <strong>NKI</strong><br />
and was much appreciated by the participants.<br />
The International Scientific Advisory Board spent two<br />
full days at the Institute to critically evaluate our research<br />
portfolio. This was also meant as a rehearsal for the site<br />
visit evaluation that will take place in the Fall of 2009<br />
in which an international site visit team appointed by<br />
the Royal Netherlands Academy of Arts and Sciences<br />
will evaluate the Cancer Institute on behalf of the<br />
Dutch Cancer Society and the Ministry of Health,<br />
the organizations that provide the core funding for<br />
The Netherlands Cancer Institute. Our International<br />
Scientific Advisory Board was very positive about the<br />
quality of our research program, but it also gave valuable<br />
advice with regard to policies for recruiting new staff<br />
members, coaching young group leaders, and fostering<br />
translational research. Many of their suggestions have<br />
already been implemented in the course of <strong>2008</strong>.<br />
Table 1<br />
Core research funding <strong>NKI</strong>-AVL from the Cancer Society and the Ministry of Health in period 1993-<strong>2008</strong> in million Euro’s<br />
Year 1993 1996 1999 2001 2002 2003 2004 2005 2006 2007 <strong>2008</strong><br />
Cancer Society 4.1 5.6 7.0 8.7 8.7 9.1 8.9 8.6 8.3 8.8 9.3<br />
Ministry Health 7.3 8.1 8.8 9.4 9.9 10.1 9.1 9.3 9.4 9.5 9.9<br />
Total* 11.4 13.7 15.8 18.1 18.6 19.2 18.0 17.9 17.7 18.3 19.4<br />
In addition to the core funding the <strong>NKI</strong> acquired support through external grants, donations, and research agreements.<br />
The contribution of this latter income to the total budget has been steadily increasing over the years and represents roughly<br />
60% of the total budget in <strong>2008</strong>.
The staff retreat that will be held in January 2009 will<br />
focus on how to further enhance the synergy between<br />
the preclinical and clinical research within the <strong>NKI</strong>-AVL.<br />
In <strong>2008</strong>, we continued to expand collaborative efforts<br />
and signed an agreement with the Technical University<br />
Twente to work together in developing minimally<br />
invasive surgical techniques. We also concluded an<br />
agreement with Astra Zeneca to serve as one of their<br />
preferred academic partners both with regard to<br />
preclinical and clinical research. We signed an<br />
agreement with the Karolinska Institutet in Stockholm<br />
and the Institut Gustave Roussy in Paris to collaborate<br />
more closely on innovative clinical trials and exchanging<br />
expertise in specific diagnostic areas. This collaborative<br />
effort between three European cancer centers will give<br />
access to powerful technological platforms of genomics/<br />
proteomics assays. These are important to refine<br />
diagnosis and measure responses to therapy, thereby<br />
assisting in defining more effective combination<br />
therapies. These initiatives should help us to advance<br />
personalized medicine. We are actively involved in<br />
establishing a broader alliance of European<br />
Comprehensive Cancer Centers of Excellence, which can<br />
also serve as a more influential companion of the<br />
pharmaceutical industry.<br />
Highlights<br />
Our institute continues to have a strong scientific<br />
output; <strong>2008</strong> was again a productive year. It is always<br />
difficult to estimate the impact of research when the<br />
results are still fresh. The research highlights<br />
summarized here serve primarily as a sampling of work<br />
currently on-going in the Institute. A more complete and<br />
detailed account of specific projects can be found in the<br />
reports of individual group leaders in this SAR and on<br />
the website (www.nki.nl).<br />
Nuno Rocha, Rik van der Kant and Coenraad Kuijl from<br />
the Neefjes lab (Division of Cell Biology) performed an<br />
in-depth cell biological study explaining the contribution<br />
of cholesterol to lysosomal behavior as detected in<br />
various genetic diseases like Gaucher and Niemann Pick<br />
disease. They showed that a unique cholesterol sensor<br />
on lysosomes attracts a protein from the ER to remove a<br />
motor protein on late endosomes. This is the first study<br />
showing how different intracellular compartments<br />
communicate to control each other’s behavior.<br />
Eva Wielders in the group of Hein te Riele (Division of<br />
Molecular Biology) has used a novel technique,<br />
oligonucleotide-directed gene modification, to recreate<br />
allelic variants of the central mismatch repair gene<br />
MSH2 in mouse embryonic stem cells. By studying the<br />
phenotype of mutant ES cells she could unambiguously<br />
classify two MSH2 variants of uncertain significance as<br />
innocent polymorphisms and a third one as a deleterious<br />
mutation causative of hereditary non-polyposis colorectal<br />
cancer. This work will help medical geneticists in the<br />
counseling of (suspected) HNPCC families.<br />
Breast cancers that are deficient in homologous<br />
recombination are exquisitely sensitive to bifunctional<br />
alkylating agents, such as were given in the high-dose<br />
chemotherapy programs in the late 20 th century.<br />
13<br />
INTRODUCTION<br />
Marieke Vollebergh in the group of Sabine Linn<br />
(Divisons of Medical Oncology and Molecular Biology),<br />
in collaboration with Petra Nederlof and Jelle Wesseling<br />
(Division of Diagnostic Oncology), has shown that<br />
sporadic tumors more likely respond to such regimens<br />
and can be recognized by either establishing a BRCA1<br />
signature in an array-CGH test or by finding an<br />
expanding growth pattern at microscopic examination of<br />
a resection specimen.<br />
The group of Bas van Steensel (Division of Gene<br />
Regulation) mapped some 1,300 large domains in the<br />
genome that associate with Lamin (LADs). These LADs<br />
represent a repressive chromatin environment,<br />
demonstrating that the human genome is divided into<br />
large, discrete domains that are units of chromosome<br />
organization within the nucleus.<br />
The group of Fred van Leeuwen (Division of Gene<br />
Regulation) developed a novel assay in yeast to<br />
determine protein turnover in vivo and applied it to<br />
histones. They found that histones throughout the<br />
genome are subject to extensive replication-independent<br />
exchange, demonstrating that histones and their posttranslational<br />
marks are not permanent residents in<br />
chromatin.<br />
Daniel Peeper’s group (Division of Molecular Genetics)<br />
has previously found that cells can respond to the<br />
activation of oncogenes by entering a long-term<br />
proliferative arrest, known as Oncogene-Induced<br />
Senescence (OIS). As such, OIS serves as a potent<br />
mechanism protecting us against cancer. Recently, they<br />
uncovered an unanticipated role for secreted proteins,<br />
including interleukins, in this process. Interleukins<br />
are known for their important role in the immune<br />
system, but Peeper’s group found that they are also<br />
indispensable for OIS. These secreted factors,<br />
collectively called the Senescence-Messaging Secretome<br />
(SMS), allow for communication (‘SMS-ing’) between<br />
cells under stress (like senescent cells that carry an<br />
activated oncogene) and their environment.<br />
Alexandra Pietersen in the lab of Van Lohuizen<br />
(Division of Molecular Genetics) described an important<br />
role for the Polycomb gene Bmi1 in the regulation of<br />
differentiation and proliferation of mammary epithelial<br />
stem cells and progenitors<br />
Anthony Uren and Jaap Kool (Berns and Van Lohuizen<br />
labs, Division of Molecular Genetics) in collaboration<br />
with the groups of Lodewyk Wessels (Division of<br />
Molecular Biology) and David Adams (Sanger Centre,<br />
UK), highlighted the power of high throughput<br />
insertional mutagenesis screens as a cancer gene<br />
network discovery platform in cancer-predisposed<br />
p19ARF and p53 deficient mice.<br />
The group of Laura van ‘t Veer (Division of Experimental<br />
Therapy) found that primary colorectal tumors that<br />
develop liver metastasis have a unique chromosomal<br />
signature that might be suitable to identify patients at<br />
risk to develop liver metastasis. The observed genomic<br />
aberrations also provide an inroad to understand<br />
the underlying mechanism and may lead to more<br />
individualized disease management.
14<br />
INTRODUCTION<br />
The group of Jan Schellens (Division of Medical<br />
Oncology) has documented a promising anticancer<br />
activity of the combination of the PARP inhibitor<br />
AZD2281 alone and in combination with carboplatin in<br />
early clinical studies. PARP inhibitors selectively target<br />
tumors that are defective in DNA repair by homologous<br />
recombination, such as breast cancer in BRCA1 or<br />
BRCA2 carriers.<br />
Adrian Begg and coworkers (Division of Experimental<br />
Therapy) tested the predictive potential of signatures<br />
known or suspected to affect response to radiotherapy.<br />
The putative stem cell marker CD44 was the most<br />
significant predictor of outcome (local control), with an<br />
acute hypoxia signature showing a strong trend. CD44<br />
also emerged as the most significant gene in a datadriven<br />
approach searching for any set of genes<br />
distinguishing cures from recurrences.<br />
Quality of research<br />
The quality of research can be monitored in several<br />
ways. Our scientific productivity as assessed by objective<br />
bibliometric parameters (citations and impact of<br />
scientific articles published by the <strong>NKI</strong> staff) has shown<br />
a steady increase since the beginning of the 1980’s<br />
(Table 2). It is satisfying to note that the Institute is<br />
retaining an internationally prominent position in<br />
cancer research, despite its limited budget that is<br />
relatively modest in comparison with many comparable<br />
institutes abroad.<br />
While a high citation score is gratifying, it is only one<br />
measure of scientific productivity. The quality of our<br />
work should also be gauged by the invitations of our staff<br />
to present at prestigious scientific meetings, awards<br />
obtained by our staff. We score high on all these<br />
accounts. We are also successful in securing grant<br />
support, e.g. Sjoerd Rodenhuis and colleagues<br />
successfully competed for a large grant together with<br />
industry from the Center for Translational Molecular<br />
Medicine (CTMM) aimed at identifying new diagnostic<br />
markers for therapy response. Several groups in the<br />
Institute also participate with university groups in a<br />
CTMM program on lung cancer. Bas van Steensel<br />
received a VICI grant from NWO, and VIDI awards were<br />
given to Karin de Visser and Elisabetta Citterio. I myself<br />
Table 2<br />
Short-term citations and impact of scientific articles published by the <strong>NKI</strong> research staff 1981 - <strong>2008</strong><br />
Publication year Publications Citations* Citations/publication Impact**<br />
1982 155 706 3.2 295<br />
1983 192 986 4.6 365<br />
1984 277 1797 5.1 616<br />
1985 270 1575 6.5 549<br />
1986 272 1628 5.8 650<br />
1987 296 2036 6.0 765<br />
1988 375 2040 5.5 742<br />
1989 283 1822 6.4 764<br />
1990 319 2704 8.5 854<br />
1991 268 2933 10.9 910<br />
1992 269 2721 10.1 911<br />
1993 245 3033 12.3 958<br />
1994 307 4380 14.2 1292<br />
1995 287 3648 12.7 1415<br />
1996 260 4056 15.6 1520<br />
1997*** 274 4174 14.1 1811<br />
1998 276 3138 14.2 1392<br />
1999 280 3474 12.4 1766<br />
2000 270 3551 13.2 1659<br />
2001**** 298 3991 13.3 1582<br />
2002 349 7436 21.3 2455<br />
2003 333 5240 15.3 2122<br />
2004 327 5267 16.1 1882<br />
2005 368 6350 17.3 2461<br />
2006 436 6330 14.5 2608<br />
2007 412 2629<br />
<strong>2008</strong># 422 2396<br />
* Citations in the two years after publication, excluding self-citations. Starting 1989 citation analysis has been carried out<br />
online. This allows detection (and elimination) of all self-citations. Before 1989 this pruning was limited to first authors.<br />
** The impact factor is the average number of citations per year of an article in a given journal. The total impact is the sum<br />
of the impact of all articles published that year.<br />
*** As from 1997 publication year of articles is the criterion, instead of Scientific Report-year listing.<br />
**** Self-citations not excluded for publications 2001 and later.<br />
# From <strong>2008</strong> the online publication date is used as criteria for the year of publication.
eceived a program award from the Dutch Cancer<br />
Society. The quality of research of the groups in each<br />
division is evaluated approximately every 5 years by an<br />
external site visit team. Because of the reshuffling in<br />
<strong>2008</strong> of research groups as a result of the relocation to<br />
the new research building and the fact that our<br />
International Scientific Advisory Board conducted an<br />
overall evaluation of the research in <strong>2008</strong>, no site visit of<br />
individual research groups was held in <strong>2008</strong>.<br />
Honors and appointments<br />
The <strong>NKI</strong>-AVL cannot award university degrees. However,<br />
many of our staff members hold special part-time chairs<br />
at Dutch Universities. This allows them to award PhD<br />
degrees to graduate students receiving their training at<br />
The Netherlands Cancer Institute. In <strong>2008</strong>, 27 staff<br />
members had professorships at one of the Dutch<br />
Universities. John Haanen was appointed as Professor of<br />
Translational Immunotherapy of Cancer at the Leiden<br />
University, and Daniel Peeper as professor of Functional<br />
Oncogenomics at the Free University in Amsterdam.<br />
Harry Bartelink received a doctorate honoris causa from<br />
the Academia of Medicine in Gdansk, Poland. Titia<br />
Sixma became member of the Royal Netherlands<br />
(Dutch) Academy of Arts and Sciences. Bas van Steensel<br />
and Daniel Peeper were elected as EMBO members. The<br />
<strong>AvL</strong>-prize <strong>2008</strong> for young, promising <strong>NKI</strong>-scientists was<br />
awarded to Wilbert Zwart for his work on tamoxifen<br />
resistance mechanisms.<br />
There were substantial changes in our clinical staff<br />
mostly due by retirements. Harry Masselink, Joos<br />
Lebesque and Bart van Bunningen retired as radiation<br />
oncologists after serving the Institute for periods<br />
ranging from 27 to 37 years. Corrie Marijnen left the<br />
Radiotherapy Department to become the new chair of<br />
the subsection Radiation Oncology at the Leiden<br />
University Medical Center. Jan Schornagel, Wim ten<br />
Bokkel Huinink, Nico van Zandwijk retired as medical<br />
oncologists and Jaap van der Sande as a neurooncologist.<br />
Fortunately, we have been able to recruit excellent<br />
successors. Sanne Schagen and Eveline Bleiker were<br />
appointed as tenured staff members in the Division of<br />
Psychosocial and Epidemiological Research, and<br />
Maarten Fornerod and Jos Jonkers became tenured staff<br />
members in the Division of Gene Regulation and<br />
Molecular Biology, respectively.<br />
Staff of the <strong>NKI</strong>-AVL fulfilled numerous functions in<br />
national and international organizations, on boards of<br />
scientific journals, as members of study sections, and as<br />
organizers or co-organizers of scientific meetings,<br />
workshops and congresses.<br />
15<br />
INTRODUCTION<br />
Outlook and acknowledgements<br />
We are confident that the <strong>NKI</strong>-AVL will continue to<br />
flourish, even in the face of a worldwide economic crisis<br />
which may force us to be even more frugal than we<br />
already are. Collectively, we have the scientific skills and<br />
expertise, as well as the drive and motivation to<br />
effectively compete for resources to support our<br />
activities. In the institute-wide evaluation that will take<br />
place later this year, we aim to convince the site visit<br />
team that the <strong>NKI</strong> is a unique institution that should be<br />
given full support by the Dutch Cancer Society and the<br />
Ministry of Health that provide the core funding for the<br />
Institute. Thus far, we have been very successful in<br />
securing support through a variety of funding<br />
mechanisms, and with the on-going initiatives to<br />
combine forces with other institutions and<br />
pharmaceutical industries within and outside the<br />
Netherlands we are in a good position to raise additional<br />
funds for our research. But we also need to access new<br />
sources to fund our research. The Antoni van<br />
Leeuwenhoek Research Fund that we founded together<br />
with the Dutch Cancer Society is one such endeavour.<br />
This fund can also help us to further increase our<br />
visibility to industry and private sponsors as an<br />
organization worth investing in. Real breakthroughs are<br />
more likely to come from of the combination of basic<br />
understanding of the underlying mechanisms and stateof-the-art<br />
implementation, and offer the best<br />
perspectives to substantially improve cure rates and<br />
long-term survival rates of cancer patients. This is an<br />
area in which the <strong>NKI</strong> has a strong record. I am<br />
convinced that breakthroughs in cancer diagnosis and<br />
treatment can be realized with approaches that are now<br />
broadly applied in the <strong>NKI</strong>.<br />
Our scientific output has been very robust over the years,<br />
indicating that the organization is stable and capable of<br />
making substantial contributions to reduce significantly<br />
the burden of cancer. This, together with the drive and<br />
enthusiasm that is so characteristic of the individuals<br />
that shape our institute, should convince donors that the<br />
<strong>NKI</strong>-AVL is an institution at the international forefront<br />
of cancer research and therefore worth supporting.<br />
In closing, I want to end by thanking those who<br />
continue to support us: The Dutch Cancer Society, the<br />
most reliable and significant sponsor of our research;<br />
the Ministry of Health, which provides a substantial core<br />
grant to the Institute and has provided the funds for a<br />
new hospital and renovation of our research facilities;<br />
and, last but not least, the many individuals who provide<br />
us with financial, moral, and practical support. Only<br />
with their help can we continue to develop new ideas<br />
that can improve the perspectives of cancer patients.<br />
I hope you are inspired by this report.<br />
Ton Berns<br />
Director of Research
16<br />
EDUCATION IN ONCOLOGY<br />
EDUCATION IN ONCOLOGY<br />
In the Netherlands Cancer Institute, student training<br />
takes place at various levels, from Master, PhD to postdoctoral<br />
level and involves medical workers, technical<br />
personnel and scientists. Research and clinical staff as<br />
well as their group members are involved in theoretical<br />
and practical training. A number of staff members have<br />
joint appointments as professors at Dutch universities<br />
and also contribute to the regular curriculum at these<br />
universities. The research divisions attract students from<br />
universities throughout the country. The <strong>NKI</strong> has a<br />
formal affiliation with the Science faculty of the<br />
University of Amsterdam (UvA) and is committed to<br />
make a contribution to Master student teaching. The<br />
institute participates in the Oncology Graduate School<br />
Amsterdam, together with the medical faculties of the<br />
UvA and the Free University (VU), referred to as<br />
Academic Medical Center (AMC) and VU medical center<br />
(VUMC), respectively. All educational activities are<br />
supervised by the Teaching Committee, which consists<br />
of Jannie Borst (chair and dean Master students),<br />
Hein te Riele (general affairs), Roderick Beijersbergen<br />
(Master course), John Hilkens, John Collard (HLO<br />
students and publicity), Titia Sixma (dean PhD students)<br />
and Fons Balm (clinical teaching). Peter Peters<br />
functions outside the education committee as dean for<br />
the post-docs.<br />
MASTER STUDENTS<br />
The program in Experimental Oncology attracts Master<br />
students of all national universities. Students generally<br />
have a background in (Medical) Biology, Health Sciences,<br />
Chemistry, Pharmacology, Medicine, or Psychology.<br />
The program offers combined practical and theoretical<br />
training in various aspects of experimental oncology.<br />
Practical training includes participation in ongoing<br />
research projects for a minimum of four months.<br />
In <strong>2008</strong>, 31 Dutch university students completed a<br />
placement of 5-12 months at the biomedical research<br />
divisions. As in previous years, the students came<br />
primarily from the Amsterdam Universities UvA (7) and<br />
VU (9), as well as Utrecht University (7), but also from<br />
Nijmegen (4), Wageningen (2), Leiden (1) and Delft (1).<br />
The new Life Science and Technology Masters is<br />
particularly suited to the biomedical research<br />
environment in the <strong>NKI</strong>. The institute also provides<br />
practical training opportunities to trainee technicians,<br />
who stay for similar periods of time as the university<br />
students and like these, often make significant<br />
contributions to research progress of the PhD students<br />
and post-docs who supervise them. The core element of<br />
theoretical training is the course in Experimental<br />
Oncology, given twice yearly (table 1). This course is<br />
compulsory for <strong>NKI</strong> Master students who do an<br />
internship, but in addition attracts many as students<br />
from throughout the country. We routinely host about<br />
40 students per course. It includes lectures and tutorials<br />
given by our highest level clinical and research<br />
professionals and is rated very highly in University<br />
evaluations.<br />
PHD STUDENTS<br />
The PhD students at the <strong>NKI</strong>-AVL participate in the<br />
Oncology graduate school Amsterdam (OOA) together<br />
with the oncology departments of the VUMC and the<br />
AMC. In <strong>2008</strong>, the institute had 144 PhD students<br />
registered with the OOA. Of these, 22 students defended<br />
a PhD thesis at a Dutch university.<br />
Students participate in research of their group and in<br />
interdepartmental work discussions. In addition, the<br />
students participate in the OOA training program, which<br />
consists of courses (table 2) and an annual retreat on the<br />
island Texel. Apart from courses on different topics in<br />
cancer research, the OOA offers a course on scientific<br />
English. Also, students with no prior background in<br />
cancer research can participate in the Experimental<br />
Oncology course.<br />
The PhD student retreat focuses entirely on the research<br />
of the graduate students themselves. At this retreat,<br />
students are not only presenting their work in the form<br />
of a poster in the first year and presentations in<br />
subsequent year, but they are also in charge of chairing<br />
sessions and discussions. In recent years, they also<br />
participate in peer review, giving a prize for the best<br />
poster and best presentation. In this manner, the retreat<br />
provides training in presentation and interaction skills,<br />
but it also provides an overview of the research in the<br />
OOA at an early stage of the student’s career. This<br />
provides a good opportunity for translational interaction<br />
and bottom-up research, allowing the graduate students<br />
Table 1 – Courses in Experimental Oncology<br />
Epidemiology Flora van Leeuwen<br />
Surgery Emiel Rutgers<br />
Radiodiagnostics Renato Valdes Olmos,<br />
Michiel Sinaasappel<br />
Pathology* Daphne de Jong<br />
Molecular diagnostics** Ron Kerkhoven,<br />
Sabine Linn<br />
Drug therapy Sjoerd Rodenhuis<br />
Radiotherapy* Marcel Verheij<br />
DNA damage response<br />
and apoptosis** Adrian Begg, Jannie Borst<br />
Signal transduction** Wouter Moolenaar<br />
Cell cycle** René Bernards,<br />
Daniel Peeper<br />
Cell Division Rob Wolthuis<br />
Cell senescence and<br />
genomic instability** Roderick Beijersbergen,<br />
Hein te Riele<br />
Cell adhesion** Ed Roos<br />
Tumor microenvironment Karin de Visser<br />
Mouse models of cancer** Jos Jonkers<br />
Immunology and<br />
immunotherapy** Ton Schumacher,<br />
John Haanen<br />
Analysis of protein structure Titia Sixma<br />
Rational drug development<br />
and drug delivery** Jos Beijnen,<br />
Alfred Schinkel<br />
* including tour<br />
** including tutorial
Table 2 – OOA graduate student courses <strong>2008</strong><br />
9-12 March The Macroscopic and Pathologic<br />
Anatomy of the mouse<br />
WH Lamers, CJF van Noorden<br />
April English Writing and Presenting<br />
Ann Bless<br />
14-16 April Epigenetics<br />
R Steenbergen, T vd Elsen, J Kooter<br />
11-20 May Signalling<br />
Wouter Moolenaar and<br />
Arnoud Sonnenberg<br />
Spring Stem cells and Cancer<br />
Jan Paul Medema<br />
14-16 October Annual Graduate Student Retreat<br />
Titia Sixma<br />
October Inflammation and Cancer<br />
Ed Roos en Karin de Visser<br />
November English Writing and Presenting<br />
Ann Bless<br />
themselves to contribute significantly to interaction<br />
between different research groups.<br />
In addition, senior graduate students can participate in a<br />
joint retreat with other cancer institutes in Europe. In<br />
<strong>2008</strong>, this event was hosted by the <strong>NKI</strong> and the excellent<br />
organization was performed by four <strong>NKI</strong> graduate<br />
students (Floor Frederiks, Bernike Kalverda, Jasper<br />
Mullenders and Daan Peric Hupkes). Participants from<br />
British CrUK institutes, the Italian IFOM and the<br />
Spanish CNIO contributed to a program of scientific<br />
lectures and posters as well as an enthusiastic social<br />
session. This retreat gives students the opportunity to<br />
compare notes among excellent cancer institutes<br />
throughout Europe.<br />
Each student has a supervisory committee that meets<br />
once a year to monitor scientific progress. The committee<br />
discusses progress with the supervisor and student<br />
separately and participates in a joint discussion of the<br />
research. The students are represented in the OIOcouncil<br />
that meets with the Dean of graduate student<br />
affairs on a regular basis, as well as upon special request.<br />
POST-DOCS<br />
Post-docs in the Netherlands Cancer Institute perform<br />
challenging research in an internationally competitive<br />
environment. They participate in work discussions both<br />
within the group and among different divisions. Within<br />
the research group there are often opportunities to<br />
receive training in supervision, manuscript writing and<br />
presentation. In many cases, postdocs in the biological<br />
sciences, medicine and technology have a preference<br />
for working in an institute or university and indeed,<br />
many of our post-docs go on to careers in science.<br />
Still, there are many other options available for an<br />
interesting career after a successful postdoc period.<br />
Many postdocs are unaware of these possibilities, or<br />
they may consider it too great a step to go into biotech,<br />
industry, journalism or a government position. Since<br />
only 20% of postdocs are likely to get a permanent<br />
position in academia, it can be of interest to have a good<br />
look around during your postdoc period and to find out<br />
17<br />
EDUCATION IN ONCOLOGY<br />
about all the possibilities for the next step in your career.<br />
This year we were very happy to be able to receive a grant<br />
from the ministry of economic affairs to professionalize<br />
our ten year <strong>NKI</strong>-AVL history of postdoc activities in<br />
order to create an organization which we called PCDI for<br />
Postdoc Career Development Initiative with a nationwide<br />
activity. We have not only offered new perspectives, but<br />
also helped improving skills that will increase career<br />
choices, both within and outside academia. The activities<br />
we have offered can be visited at www.pcdi.nl.<br />
The PCDI organized two postdoc retreats at Kapellerput,<br />
which were held from 9-11th of April and from 5-7th<br />
of November <strong>2008</strong>. Each time we reached hundred<br />
participants and were overbooked. Thanks to the<br />
organizing committees we had great inspirational<br />
speakers, among which Bruce Alberts, Hans Clevers,<br />
Peter Friedl, Bart Noordam but also young successful<br />
scientists, business managers and entrepreneurs.<br />
The workshops on transferable skills were well received,<br />
and we look back at stimulating networking days with<br />
enthusiastic ex-postdocs and other representatives<br />
working in academia, companies and non-profit<br />
organizations. The retreats scored in an anonymous<br />
evaluation between very good and excellent, which will<br />
hopefully inspire the participating postdocs and last year<br />
PhD students to make the right choices with regard to<br />
their career.
18<br />
BIOCHEMISTRY<br />
Division head, group leader Titia Sixma<br />
Titia Sixma PhD Group leader<br />
Alex Fish PhD Post-doc<br />
Rick Hibbert PhD Post-doc<br />
Prakash Rucktooa PhD Post-doc<br />
Andrea Alfieri PhD Post-doc<br />
Alex Faesen MSc PhD student<br />
Mark Vargas MSc PhD student<br />
Annet Reumer MSc PhD student<br />
Francesca Mattiroli MSc PhD student<br />
Judith Smit MSc PhD student<br />
Pim Van Dijk Technical staff<br />
Herrie Winterwerp Technical staff<br />
DIVISION OF BIOCHEMISTRY<br />
STRUCTURAL BIOLOGY<br />
Development of cancer is generally due to errors that occur in cellular pathways.<br />
Structural biology can help to understand these errors at the atomic level, by studying<br />
the molecules involved. We use X-ray crystallography as a tool to provide threedimensional<br />
structures and we interpret the structural data using a variety of<br />
biochemical and biophysical techniques. These studies provide more insight in the<br />
molecular processes and they can also provide targets for drug design studies. In our<br />
group we focus mainly on proteins involved in ubiquitin conjugation in chromatin<br />
regulation, on DNA mismatch repair and nicotinic receptor signaling.<br />
DNA mismatch repair DNA mismatch repair plays a crucial role in ensuring<br />
genomic stability. Defects in the mismatch repair cascade in humans predispose to<br />
hereditary non-polyposis colorectal cancer and are associated with a variety of<br />
sporadic cancers. DNA mismatch repair is initiated by the protein MutS (in<br />
Escherichia coli) or its MSH homologs in eukaryotes. MutS recognizes and binds to<br />
mismatches or unpaired bases that have escaped the proofreading capacity of the<br />
DNA replication machinery or are present in the genome during recombinatorial<br />
events between non-fully complementary DNA strands. Mismatch binding triggers<br />
the uptake of ATP in the MutS nucleotide-binding domain and it is this mismatchdependent<br />
ATP state that authorizes repair by recruitment of additional mismatch<br />
repair components. In collaboration with Robert van den Heuvel in Utrecht we have<br />
used native mass spectrometry to study MutS DNA complexes. In a collaboration<br />
with Serge Cohen and Tassos Perrakis a novel method was developed that will allow<br />
us to analyze the detailed nucleotide status of these large complexes. In collaboration<br />
with Peter Friedhoff and Martin Marinus we studied a dimeric mutant of the MutS<br />
protein. This mutant allows detailed kinetic studies and structure determination of<br />
the full-length protein, alone and in complexes. Analysis of these data is beginning to<br />
yield interesting insight into the mechanism of mismatch discrimination (figure 1).<br />
Figure 1: Surface plasmon<br />
resonance analysis of DNA<br />
mismatch repair protein MutS<br />
biniding to mismatched DNA.<br />
Use of a dimer mutant allows<br />
kinetic fitting of DNA binding.<br />
Interestingly, it is not the search<br />
process, but the off-rate that<br />
provide discrimination between<br />
mismatch and homoduplex DNA.<br />
Ubiquitin and SUMO conjugation Ubiquitin conjugation is critical for signaling in<br />
almost all cellular processes, including DNA repair, apoptosis, cell cycle, chromatin<br />
regulation and endocytosis. Since these processes are of so important for cellular<br />
stability, deregulation of ubiquitin-dependent processes often leads to cancer.<br />
Structure analysis of the proteins involved in ubiquitin and SUMO conjugation could<br />
contribute to the development of novel drugs wich are directed towards critical
pathways in ubiquitin and SUMO conjugation. The process of conjugation by<br />
ubiquitin-(like) proteins involves covalent linking of one or more of the 76-aminoacids<br />
within ubiquitin to a target protein by an E1/E2/E3 cascade of enzymes. Correct<br />
ubiquitination requires the complex spatial arrangement of ubiquitin, E2, E3 proteins<br />
and the target simultaneously in a precise but flexible manner. Although the overall<br />
mechanism has been defined, many questions on selectivity and specificity remain.<br />
We study distinct E2/E3 complexes involved in conjugation of ubiquitin and the<br />
related modifier SUMO as well as proteins involved in de-ubiquitination.<br />
One interesting aspect of SUMO compared to ubiquitin is its specificity for certain<br />
sequences. In collaboration with Klaus Schwamborn at Pepscan a chip was developed<br />
that allows studying this specificity in an efficient manner (Schwamborn et al, <strong>2008</strong>).<br />
The E2 ubiquitin conjugating enzyme for sumoylation, Ubc9, can itself be modified<br />
by SUMO (figure 2).<br />
Figure 2: The activity for the E2 for sumoylation, Ubc9, can be modulated by SUMO itself in two different<br />
manners: Right: Binding of SUMO to Ubc9 in a non-covalent interaction, the so-called back-side binding,<br />
affects SUMO chain formation (Knipscheer et al, EMBO J. 2007). Left: The covalent SUMO modification<br />
of mammalian Ubc9 on K14 affects the target choice, increasing the rate of modification of the SIM<br />
containing target Sp100, but limits its activity against RanGAP (Knipscheer et al, Mol Cell <strong>2008</strong>)<br />
In collaboration with Andrea Pichlers group in Vienna, we showed that this<br />
modification is important for target choice of the E2 enzyme (Knipscheer et al,<br />
<strong>2008</strong>). It can enhance modification of the Sp100 transcription factor and we showed<br />
that this enhanced modification is due to binding of the covalently bound SUMO to<br />
the target via a SUMO Interaction Motif (SIM). Surprisingly the site of SUMO<br />
modification on Ubc9 is equivalent to the site of SUMO modification of E2-25K, an<br />
E2 enzyme for ubiquitin, but the consequence of the latter modification is radically<br />
different (Pichler et al, 2005). Our crystal structure suggested that this difference<br />
could be due to intramolecular interaction of the attached SUMO with a Ubc9specific<br />
beta-hairipin and indeed, specific removal of the hairpin, by replacing it with<br />
a di-glycine sequence causes a shift in the effect of E2 modification to resemble that<br />
in E2-25K (Knipscheer et al, <strong>2008</strong>).<br />
DNA translesion synthesis (TLS) allows for tolerance to DNA damage and synthesis<br />
beyond the mismatch during replication and avoids replication stallimg. This process<br />
is initiated by ubiquitin modification of PCNA by the ubiquitin conjugating E2/E3<br />
enzyme complex Rad6/Rad18. In our studies of the Rad6/Rad18 activity we used<br />
thermodynamic analysis of the DNA binding properties. Studying the ZnF domain<br />
we were able to compare the binding properties of ubiquitin and short chains of<br />
ubiquitin (Crosetto et al, <strong>2008</strong>).<br />
Nicotinic Acetylcholine receptor homolog AchBP In collaboration with the<br />
groups of Guus Smit and Iwan de Esch at the Free University in Amsterdam we<br />
study the Acetylcholine Binding Proteins (AChBP) as tools for understanding the<br />
ligand binding in pentameric ligand-gated ion-channels. AChBP has strong sequence<br />
similarity to the a-subunits of the nicotinic acetylcholine receptor ligand-binding<br />
domain and our AChBP crystal structures are the established high-resolution model<br />
for the ligand-binding domains in this class of ion channels.<br />
Based on in silico screening we found a number of novel molecules that bind AChBP.<br />
Structural analysis showed that binding was indeed present, but with considerable<br />
variability in the binding. The analysis of these compounds on nAChRs indicate that<br />
these compounds compete with bungarotoxin, implying binding to the ligand<br />
binding site. In addition, however, they seem to bind to the transmembrane pore,<br />
precluding further development as lead compounds. Further studies focus on<br />
additional hits from the in silico screen.<br />
Publications<br />
19<br />
BIOCHEMISTRY<br />
Bertaccini EJ, Lindahl E, Sixma T,<br />
Trudell JR. Effect of cobratoxin binding on<br />
the normal mode vibration within<br />
acetylcholine binding protein. J Chem Inf<br />
Model. <strong>2008</strong>;48:855-60<br />
Cohen SX, Ben Jelloul M, Long F, Vagin A,<br />
Knipscheer P, Lebbink J, et al. ARP/wARP<br />
and molecular replacement: the next<br />
generation. Acta Crystallogr D Biol<br />
Crystallogr. <strong>2008</strong>;64:49-60<br />
Crosetto N, Bienko M, Hibbert RG,<br />
Perica T, Ambrogio C, Kensche T, et al.<br />
Human WRNIP1 is localized in replication<br />
factories in a UBZ-dependent manner. J<br />
Biol Chem. <strong>2008</strong><br />
Knipscheer P, Flotho A, Klug H, Olsen JV,<br />
van Dijk WJ, Fish A, et al. Ubc9<br />
sumoylation regulates SUMO target<br />
discrimination. Mol Cell. <strong>2008</strong>;31:371-82<br />
Schwamborn K, Knipscheer P, van Dijk E,<br />
van Dijk WJ, Sixma TK, Meloen RH, et al.<br />
SUMO assay with peptide arrays on solid<br />
support: insights into SUMO target sites.<br />
J Biochem. <strong>2008</strong>;144:39-49<br />
van Leuken RJ, Luna-Vargas MP, Sixma TK,<br />
Wolthuis RM, Medema RH. Usp39 is<br />
essential for mitotic spindle checkpoint<br />
integrity and controls mRNA-levels of<br />
aurora B. Cell Cycle. <strong>2008</strong>;7:2710-9
20<br />
BIOCHEMISTRY<br />
BIOCHEMISTRY<br />
Group leader Anastassis Perrakis<br />
Anastassis Perrakis PhD group leader<br />
Patrick Celie PhD Post-doc<br />
Serge Cohen PhD Post-doc<br />
Valeria De Marco PhD Post-doc<br />
Dene Littler PhD Post-doc<br />
Krista Joosten PhD Post-doc<br />
Wijnand Mooij PhD Post-doc<br />
Eirini Mitsiki PhD Post-doc<br />
Jens Hausmann MSc PhD student<br />
Evangelos Christodoulou Technical staff<br />
Tatjana Heidebrecht Technical staff<br />
Diederick De Vries Technical staff<br />
Publications<br />
van de Weerdt BC, Littler DR, Klompmaker R,<br />
Huseinovic A, Fish A, Perrakis A, et al.<br />
Polo-box domains confer target specificity to<br />
the Polo-like kinase family. Biochim<br />
Biophys Acta. <strong>2008</strong> Jun;1783(6):1015-22.<br />
Repanas K, Fuentes G, Cohen SX, Bonvin AM,<br />
Perrakis A. Insights into the DNA cleavage<br />
mechanism of human LINE-1 retrotransposon<br />
endonuclease. Proteins. <strong>2008</strong><br />
Perrakis A, Romier C. Assembly of protein<br />
complexes by coexpression in prokaryotic<br />
and eukaryotic hosts: an overview. Methods<br />
Mol Biol. <strong>2008</strong>;426:247-56<br />
Langer G, Cohen SX, Lamzin VS, Perrakis A.<br />
Automated macromolecular model building<br />
for X-ray crystallography using ARP/wARP<br />
version 7. Nat Protoc. <strong>2008</strong>;3:1171-9<br />
Joosten K, Cohen SX, Emsley P, Mooij W,<br />
Lamzin VS, Perrakis A. A knowledgedriven<br />
approach for crystallographic protein<br />
model completion. Acta Crystallogr D Biol<br />
Crystallogr. <strong>2008</strong>;64:416-24<br />
Cohen SX, Ben Jelloul M, Long F, Vagin A,<br />
Knipscheer P, Lebbink J, et al. ARP/<br />
wARP and molecular replacement: the next<br />
generation. Acta Crystallogr D Biol<br />
Crystallogr. <strong>2008</strong>;64:49-60<br />
STRUCTURAL BIOLOGY<br />
This year our biology focus remained around mechanisms for correct DNA<br />
inheritance: DNA replication and the subsequent chromatide separation between the<br />
daughter cells. This interest is demonstrated by our work in the DNA replication<br />
license and in spindle assembly checkpoint proteins.<br />
Our involvement on three in-house collaborations remained active, trying to<br />
determine structures that are of importance to in-house research: we work with<br />
Wouter Moolenaar on ATX, with Piet Borst on JBP1 and with Ton Schumacher on<br />
MHC class I structures.<br />
Work in computation methods went on, with many steps taken towards new<br />
algorithms for crystallographic model completion and consolidating the control<br />
structures of the ARP/wARP software. A new side product of our computation effort<br />
was a tool to facilitate the design of truncated proteins suitable for protein expression<br />
experiments.<br />
DNA replication licensing and the role of the Cdt1/Geminin complex This year<br />
we finalized our work on the function of the Geminin: Cdt1 complex, an inhibitor of<br />
the formation of the pre-replication complex, which is necessary to be assembled<br />
prior to DNA replication in eukaryotic cells. Based on our crystal structure of a<br />
human truncated Geminin/Cdt1 complex and X-ray solution scattering experiments<br />
(SAXS) we demonstrated that the complex can exist both as a heterotrimer and a<br />
heterohexamer under physiological conditions, possibly representing a conformation<br />
switch. In vivo experiments in mammalian cells and in Xenopus oocyte extracts<br />
conclusively support our conclusions for the function of this molecular switch.<br />
Structural studies of proteins involved in mitotic progression Our interest in<br />
this area was initiated by the work on Polo-like kinases, together with the group of<br />
Rene Medema. We have recently shown with biophysical measurements combined<br />
with cell-based assays that the polo binding domains (PBDs) of Plk1-3 show<br />
unexpected differences. We demonstrated a partial functional overlap but also<br />
distinct roles of the different PBDs.<br />
Last year we have initiated a high-throughput approach towards characterising<br />
different proteins involved in mitotic progression and obtained the first crystals.<br />
We have made good progress improving the crystals of a regulatory domain of BubR1<br />
and we are about to determine the structure of a regulatory domain of TTK/Mps1.<br />
We have also determined the structure of the DNA-binding domain the FoxM1<br />
transcription factor (figure 3).<br />
Figure 3: (A) A cartoon representation of the structure of the complex of the FoxM1 DNA binding domain<br />
bound to DNA and the assumed position of the N- and C-terminal domains. (B) Binding of FOXM1 to<br />
Random DNA, a singe recognition sequence and a double recognition sequence and (C) the occurrence of<br />
FOXM1 DNA recognition sequnces in the 3,000 bp flanking regions of human genes.
FoxM1 controls the production of genes involved in cell cycle progression and<br />
control, such as Cyclin B2, Plk1 and securin. FoxM1 is highly expressed during<br />
development, but it is also known to be present in numerous cancer cell-lines. More<br />
excitingly, in the laboratory the artificial-inhibition of FoxM1 has been shown to<br />
noticeably reduce cell-viability in primary breast cancer cell-lines. This behaviour<br />
makes a clear understanding of how FoxM1 functions at a molecular level highly<br />
desirable. The structure of the domain of FoxM1 responsible for DNA-binding, tells<br />
us much about its sequence specificity. We will extend this work to learn more about<br />
the regulation of FoxM1’s transcriptional activity, which in the longer-term can aid the<br />
development of inhibitors against this potential therapeutic target.<br />
Structural studies of autotaxin Autotaxin (NPP1) hydrolyzes circulating<br />
lysophosphatidylcholine. Engineering of a human cell line (HEK293 derivative) that<br />
is stably expressing glycosylation mutants of rat autotaxin was the key for<br />
crystallization success, resulting to crystals diffracting to high resolution. Structure<br />
solution has been hampered by technical issues, such as low protein yield of Seleno-<br />
Methionine substituted ATX and small crystal size. We are still in the process of<br />
determining the structure.<br />
Structural studies of JBP1 The JBP1 protein, discovered by the Piet Borst group,<br />
binds to DNA that contains base J (J-DNA). It has been shown to be essential for<br />
survival in major protozoal human pathogens such as T. brucei (sleeping sickness), T.<br />
cruzi (Chagas’ disease) and Leishmania species (various types of Leishmaniasis).<br />
Recently we were able to crystallize the DNA-binding domain of JBP1 in complex<br />
with J-DNA and we will proceed with structure determination this year.<br />
Structural studies of MHC class I molecules Major histocompatibility complex<br />
(MHC) class I molecules associate with a variety of peptide ligands and present these<br />
ligands on the cell surface for recognition by cytotoxic T cells. Together with the<br />
groups of Ton Schumacher (H7) and Huib Ovaa (H3) we have generated a protocol<br />
for the in crystallo exchange of a photo-cleavable Se-Met labelled peptide bound to a<br />
class I molecule, with a new peptide, alleviating the need to refold and crystallize<br />
every new class I - peptide complex. We have determined a few structures that<br />
demonstrate that this strategy can produce good quality MHC class I - peptide<br />
complexes.<br />
Methods for high throughput Structural Biology The PiMS protein information<br />
management system work was finished early this year, allowing the handling of<br />
crystallization data. The end of the BIOXHIT network for methods development in<br />
X-ray crystallography marks the end of our work in this area.<br />
We carried on with an in-house development focused on the creation of a collection<br />
of versatile ligation independent cloning (LIC) vectors suitable for E. coli, insect and<br />
mammalian cells expression. This development allows a single PCR product to be<br />
directly transferred to the vector of choice for all available expression systems. As part<br />
of the 3D-Repertoire network we are participating in a large-scale test for the<br />
recombinant production of binary complexes, using our vectors. As part of the<br />
SPINE-2 network we have also benchmarked our vectors together with similar<br />
systems for recombinant expression, with encouraging results.<br />
As part of the effort to be able to design easily cloning experiments for the expression<br />
of truncation variants of our proteins of interest, we have also created the<br />
proteinCCD server for enabling the design of oligo-nucleotides for such experiments<br />
(http://xtal.nki.nl/CCD)<br />
Methods for X-ray crystallography The development within the ARP/wARP<br />
framework, focused on developing ‘conditional restraints’ for enhancing hybrid<br />
model refinement at a low resolution, density based validation through statistical<br />
targets, some fundamental work on algorithms for extracting density features from<br />
electron density regions of predefined shapes, and a new loop building algorithm.<br />
All these development will allow better model building in the future versions of<br />
ARP/wARP.<br />
21<br />
BIOCHEMISTRY<br />
Figure 4: Crystals of ATX, FoxM1/DNA,<br />
JBP/DNA with and without polarize filters,<br />
two MHC class I complexes after soaking,<br />
Mps1 and BubR1 kinases regulatory<br />
domains.
22<br />
MOLECULAR CARCINOGENESIS<br />
CELL BIOLOGY I<br />
Division head, group leader Arnoud Sonnenberg<br />
Arnoud Sonnenberg PhD Group leader<br />
Ruben Postel PhD Post-doc<br />
Pablo Secades PhD Post-doc<br />
Quint Le Duc MSc PhD student<br />
Evelyne Frijns MSc PhD student<br />
Mirjam Ketema MSc PhD student<br />
Coert Margadant MSc PhD student<br />
Norman Sachs MSc PhD student<br />
Maaike Kreft Technical staff<br />
Ingrid Kuikman Technical staff<br />
Publications<br />
van den Bout I, van Rheenen J, van<br />
Angelen AA, de Rooij J, Wilhelmsen K,<br />
Jalink K, Divecha N, Sonnenberg A.<br />
Investigation into the mechanism regulating<br />
MRP localization. Exp Cell Res<br />
<strong>2008</strong>;314:330-41<br />
van den Bout I. Identification and<br />
functional analysis of genes regulated by b1<br />
and b3 integrins. Academic thesis,<br />
University of Amsterdam <strong>2008</strong><br />
Huveneers S, Arslan S, van de Water B,<br />
Sonnenberg A, Danen EH. Integrins<br />
uncouple Src-induced morphological and<br />
oncogenic transformation. J Biol Chem<br />
<strong>2008</strong>;283:13243-51<br />
Figure 1: Localization of zebrafish<br />
Nesprin-3a at the nuclear envelope in<br />
transiently transfected keratinocytes.<br />
Confocal images of cells expressing GFPnesprin-3a<br />
stained with DAPI to visualize<br />
nuclei.<br />
DIVISION OF CELL BIOLOGY I<br />
RECEPTORS FOR MATRIX ADHESION<br />
Torsin A influences the interaction between nesprin-3 and Sun proteins We<br />
have previously identified a new member of the nesprin protein family, nesprin-3,<br />
that binds to the cytoskeletal linker protein plectin, thereby establishing a link<br />
between the intermediate filament system and the nucleus. The localization of<br />
nesprin-3 at the outer nuclear membrane depends on its interaction with Sun<br />
proteins present at the inner nuclear membrane. We have recently identified an<br />
additional binding partner of nesprin-3, torsin A, which might influence the<br />
localization of nesprin-3 by regulating the interaction of nesprin-3 with Sun proteins.<br />
Torsin A is an AAA+ ATPase, which resides in the lumen of the endoplasmic<br />
reticulum and the nuclear envelope, and mutation of torsin A causes the movement<br />
disorder early onset torsion dystonia. The interaction between nesprin-3 and torsin A<br />
does not require the Sun binding motif of the nesprin-3 C-terminal KASH domain<br />
and is independent of the ATPase activity of torsin A. However, the ATPase activity of<br />
torsin A seems to be involved in establishing the binding of nesprin-3 to Sun<br />
proteins, since co-precipitation of Sun2 with nesprin-3 is reduced in the presence of<br />
ATPase-deficient torsin A mutants. We are currently investigating the effect of torsin<br />
A knock-down on the localization of nesprin-3 and its interaction with Sun proteins.<br />
Furthermore, a family member of torsin A, torsin B, can interact with nesprin-3 in a<br />
similar manner, indicating a potential redundancy in function.<br />
Function of nesprin-3 in zebrafish In order to reveal the function of nesprin-3 in<br />
vivo, we used the zebrafish as a model system. Cloning the full-length zebrafish<br />
nesprin-3 mRNA revealed (as in the mouse) two variants: nesprin-3a and nesprin-3b.<br />
The zebrafish nesprin-3a transcript encodes a protein of 1100 amino acids, which<br />
contains the conserved plectin-binding domain at the N-terminus as well as the<br />
C-terminal conserved KASH domain at the C-terminus. When overexpressed in cells<br />
zebrafish nesprin-3a-GFP is localized at the nuclear envelope. The nesprin-3b<br />
transcript appears to have used an alternative splice site in exon 1, resulting in an inframe<br />
deletion of 7 amino acids in the plectin-binding domain. Additionally, we<br />
observed that both the nesprin-3a and nesprin-3b transcripts become expressed<br />
dynamically during the first days of development. Furthermore, in situ hybridization<br />
with dig-labelled antisense nesprin-3 RNA revealed maternal and ubiquitous nesprin-3<br />
mRNA expression during early embryonic development. Morpholino-mediated<br />
knockdown of zygotic nesprin-3 in zebrafish resulted in blistering of the epidermis<br />
already 2-3 days post fertilization. This suggests that loss of nesprin-3 in vivo affects<br />
the complete cell morphology in epidermal cells. A detailed analysis of the epidermal<br />
cell phenotype in these nesprin-3 knock-down zebrafish embryos is ongoing.<br />
Modulating podocyte adhesion Cell adhesion to the extracellular matrix can be<br />
regulated by altering the adhesive properties of integrins. A candidate protein in<br />
modulating the adhesive strength of certain integrins is the tetraspanin CD151.<br />
CD151 tightly associates with the class of laminin-binding integrins, especially a3b1.<br />
Recently, we generated CD151 knockout mice which develop severe kidney failure<br />
similar to that in patients with mutations in the CD151 gene primary glomerular<br />
disease. Electron microscopy analysis revealed that CD151 as well as the integrin a3b1<br />
are present at the basal side of podocyte foot processes. These highly specialized<br />
structures anchor podocytes to the underlying basement membrane to withstand the<br />
permanent transcapillary filtration pressure in the kidney glomerulus. Mice lacking<br />
either CD151 or a3b1 specifically in podocytes show a remarkably similar kidney<br />
phenotype to that seen in mice in which CD151 was totally knocked out, suggesting<br />
that podocyte defects are the cause for the disease. In vitro analysis indeed revealed<br />
that podocytes without CD151 detach from a laminin matrix at far lower forces than<br />
required to detach podocytes with CD151. Whereas re-expression of wild-type CD151<br />
rescued the adhesion deficit, mutant CD151 incapable of binding a3b1 did not.<br />
Podocyte adhesion via a3b1 alone may be sufficient to withstand moderate filtration
pressures, but CD151 is necessary to cope with prolonged and/or high filtration<br />
pressure. Furthermore, we show that mice lacking CD151 develop kidney failure only<br />
on a pure FVB/N strain, but not on a pure C57Bl6/J strain. The latter strain is known<br />
to have a lower blood and consequently filtration pressure. CD151 mutations in<br />
humans may therefore predispose to kidney failure which is manifested when<br />
transcapillary filtration pressure rises, for example due to hypertension. We are<br />
currently investigating this hypothesis.<br />
Figure 2: Glomerular abnormalities in CD151KO mice.<br />
Electron micrograph of a 3-month-old CD151KO mouse<br />
glomerulus reveals abnormal capillary loops characterized<br />
by thickening of the basement membrane (*) and loss of<br />
the regular pattern of podocyte foot processes (arrows).<br />
These initial lesions are responsible for the later kidney<br />
failure.<br />
Conditional deletion of the Itgb4 integrin gene in Schwann cells leads to<br />
delayed peripheral nerve regeneration Several different integrins participate in<br />
the complex interactions that promote repair of the peripheral nervous system. In<br />
collaboration with Dr. I van der Zee (NCMLS, Nijmegen), we have investigated the<br />
role of the integrin a6b4 in peripheral nerve regeneration in mice by cre-mediated<br />
deletion of the Itgb4 (b4) gene in Schwann cells. After a crush lesion of the sciatic<br />
nerve, the recovery of motor, but not that of sensory, nerve function in b4 (-/-) mice<br />
was delayed. Immunostaining of neurofilament-200 showed that there also is a<br />
significant reduction in the number of newly outgrowing nerve sprouts in b4 (-/-)<br />
mice. Morphometric quantitative measurements revealed that fewer axons are<br />
myelinated in the nonlesioned b4 (-/-) nerves. After a sciatic nerve crush lesion, b4<br />
(-/-) mice did not only have fewer myelinated axons compared with lesioned wild-type<br />
nerve, but their axons also showed a higher g-ratio and a thinner myelin sheath,<br />
pointing at reduced myelination. This study revealed that the b4 protein remains<br />
expressed in the early stages of peripheral regeneration, albeit at levels lower than<br />
those before the lesion was inflicted, and showed that laminin deposition is not<br />
altered in the absence of b4. These results together demonstrate that integrin a6b4<br />
plays an essential role in axonal regeneration and subsequent myelination.<br />
Kindlin-1 is an essential activator of b1 integrin Kindler syndrome (KS) is a rare<br />
autosomal recessive skin disorder mainly characterized by severe skin blistering. KS<br />
patients all carry loss-of-function mutations on both alleles of a gene designated<br />
KIND1, encoding the protein kindlin-1. Kindlin-1 is part of a protein family including<br />
kindlin-2 (Mig-2) and kindlin-3 (URP-1). Whereas expression of kindlin-2 is more<br />
ubiquitous, expression of kindlin-3 is restricted to haematopoietic cells, and kindlin-1<br />
is predominantly expressed in the colon and the epidermis. Both kindlin-2 and<br />
kindlin-3 have been shown to be essential for integrin activation. We have<br />
investigated the function of kindlin-1 using normal human keratinocytes (NHK) and<br />
kindlin-1 deficient cells isolated from 2 different KS patients. In both KS patients<br />
there was a duplication of the basement membrane and there were microblisters<br />
along the epidermal-dermal junction. We have previously established that deletion of<br />
the integrin a3b1 in the epidermis causes the same phenotype in mice. In vitro, we<br />
find that kindlin-1 is colocalized with b1 integrins in focal adhesions and is associated<br />
with the actin, but not the tubulin or keratin cytoskeletal systems. Cell adhesion, cell<br />
spreading, cytoskeletal organization, and proliferation on collagens and on laminins<br />
are severely impaired in KS cells as compared to NHK. The defects are partially<br />
rescued by outside-in activation of b1 integrins with the activating antibody TS2/16.<br />
Further, the defects are reversed by stable reconstitution of KS cells with kindlin-1.<br />
Summarizing, we have identified kindlin-1 to be an essential activator of b1 integrins<br />
in the skin. Studies to identify the mechanism of activation are ongoing.<br />
Publications (continued)<br />
23<br />
CELL BIOLOGY I<br />
Huveneers S, Truong H, Fässler R,<br />
Sonnenberg A, Danen EHJ. Binding of<br />
soluble fibronectin to integrin a5b1 – link to<br />
focal adhesion redistribution and contractile<br />
shape. J Cell Sci <strong>2008</strong>;121:2452-62<br />
Huveneers S. The regulation of Rho and<br />
Src signaling Academic thesis, University of<br />
Amsterdam, <strong>2008</strong><br />
Margadant C, Frijns E, Wilhelmsen K,<br />
Sonnenberg A. Regulation of<br />
hemidesmosome disassembly by growth<br />
factor receptors. Curr Op Cell Biol<br />
<strong>2008</strong>;20:589-96<br />
Braam SR, Zeinstra L, Litjens S,<br />
Ward-van Oostwaard D, van den Brink S,<br />
van Laake L, Lebrin F, Kats P,<br />
Hochstenbach R, Passier R, Sonnenberg<br />
A, Mummery CL. Recombinant vitronectin<br />
is a functionally defined substrate that<br />
supports human embryonic stem cell self<br />
renewal via avb5 integrin. Stem Cells<br />
<strong>2008</strong>;26:2257-65<br />
Yañez-Mó M, Barreiro O, Gonzalo P,<br />
Batista A, Megias D, Genis L, Sachs N,<br />
Sala-Valdés M, Alonso MA, Montoya MC,<br />
Sonnenberg A, Arroyo AG,<br />
Sánchez-Madrid F. MT1-MMP<br />
collagenolytic activity is regulated through<br />
association with tetraspanin CD151 in<br />
primary endothelial cells. Blood<br />
<strong>2008</strong>;112:3217-26<br />
Nery FC, Zeng J, Niland BP, Hewett J,<br />
Farley J, Irimia D, Li Y, Wiche G,<br />
Sonnenberg A, Breakefield XO. TorsinA<br />
binds the KASH domain of nesprins and<br />
participates in linkage between nuclear<br />
envelope and cytoskeleton. J Cell Sci<br />
<strong>2008</strong>;121:3476-86<br />
van der Zee CEEM, Kreft M, Beckers G,<br />
Kuipers A, Sonnenberg A. Conditional<br />
deletion of the Itgb4 integrin gene in<br />
Schwann cells leads to delayed peripheral<br />
nerve regeneration. J Neuroscience<br />
<strong>2008</strong>;28:11292-303<br />
Kanters E, van Rijssel J, Hensbergen PJ,<br />
Hondius D, Mul FP, Deelder AM,<br />
Sonnenberg A, van Buul JD, Hordijk P.<br />
Filamin B mediates ICAM-1-driven<br />
leukocyte transendothelial migration. J Biol<br />
Chem <strong>2008</strong>;283;31830-9
24<br />
CELL BIOLOGY I<br />
Division head, group leader Wouter Moolenaar<br />
Wouter Moolenaar PhD Group leader<br />
Maikel Jongsma PhD Post-doc<br />
Dalila Elouarrat MSc PhD student<br />
Anna Houben MSc PhD student<br />
Bas Ponsioen MSc PhD student<br />
Leonie Van Zeijl MSc PhD student<br />
Trudi Hengeveld Technical staff<br />
Adrian Rzadkowski Technical staff<br />
LIPID GROWTH FACTOR SIGNALING<br />
Our group has a longstanding interest in lipid growth factors, particularly<br />
lysophosphatidic acid (LPA) and sphingosine 1-phosphate (S1P), their signaling<br />
properties and their role in health and disease. LPA and S1P signal through specific<br />
G protein-coupled receptors to stimulate cell migration, proliferation and survival.<br />
LPA receptor signaling has been implicated in wound healing, embryonic<br />
development and cancer. LPA is produced by a secreted lysophospholipase D<br />
(lysoPLD), named autotaxin (ATX), an autocrine motility factor for tumor cells and<br />
implicated in tumor progression and angiogenesis. Our current research focuses on<br />
the characterization of novel LPA receptors as well as the regulation of ATX and its in<br />
vivo functions. We have developed ATX activity assays, which have successfully been<br />
used for high-throughput screening of small-molecule compound libraries. Since<br />
ATX is found overexpressed in various cancers, whilst LPA promotes tumor<br />
progression in mouse models, ATX qualifies as a potential target for therapy. Our<br />
work should lead to novel ways of interfering with LPA receptor signalling cells and<br />
with inappropriate LPA production in the cellular microenvironment.<br />
A second line of research is devoted to the regulation of cell-cell communication via<br />
gap junctions, which is often defective in tumor cells.<br />
LPA receptor signaling: Rho activation, gene expression and oncogenic<br />
potential<br />
The LPA-Rho activation pathway: identification of a new player<br />
LPA receptors strongly couple to the G(13)-RhoA pathway to regulate the actin<br />
cytoskeleton. Through live-cell imaging studies, we recently discovered that LPAinduced<br />
RhoA activation is consistently accompanied by rapid recruitment of a<br />
‘Chloride Intracellular Channel’ protein (CLIC) to the plasma membrane (figure 3).<br />
CLIC family members are soluble proteins that have been proposed to auto-insert<br />
into membranes to form chloride channels. Using patch-clamp technology, we are<br />
currently examining whether LPA-induced CLIC translocation results in enhanced<br />
chloride currents across the plasma membrane (collaboration Kees Jalink).<br />
Figure 3: Rapid recruitment of CLIC4 to the plasma membrane in response to RhoA-activating receptor<br />
agonists (LPA or TRP).<br />
Expression profiling<br />
To examine how LPA receptor signaling influences global gene transcription, we<br />
subjected mouse embryonic fibroblasts (MEFs) to microarray analysis using 32K<br />
oligo-arrays. About 900 PA-regulated genes could be clustered into groups based on<br />
their temporal patterns and biological functions. In addition to genes involved in<br />
growth stimulation and cytoskeletal reorganization, LPA induced many genes that<br />
encode secreted factors, including chemokines, growth factors, cytokines, proangiogenic<br />
factors, components of the plasminogen activator system and<br />
metalloproteases, as confirmed by quantitative RT-PCR. The LPA-associated gene<br />
signature showed a remarkable overlap with that induced by epidermal growth factor.<br />
Our study highlights the importance of LPA in programming fibroblasts not only to<br />
proliferate but also to produce many paracrine mediators of tissue remodeling,<br />
angiogenesis, inflammation and tumor progression.<br />
Oncogenic potential of LPA receptors<br />
Using a functional genetic screen, Panthea Taghavi and colleagues (Division of<br />
Molecular Genetics) have identified the LPA2 receptor as an oncogene that<br />
collaborates with c-Myc and Tbx2 in MEF transformation. LPA1 and LPA4, but not<br />
LPA3, could reproduce the transforming effect of LPA2. LPA-induced cell
transformation was dependent on the Gi-linked ERK and PI3K signaling pathways.<br />
The transforming ability of LPA1, LPA2 and LPA4 was confirmed by tumor<br />
formation assays in nude mice. These results show that LPA signaling can promote<br />
cell transformation and tumor formation in the conjunction with c-Myc and Tbx2.<br />
Through a similar genetic screen, Roderik Kortlever and colleagues (Division of<br />
Molecular Carcinogenesis) showed that the LPA2 receptor induces senescence bypass<br />
in MEFs in the presence of wild-type p53. This result uncovers an unexpected link<br />
between LPA receptor signaling and the p53 tumor-suppressive pathway.<br />
Autotaxin, a secreted lysophospholipase D implicated in vascular<br />
development and tumor progression<br />
Regulation of ATX and discovery of small-molecule inhibitors<br />
ATX (or NPP2) is a unique ecto-nucleotide phosphodiesterase/pyrophosphatase<br />
(NPP) that functions as a lysoPLD to produce LPA from extracellular<br />
lysophosphatidylcholine. Through genetic studies in mice, we have established that<br />
ATX is essential for vascular development. To identify small-molecule inhibitors of<br />
ATX, we have performed high-throughput screening of chemical compound libraries<br />
(collaboration Huib Ovaa and David Egan). Several positive ‘hits’ were obtained that<br />
inhibited ATX activity as well as ATX-mediated tumor cell migration. Among these<br />
hits are several new chemical structures that have been optimized to reach IC(50)<br />
values in the nanomolar range. Specificity, selectivity and toxicity of these pharmacological<br />
inhibitors are being further evaluated in cell-based and animal studies.<br />
ATX isoforms<br />
The so-called ‘melanoma-derived’ isoform of ATX, termed ATXm, contains a<br />
52-residue polybasic insert of unknown function in the catalytic domain. We find that<br />
the ATXm insert is cleaved by the protease furin, yet proteolytic cleavage does not<br />
affect the catalytic activity of ATXm. It appears that ATXm cleavage occurs at or near<br />
the cell surface, while both cleavage products remain covalenty linked to keep ATXm<br />
functionally intact. The polybasic insert confers increased affinity for heparin on<br />
ATXm, but proteolytic cleavage did not alter heparin binding affinity. We hypothesize<br />
that cleavage of ATXm may serve to modulate the interaction of ATXm with an as-yetunidentified<br />
binding partner. We are currently exploring this scenario.<br />
ATX function in vivo<br />
Having shown that ATX is essential for vascular development, our next step is to<br />
establish the importance of ATX in metastasis and tumor angiogenesis, making use<br />
of various mouse models. We find that knockdown of endogenous ATX in melanoma<br />
cells reduces their metastatic capacity in a mouse model, supporting the view that<br />
secreted ATX alters the microenvironment to promote tumor progression. Through<br />
various collaborations with outside groups, we hope to learn more about ATX<br />
function in specific tissues and to uncover the origin of ATX in the circulation. Newly<br />
discovered small-molecule inhibitors of ATX will be tested in experimental animals<br />
for their anti-tumor potential. In addition, we set out to analyze the role of ATX in<br />
vascular development of zebrafish embryos, using the morpholino-based knockdown<br />
approach (collaboration Stefan Schulte-Merker, Hubrecht Institute, Utrecht).<br />
Preliminary results suggest that ATX-deficient zebrafish embryos suffer from<br />
vascular defects reminiscent of those observed in ATX knockout mice.<br />
Regulation of gap junctional communication Direct cell-cell communication<br />
through gap junctions is essential for coordinated cell behavior; it is usually defective<br />
in tumor cels. Gap junctions consist of clustered connexin subunits that form cell-tocell<br />
channels for small molecules, with connexin43 (Cx43) being the most widespread<br />
connexin. In fibroblasts, Cx43 gap junctional communication is inhibited by LPA and<br />
other G-protein-coupled receptor agonists. We established the closure and reopening<br />
of Cx43 channels is controlled by phosphatidylinositol 4,5-bisphosphate (PIP 2).<br />
Our subsequent studies identified the ubiquitin E3 ligase Nedd4 as an essential<br />
component in the pathway from activated receptor to Cx43 channel closure.<br />
Following receptor activation, Nedd4 binds to Cx43 in a manner that depends on<br />
Cx43 residue Tyr265. The challenge now is to define the link between PIP 2 hydrolysis<br />
and Nedd4 recruitment to Cx43 gap junctional channels.<br />
Publications<br />
25<br />
CELL BIOLOGY I<br />
Kortlever RM, Brummelkamp TR,<br />
van Meeteren LA, Moolenaar WH,<br />
Bernards R. Suppression of the p53-<br />
Dependent Replicative Senescence Response<br />
by lysophosphatidic acid signaling. Mol<br />
Cancer Res. <strong>2008</strong>; 6:1452-60<br />
Stortelers C, Kerkhoven R,<br />
Moolenaar WH. Multiple actions of LPA<br />
on fibroblasts revealed by transcriptional<br />
profiling. BMC Genomics <strong>2008</strong>; 9:387<br />
Taghavi P, Verhoeven E, Jacobs J,<br />
Lambooij J, Stortelers C, Tanger E,<br />
Moolenaar WH, van Lohuizen M. In vitro<br />
genetic screen identifies a cooperative role for<br />
LPA signaling and c-Myc in cell<br />
transformation. Oncogene <strong>2008</strong>; 27:6806-16<br />
Van Meeteren L, Brinkmann V,<br />
Saulnier-Blache JS, Lynch KR,<br />
Moolenaar WH. Anticancer activity of<br />
FTY720: Phosphorylated FTY720 inhibits<br />
autotaxin, a metastasis-enhancing and<br />
angiogenic lysophospholipase D. Cancer<br />
Lett. <strong>2008</strong>; 266:203-8
26<br />
CELL BIOLOGY I<br />
Group leader John Collard<br />
John Collard PhD Group leader<br />
Audrey Gerard PhD Post-doc<br />
Sandra Iden PhD Post-doc<br />
Saskia Ellenbroek MSc PhD student<br />
Amra Hajdo-Milasinovic MSc PhD student<br />
Sander Mertens MSc PhD student<br />
Saskia Amra Sander MSc PhD student<br />
Rob Van der Kammen Technical staff<br />
Publications<br />
Strumane K, Rygiel TP, van der Valk M,<br />
and Collard JG. Tiam1-deficiency impairs<br />
mammary tumor formation in MMTV-cneu<br />
but not in MMTV-c-myc mice. J<br />
Cancer Res Clin Oncol, 2009; 135:69-80<br />
Gérard A, van der Kammen RA, Janssen H,<br />
Ellenbroek SI, Collard JG. The Rac<br />
activator Tiam1 controls efficient T-cell<br />
trafficking and route of trans-endothelial<br />
migration. Blood, 2009 (in press)<br />
Iden S, Collard JG. Cross-talk between<br />
small GTPases and polarity proteins in cell<br />
polarization. Nat Rev Mol Cell Biol, <strong>2008</strong>;<br />
9:846-859<br />
Strumane K, Song J-Y, Baas I, Collard JG.<br />
Increased Rac activity is required for the<br />
progression of T-lymphomas induced by<br />
Pten-deficiency. Leukemia Research, <strong>2008</strong>;<br />
32:113-120<br />
Rygiel TP, Mertens AE, Strumane K,<br />
van der Kammen RA, Collard JG. The Rac<br />
activator Tiam1 prevents keratinocyte<br />
apoptosis by controlling ROS-mediated<br />
ERK phosphorylation. J Cell Sci, <strong>2008</strong>;<br />
121:1183-1192<br />
GENETIC CONTROL OF INVASION AND METASTASIS<br />
The aim of our research is to identify and characterize genes that play an essential<br />
role in the acquisition of an invasive and metastatic phenotype of tumorigenic cells.<br />
Insight into the molecular mechanisms that underlie the formation and progression<br />
of tumors may lead to the development of novel diagnostic tools or improved<br />
therapeutic strategies.<br />
Function-based screens for invasion-inducing genes Using functional screens<br />
we have identified different invasion-inducing genes by retroviral insertional<br />
mutagenesis in combination with in vitro selection of invasive T-lymphoma cells.<br />
These included the a 5- subunit of the a 5b 1 integrin, the LPA2 receptor, Rap1 and<br />
Tiam1, all of which have been implicated in cell-matrix adhesion and/or signaling<br />
pathways mediated by Rho GTPases. Interestingly, the Tiam1 gene encodes an<br />
activator of the Rho-like GTPase Rac. Currently, we are studying by which molecular<br />
mechanisms in particular Rho proteins play a role in normal development and tumor<br />
formation and progression.<br />
Rho family proteins are controlled by various regulatory proteins The invasioninducing<br />
Tiam1 gene encodes an activator or guanine nucleotide exchange factor<br />
(GEF) for the Rho-like GTPase Rac. Rho family proteins, which include Cdc42, Rac<br />
and RhoA, control a wide range of biological processes such as cell adhesion, cell<br />
migration and cell polarity. In particular, they act in signaling pathways that regulate<br />
the reorganization of the actin cytoskeleton in response to receptor stimulation.<br />
Regulators of the activities of Rho proteins are GEFs and GTPase activating proteins<br />
(GAPs). GEFs activate small GTPases by exchanging GDP for GTP, whereas GAPs<br />
inactivate them by enhancing the intrinsic rate of hydrolysis of bound GTP to GDP.<br />
In the cytoplasm, GTPases are kept in the GDP-bound form by association with<br />
guanine nucleotide dissociation inhibitors (RhoGDIs). RhoGDIs bind and mask the<br />
hydrophobic C-terminal region of GTPases, which is responsible for targeting them<br />
to the plasma membrane.<br />
Tiam1 regulates Rac-mediated downstream signaling GEFs are regulated by<br />
intra-molecular inhibition, changes in intracellular localization, post-translational<br />
modifications and interaction with other proteins. All of these regulatory<br />
mechanisms seem to be involved in Tiam1 regulation. In particular, complex<br />
formation with various scaffold proteins is an important mechanism to determine<br />
signaling towards and downstream of Rac. For instance, oncogenic Ras controls Rac<br />
signaling through direct interaction with the RBD domain of Tiam1. We found that<br />
Tiam1 is indispensable for a3b1-mediated Rac activation and that Tiam1-deficient<br />
keratinocytes do not adhere to and spread on a glass substrate, because they are<br />
unable to deposit their own laminin-5 substrate. Tiam1-deficient keratinocytes are<br />
impaired in activation of Rac upon adhesion to an exogenous laminin-5 substrate,<br />
whereas other integrin-mediated signaling pathways are intact. Tiam1-deficiency<br />
thereby hampers keratinocyte migration in vitro and re-epithelialization of excision<br />
wounds in mouse skin in vivo. Furthermore, Tiam1 interacts with the p21-Arc subunit<br />
of the Arp2/3 complex. As a result, Tiam1 co-localizes with the Arp2/3 complex at<br />
sites of actin polymerization such as epithelial cell-cell contacts and membrane<br />
ruffles. The Arp2/3 complex acts as a scaffold to localize Tiam1 and thereby Rac<br />
activity, which both are required for activation of the Arp2/3 complex. The Arp2/3<br />
complex drives actin polymerization primarily at the leading edge of cells. In<br />
addition, Tiam1 interacts with the Par polarity complex and thereby regulates cell<br />
polarity as discussed below.<br />
Tiam1-Rac signaling and cell polarity Tiam1 associates with Par3 of the Par<br />
polarity complex and thereby regulates polarity processes in various cell types and<br />
different contexts. Tiam1 localizes to adherens junctions in epithelial cells and ectopic<br />
expression of Tiam1 promotes E-cadherin-based cell-cell adhesions whereas knock<br />
down of Tiam1 leads to loss of cell-cell adhesions and epithelial-mesenchymal<br />
transition. Tiam1 controls tight junction (TJ) formation by activation of the Par<br />
polarity complex, which is required for the establishment of apical-basal cell polarity
in epithelial cells. In the absence of cell-cell adhesions, epithelial cells develop frontrear<br />
polarization and migrate in a directional persistent fashion involving signaling<br />
by the Tiam1-Par complex. Lack of Tiam1, knock down of Par3, or reduced PKCz<br />
activity impair front-rear polarization, persistent migration and chemotaxis of<br />
keratinocytes. Tiam1 in conjunction with the Par complex also affects directional<br />
protrusion outgrowth in astrocytes. In lymphocytes, Tiam1 is required for<br />
chemokine- and S1P induced Rac activation and subsequent cell directional cell<br />
migration. As a result, Tiam1-deficient T-cells show reduced chemotaxis in vitro, and<br />
impaired homing, egress and contact hypersensitivity in vivo. Analysis of the T-cell<br />
trans-endothelial migration cascade revealed that Par/PKCz/Tiam1/Rac signaling is<br />
dispensable for T-cell arrest but is essential for the stabilization of polarization and<br />
efficient crawling of T-cells on endothelial cells. Intriguingly, T-cells that lack Tiam1<br />
predominantly transmigrate through individual endothelial cells (transcellular<br />
migration) rather than at endothelial junctions (paracellular migration), suggesting<br />
that T-cells are able to change their route of trans-endothelial migration according to<br />
their polarization status and crawling capacity.<br />
Tumorigenicity in Tiam1 mutant mice Although Tiam1 is expressed during<br />
embryonic development, Tiam1-deficient (Tiam1 -/- ) mice develop, grow, and<br />
reproduce normally. In mouse skin, Tiam1 is present in basal and suprabasal<br />
keratinocytes of the epidermis as well as in hair follicles. We found that Tiam1 -/- mice<br />
display resistance to DMBA/TPA-induced skin tumor formation. Although the<br />
number and growth of tumors is strongly reduced in Tiam1 -/- mice, the frequency of<br />
malignant conversion of the skin tumors that do develop is increased. We also<br />
studied the function of Tiam1 in tumorigenesis induced by other oncogenic signaling<br />
pathways and in different cell types. For instance, we found that Tiam1 is a Wntresponsive<br />
gene and that its protein levels are increased in intestinal tumors<br />
produced in APC-min mutant mice as well as in human colon tumors. Furthermore,<br />
the initiation and growth of intestinal tumors was reduced in Tiam1-deficient APCmin<br />
mice, suggesting that Tiam1 promotes b-catenin/TCF-induced intestinal tumor<br />
formation. In contrast, we do not find differences in T-lymphomagenesis induced by<br />
constitutive activation of the PI3-kinase pathway in wild-type and Tiam1-deficient<br />
mice. Mammary tumor formation and progression induced by MMTV-c-neu appear<br />
to be delayed and reduced in the absence of Tiam1, whereas mammary tumors<br />
induced by MMTV-myc are not dependent on the presence of Tiam1. These studies<br />
indicate that Tiam1 may contribute to the formation and progression of tumors<br />
induced by various -but not all-oncogenic signaling pathways. During tumor<br />
initiation, Tiam1-mediated Rac activation promotes survival signals and thereby<br />
prevents apoptosis whereas the growth and the progression of tumors can be<br />
promoted or inhibited by the presence of Tiam1 depending on the oncogenic pathway<br />
involved and tumor cell type studied.<br />
Opposing functions of Rac1 and Rac3 Rac1 and Rac3 are highly homologous<br />
regulatory proteins of the Rho GTPase family. Rac1 is ubiquitously expressed and<br />
regulates adhesion, migration and differentiation in various cell types. In contrast,<br />
Rac3 is primarily expressed in the brain. We found opposing functions for Rac1 and<br />
Rac3 in neuronal cells. Knock down of Rac1 by siRNA leads to decreased cell-matrix<br />
adhesions and cell rounding in neuronal cells whereas knock down of Rac3<br />
stimulates cell adhesion and neuritogenesis. The Rac1-opposing function of Rac3 is<br />
not mediated by or dependent on components of the RhoA signaling pathway.<br />
Further analyses revealed that Rac1 and Rac3 interact with Git1, a multifunctional<br />
ArfGAP protein which regulates cell-matrix adhesion, cell spreading and endocytosis.<br />
However, in contrast to Rac1, the Rac3/Git1 interaction is not mediated by bPix and<br />
Rac3 severely attenuates Git1/paxillin interaction required for cell spreading.<br />
Interestingly the ArfGAP domain of Git1 is required for Rac3 downstream signaling<br />
and Arf6 activity is strongly reduced in Rac3 expressing cells. Our data suggests that<br />
Rac3 and Rac1 oppose each other by differently modulating Git1 function.<br />
27<br />
CELL BIOLOGY I<br />
Figure 4: Percentage of WT and Tiam1-/-<br />
T-cells showing trans-endothelial migration<br />
via the transcellular route by migrating<br />
through individual endothelial cells.<br />
Figure 5: Crawling and route of transendothelial<br />
migration depend on PKC/<br />
Tiam1-mediated polarization and crawling.<br />
Polarized T-cells crawl on top of endothelial<br />
monolayers and preferentially transmigrate<br />
through endothelial junctions (paracellular<br />
migration). Loss of Tiam1 and disturbed<br />
Par/PKC-signaling prevents chemokineinduced<br />
Rac activation leading to unstable<br />
polarization and thereby impaired T-cell<br />
crawling on top of endothelial monolayers.<br />
As a result these T-cells preferentially cross<br />
the endothelial monolayer through<br />
individual endothelial cells (transcellular<br />
migration).
28<br />
CELL BIOLOGY I<br />
Group leader Ed Roos<br />
Ed Roos PhD Group leader<br />
Joost Meijer MSc PhD student<br />
Yvonne Wijnands Technical staff<br />
Publications<br />
Alvarez B, Stroeken PJ, Edel MJ, Roos E.<br />
Integrin Cytoplasmic Domain-associated<br />
Protein-1 (ICAP-1) promotes migration of<br />
myoblasts and affects focal adhesions.<br />
J Cell Physiol <strong>2008</strong>;214:474-82<br />
Meijer J, Ogink J, Kreike B, Nuyten D,<br />
De Visser KE, Roos E. The chemokine<br />
receptor CXCR6 and its ligand CXCL16<br />
are expressed in carcinomas and inhibit<br />
proliferation. Cancer Res <strong>2008</strong>;68:4701-08<br />
Meijer J, Ogink J, Roos E. Effect of the<br />
chemokine receptor CXCR7 on proliferation<br />
of carcinoma cells in vitro and in vivo.<br />
Br J Cancer <strong>2008</strong>;99:1493-1501<br />
Figure 6: Role CXCR4 in metastasis During<br />
outgrowth of metastases, cells are deprived of<br />
glucose because of insufficient blood supply.<br />
This becomes critical at a size of ~1000 cells,<br />
long before the onset of angiogenesis that<br />
comes about when tumors contain ~10 6<br />
cells. A signal transduced by a CXCR4containing<br />
complex induces adaptation of<br />
cells, e.g. by changes in tumor cell<br />
metabolism and by an increase in glucose<br />
transporters in the plasma membrane. This<br />
allows cells to survive and even proliferate in<br />
low glucose conditions.<br />
MECHANISMS OF METASTASIS<br />
The chemokine receptor CXCR4 in carcinoma metastasis We showed previously<br />
that CXCR4, the receptor for the chemokine SDF-1 (CXCL12), is required for<br />
metastasis of a colon carcinoma but not involved in invasion. We next observed that<br />
CXCR4 is required for continuation of growth when micrometastases are ~1000 cells<br />
in size. The same is true for dispersed single cells in a s.c. Matrigel plug. To show<br />
this, we used a CXCL12-KDEL ‘intrakine’ that downregulates CXCR4 but also the<br />
newly discovered receptor CXCR7. Using RNAi and other intrakines we have now<br />
demonstrated that CXCR4 is involved and not CXCR7. CXCR4 was also necessary for<br />
outgrowth of KEP1 mammary carcinoma, a mouse model for human invasive lobular<br />
mammary carcinoma. After reaching a size of ~1000 cells, CXCR4-supressed KEP1<br />
tumors regressed. Surprisingly, CXCL12 is not involved, as demonstrated with<br />
inhibitors and the K1R-CXCL12 antagonist. We have now established that CXCR4 is<br />
essential for resistance against glucose deprivation, and that this depends on an<br />
alternative CXCR4 ligand. This also involves several other surface proteins. Even in<br />
medium without glucose and serum, KEP1 cells can survive when this ligand is<br />
added. Apparently, tumor cells are not able to adapt to glucose deprivation when not<br />
induced to do so by a signal from a CXCR4 signaling complex. It should be noted<br />
that angiogenesis does not come about before tumors are 1 mm in diameter and<br />
contain ~10 6 cells. Before that, tumors have no blood supply and can only grow when<br />
they adapt their metabolism. We are presently exploring the option to generate<br />
specific inhibitors of this phenomenon that should have therapeutic potential.<br />
Chemokines as growth factors for carcinomas We have found that several<br />
chemokine receptors can influence growth of carcinomas. CXCR7 is expressed on<br />
many carcinomas and mediates strong enhancement of proliferation induced by<br />
CXCL12. On some cells with lower CXCR7 levels, CXCR4 can also mediate this<br />
effect. In contrast to reports by others, however, we observed no effect at all of stable<br />
and complete CXCR7 suppression on growth of s.c. tumors and lung metastases.<br />
Microarray analysis revealed expression of the chemokine receptor CXCR6 and its<br />
ligand CXCL16 by all 170 human mammary carcinomas tested and in many<br />
carcinoma cell lines, the latter confirmed by FACS analysis. So far, they had only<br />
been detected on T-lymphocytes and macrophages, respectively. Soluble CXCL16<br />
enhanced proliferation via CXCR6 in cells in which endogenous CXCL16 was<br />
suppressed by RNAi. CXCL16 is special in that it is a transmembrane protein from<br />
which the soluble chemokine can be cleaved off. This transmembrane form is<br />
present on carcinoma cells. Unexpectedly, it reduced growth, since suppression of<br />
either CXCR6 or CXCL16 by an intrakine and RNAi greatly promoted proliferation in<br />
vitro and in vivo. The effect was confirmed using inhibitory CXCL16 antibodies and<br />
by transfection of CXCL16 into CXCL16-negative cells. It was blocked by pertussis<br />
toxin and therefore mediated by CXCR6. It is remarkable that carcinomas maintain<br />
expression of an autocrine receptor-ligand pair that suppresses growth. This suggests<br />
an essential function in tumor development.<br />
Synaptotagmin-3: role in migration, antigen presentation and outgrowth of<br />
carcinoma metastases We have previously shown that synaptotagmin-3 (Syt3) is<br />
required for recycling of CXCR4 through MVB. Since antigen presentation by MHC<br />
class II also involves passage through MVB, we tested involvement of Syt3 and found<br />
that Syt3 inhibition suppressed the formation of MHC II-antigen complexes. Indeed,<br />
transport of MHCII and in particular the associated invariant chain (CD74) to MVB<br />
was blocked.<br />
Syt3 and CD74 were found to be highly expressed by carcinomas. Based on the above<br />
results, we tested their possible involvement in the function of CXCR4 in outgrowth<br />
of metastases and of single tumor cells in Matrigel plugs. Indeed, inhibition of Syt3<br />
had the same effect as suppression of CXCR4: tumors did not become larger than<br />
~1000 cells. Growth of CT26 colon carcinomas was arrested whereas KEP1<br />
mammary carcinomas regressed. Next, we found that knockdown of CD74 led to<br />
greatly reduced surface levels of CXCR4, even though total protein levels were not<br />
changed. Thus, we have shown that CD74 is required to retain CXCR4 on the<br />
surface, quite likely by promoting recycling from MVB back to the plasma membrane.
BIOPHYSICS OF CELL SIGNALING<br />
Our lab employs biophysical techniques to study cell signaling events with high<br />
spatial and temporal resolution. Electrophysiological (e.g. patch clamping) and<br />
advanced bio-photonic techniques (e.g. Fluorescence Resonance Energy Transfer<br />
(FRET), Fluorescence Lifetime Imaging (FLIM), Fluorescence Cross Correlation<br />
Spectroscopy (FCCS) and photorelease of caged compounds) are used in research<br />
projects in our group as well as in a number of collaborations within and outside our<br />
institute. Our lab also contributes to the development of hardware, software and<br />
FRET sensors for various intracellular messengers.<br />
Phosphatidylinositol bisphosphate (PIP 2) as a membrane-delimited<br />
messenger Phosphoinositides in the plasma membrane regulate a wide variety of<br />
cellular responses. We developed FRET-based assays for the lipids PIP 2 and PIP 3, and<br />
use these to study the regulation of agonist-induced PIP 2 breakdown and its<br />
subsequent resynthesis by PIPkinases. Novel techniques such as rapamycin-induced<br />
recruitment of PIP 2-phosphatases to the membrane now also allow precise and<br />
dosed breakdown of PIP 2 in living cells. Results from this ongoing line play an<br />
important role in our other research lines and in collaboration with Dr W.<br />
Moolenaars lab on the regulation of Gap Junctions.<br />
The cation channel TRPM7 is a key regulator of podosomes Podosomes and<br />
invadopodia are adhesive structures involved in migration and invasion. They consist<br />
of an actin-rich core with associated regulatory and contractile proteins. Recently we<br />
found that TRPM7, a non-selective cation channel with inherent kinase activity that is<br />
involved in sensing mechanical forces, is a key regulator of podosomes. TRPM7overexpressing<br />
cells have much increased adhesion and show enhanced migration in<br />
transWell assays as well as matrigel invasion assays. TRPM7 mediates agonistinduced<br />
cell adhesion through Ca 2+ influx and myosin heavy chain phosphorylation.<br />
We discovered that PLC-activating agonists stimulate TRPM7 channels. Currently, we<br />
study TRPM7 activation in detail by combining biophysical readout techniques with<br />
mutational analysis of the C terminus. We found that TRPM7 and the Ca 2+ -sensitive<br />
PLC isoform PLC 1 form a feedback loop that involves Ca 2+ influx through TRPM7.<br />
In collaboration with the group of Dr FN van Leeuwen (Nijmegen) we have analyzed<br />
recruitment and localization of GFP-tagged versions of Drebrin, actinin 4 and SIPA1.<br />
Using FRAP experiments, we found that whereas podosomes are stable cell adhesion<br />
structures that live on average for ~15 minutes, the individual GFP-tagged<br />
components are highly dynamic, exchanging with the cytosolic pool on average about<br />
once every 10 s. Currently we address the regulation of this rapid exchange.<br />
Furthermore, we study the podosome complex by studying FRET between GFP- and<br />
mRFP-tagged constituent proteins. We also study regulation of the closely related<br />
channel TRPM6, which is involved in Mg 2+ homeostasis.<br />
Cytosol-membrane shuttling of Epac1 regulates cell adhesion During our<br />
recent studies aimed at constructing new FRET sensors for cAMP based on Epac1<br />
(short for Exchange Protein Activated by cAMP), we observed that stimulation of cells<br />
with certain agonists causes translocation of Epac1 from the cytosol to the plasma<br />
membrane. Further analysis of this phenomenon revealed that depending on the<br />
agonist, two different signaling cascades can independently control Epac1<br />
translocation to specific sites at the membrane. The relocalization of Epac1 to the<br />
membrane is thought to be crucial for the localized activation of its effector, Rap1,<br />
which regulates integrins (collaboration with Dr JL Bos, Utrecht).<br />
Figure 7: Podosomes in N1E-115/<br />
TRPM7 cells as visualized by Total<br />
Internal Reflection Fluorescence (TIRF)<br />
microscopy. TIRF is particularly suited<br />
for imaging cell adhesion structures as it<br />
is sensitive exclusively to fluorescence<br />
within ~150 nm of the substrate. Scale<br />
bar, 5 µm.<br />
Group leader Kees Jalink<br />
Kees Jalink PhD Group leader<br />
Michiel Langeslag PhD Post-doc<br />
Bas Ponsioen MSc PhD student<br />
Daan Visser MSc PhD student<br />
Jeffrey Klarenbeek MSc Technical staff<br />
Ilona Vuist Trainee<br />
Publications<br />
29<br />
CELL BIOLOGY I<br />
Zwier JM, Oomen L, Brocks L, Jalink K,<br />
Brakenhoff GJ. Quantitative image<br />
correction and calibration for confocal<br />
fluorescence microscopy using thin reference<br />
layers and SIPchart-based calibration<br />
procedures. J Microsc <strong>2008</strong>;231:59-69<br />
Vliem MJ, Ponsioen B, Schwede F,<br />
Pannekoek WJ, Riedl J, Kooistra MR,<br />
Jalink K, Genieser HG, Bos JL, Rehmann H.<br />
8-pCPT-2'-O-Me-cAMP-AM: An Improved<br />
Epac-Selective cAMP Analogue<br />
Chembiochem <strong>2008</strong>;9:2052-4<br />
Van der Krogt GN, Ogink J, Ponsioen B,<br />
Jalink K. A comparison of donor-acceptor<br />
pairs for genetically encoded FRET sensors:<br />
application to the Epac cAMP sensor as an<br />
example. PLoS ONE <strong>2008</strong>;3:e1916<br />
Oomen LC, Sacher R, Brocks HH,<br />
Zwier JM, Brakenhoff GJ, Jalink K.<br />
Immersion oil for high-resolution live-cell<br />
imaging at 37 degrees C: optical and<br />
physical characteristics. J Microsc<br />
<strong>2008</strong>;232:353-61<br />
Jalink K, Van Rheenen J. FilterFRET:<br />
quantitative imaging of FRET by sensitized<br />
emission. In: FRET and FLIM methods,<br />
Vol 33, 289-349. Th. Gadella, Ed. Elzevier,<br />
New York (in press)
30<br />
CELL BIOLOGY I<br />
Group leader Wim Van Blitterswijk<br />
Wim Van Blitterswijk PhD Group leader<br />
Marcel Verheij MD PhD Group leader<br />
Maaike Alderliesten PhD Post-doc<br />
Albert Van Hell PhD Post-doc<br />
John De Widt BSc Technical staff<br />
Shuraila Zerp BSc Technical staff<br />
Jeffrey Klarenbeek MSc Technical staff<br />
Publications<br />
Verheij M, van Blitterswijk WJ.<br />
Sphingomyelin metabolism: Implications<br />
for cell signaling and drug uptake. In:<br />
Reviews in Cancer Biology & Therapeutics<br />
2007 (Kasis UN, Notario V, Haimovitz-<br />
Friedman A, Bar-Eli M, Eds,), Chapter 3,<br />
Transworld Research Network, pp 55-71<br />
Zerp SF, Vink SR, Ruiter GA, Koolwijk P,<br />
Peters E, van der Luit AH, de Jong D,<br />
Budde M, Bartelink H, van Blitterswijk WJ,<br />
Verheij M. Alkylphospholipids inhibit<br />
capillary-like endothelial tube formation in<br />
vitro: Anti-angiogenic properties of a new<br />
class of anti-tumor agents. Anticancer Drugs<br />
<strong>2008</strong>;19:65-75<br />
Baldanzi G, Cutrupi S, Chianale F,<br />
Gnocchi V, Rainero E, Porporato P,<br />
Filigheddu N, van Blitterswijk WJ,<br />
Parolini O, Bussolino F, Sinigaglia F,<br />
Graziani A. Diacylglycerol kinase-a<br />
phosphorylation by Src is required for<br />
activation, membrane recruitment and<br />
Hgf-induced cell motility. Oncogene<br />
<strong>2008</strong>;27:942-56<br />
Los AP, de Widt J, van Blitterswijk WJ,<br />
Divecha N. Is there a role for diacylglycerol<br />
kinase-zeta in cell cycle regulation? Adv<br />
Enzyme Regul <strong>2008</strong>;48:31-9<br />
Van Blitterswijk WJ, Verheij M. Anticancer<br />
alkylphospholipids: mechanisms of action,<br />
cellular sensitivity and resistance, and<br />
clinical prospects. Curr Pharm Des<br />
<strong>2008</strong>;14:2061-74<br />
LIPID METABOLISM IN SIGNAL TRANSDUCTION<br />
Membrane lipids have both structural and signaling functions. Plasma membrane<br />
sphingolipids play a role in apoptosis and they tend to self-associate and cluster with<br />
cholesterol in dynamic microdomains known as lipid rafts. Inositol phospholipids<br />
have signaling functions in vesicular trafficking, cellular stress and survival. In this<br />
context we study mechanisms of tumor cell sensitivity and (cross-)resistance to<br />
various apoptotic stimuli. In a second project, we investigate how exogenous shortchain<br />
sphingolipids enhance the anti-cancer action of doxorubicin and related<br />
amphiphilic drugs in vitro and in vivo.<br />
Figure 8:<br />
Interconversion of phosphoinositides by<br />
PI3-kinase, PTEN and SHIP<br />
Apoptosis sensitivity to anti-tumor alkylphospholipids (APLs) Synthetic APLs<br />
such as edelfosine (1-O-octadecyl-2-O-methyl-glycero-3-phosphocholine) and<br />
perifosine (a piperidine analog) induce apoptosis in tumor cells, leaving normal cells<br />
relatively unaffected. APLs are used as anti-cancer agents in the clinic and enhance<br />
radiation- and chemotherapy-induced cell death. APLs incorporate in cell<br />
membranes, are internalized by raft-dependent endocytosis and/or through a lipid<br />
transporter. They then interfere with phospholipid biosynthesis/ signaling and the<br />
Akt/PKB survival pathway.<br />
S49 lymphoma cells can develop resistance to APL (S49 AR cells), which is<br />
accompanied by cross-resistance to other apoptotic stimuli such as DNA damage and<br />
Fas/CD95 ligation. Resistant cells were defective in raft-mediated endocytosis and in<br />
sphingomyelin synthesis (SMS1), and showed downregulated SHIP1, a PIP 3<br />
5-phosphatase, resulting in upregulation of the PI3-kinase/PKB survival pathway and<br />
increased tyrosine-phosphorylated proteins. We currently investigate the common<br />
mechanism underlying the cross-resistance to multiple apoptotic stress stimuli.<br />
For clinical applications of APLs, see the report by M Verheij, Division of Radiotherapy.<br />
Short-chain sphingolipids enhance doxorubicin uptake by tumor cells<br />
Doxorubicin (DOX) is often administered as a liposomal formulation (Caelyx ® ), from<br />
which it is gradually released into the interstitial space and then taken up by tumor<br />
cells. We discovered that certain short-chain sphingolipids such as N-octanoylglucosylceramide<br />
(GC),co-formulated in the liposomes, accelerate the cellular uptake<br />
and accumulation of DOX in various carcinoma cell lines (e.g. A431), but not in<br />
normal cardiac myoblasts. We tested these liposomes in nude mice that were<br />
subcutaneously inoculated with A431 cells. In a dose-escalation study, the anti-tumor<br />
efficacy of GC-modified liposomal DOX was superior to standard liposomal DOX.<br />
Increased drug delivery to tumor cells was also observed by intravital microscopy in<br />
mice with a tumor implanted in a dorsal skin flap window chamber (collaboration<br />
with G Koning, Erasmus MC, Rotterdam). This new liposomal formulation of DOX,<br />
which we have patented, undergoes further testing and seems promising for clinical<br />
application.<br />
Figure 9: APL-resistant S49 AR cells show decreased<br />
SHIP1 and Fas expression and constitutively activated<br />
PKB/Akt (Western blots). Bottom panel shows decreased<br />
cell surface expression of Fas (immunofluorescence).
CELL BIOLOGY II<br />
ANTIGEN PRESENTATION BY MHC CLASS I AND MHC CLASS<br />
II MOLECULES<br />
Our aim is to understand how the adaptive immune system is regulated and to find<br />
both targets and leads for modifying it. MHC class I and MHC class II molecules<br />
control the activities of T cells. These T cells can then stimulate the immune system<br />
or activate it to kill tumor cells and other cells. This is used in various immunotherapy<br />
strategies to eliminate cancers. Our aim is to supply the fundamental framework<br />
for such strategies. We use a large variety of state-of-the-art technologies like single<br />
cell biochemistry, chemistry and high-throughput screening to achieve this goal.<br />
Degrading the degradation machinery; the proteasome The proteasome is a very<br />
large and stable intracellular protease critical in antigen presentation by MHC class I<br />
molecules but also in the control of cell division and cancer. How the proteasome is<br />
constructed is reasonably clear but how it is degraded is obscure. In collaboration<br />
with the yeast group of Fred van Leeuwen, we have established a system where the<br />
fluorescent label on proteasomes can be genetically switched. This yields a living<br />
pulse chase system that can be used to determine half-lives of (tagged) proteins.<br />
We are crossing in yeast mutant strains to identify the cell ’s biological pathways<br />
controlling degradation of the proteasome. Using two switchable tag systems, the<br />
stability of large protein complexes can be determined. We have established this<br />
system and are analyzing the stability within the 20S core of the proteasome and the<br />
stability of the 26S proteasome by inserting the switch cassettes at different sites in<br />
the molecule.<br />
Gap junctions, apoptosis and cross-presentation The proteasome generates<br />
peptides for MHC class I molecules. How classical antigen presentation by MHC<br />
class I molecules is achieved is well established. However, peptides can be presented<br />
by antigen presenting cells such as dendritic cells when made by other cells. This<br />
process is called cross-presentation. We have shown that one way to get crosspresentation<br />
is by transferring peptides from an infected cell to other cells through<br />
gap junctions. We have tested whether antigen (cross)-presentation proceeds in cells<br />
sentenced to apoptosis. We show that many processes continue when the cell is ‘in<br />
apoptosis’ including direct antigen presentation and cross-presentation after gap<br />
junction transfer of peptides. This provides one explanation for presentation of<br />
antigens from apoptotic cells.<br />
Radiation and adaptive immune responses, new combination therapies We<br />
have shown that ionizing radiation induces various responses on cells including<br />
protein degradation, mTOR activation and production of new proteins. Together this<br />
results in higher MHC class I expression and better CTL responses against tumors in<br />
tissue culture and in mice. We have identified four ‘radiation-specific’ peptides<br />
including that of an Fbox protein. This Fbox protein supports cell growth under low<br />
radiation exposure and silencing arrests cells in the G1-S phase. Since Fbox proteins<br />
target substrates for ubiquitination and destruction, we are currently defining the<br />
target(s) using proteomics combined with over-expression studies. This will yield<br />
potential proteins responsible for selective sensitivity to radiotherapy in patients<br />
which will be tested once the target is identified.<br />
Endosomes and MHC class II antigen presentation MHC class II molecules<br />
control the formation of antibodies and other aspects of the immune system like CTL<br />
responses against infections and tumors. These MHC class II molecules meet<br />
peptides for antigen presentation somewhere in the endocytic pathway. How<br />
endosomes move and fuse is only partly understood. We have identified how the<br />
dynein motor protein binds to endosomes (through the Rab7-RILP motor receptor)<br />
and how this interaction is controlled (through ORP1L that can interact with the ER<br />
protein VAP). The VAP-ORP1L interaction is controlled by cholesterol in late<br />
31<br />
CELL BIOLOGY II<br />
Division head, group leader Jacques Neefjes<br />
Jacques Neefjes PhD Group leader<br />
Coen Kuijl PhD Post-doc<br />
Victoria Menendez PhD Post-doc<br />
Veronica Monserrat PhD Post-doc<br />
Tiziana Scanu PhD Post-doc<br />
Tineke Van den Hoorn MSc PhD student<br />
Marlieke Jongsma MSc PhD student<br />
Baoxu Pang MSc PhD student<br />
Petra Paul MSc PhD student<br />
Xiaohang Qiao MSc PhD student<br />
Izhar Salomon MSc PhD student<br />
Rik Van der Kant MSc PhD student<br />
Lennert Janssen BSc Technical staff
32<br />
CELL BIOLOGY II<br />
Publications<br />
Hodge JW, Guha C, Neefjes J, Gulley JL.<br />
Synergizing radiation therapy and<br />
immunotherapy for curing incurable<br />
cancers. Opportunities and challenges.<br />
Oncology (Williston Park) <strong>2008</strong>;22:1064-70<br />
Koppers-Lalic D, Verweij MC,<br />
Lipinska AD, Wang Y, Quinten E,<br />
Reits EA, Koch J, Loch S, Rezende MM,<br />
Daus F, Bienkowska-Szewczyk K,<br />
Osterrieder N, Mettenleiter TC,<br />
Heemskerk MH, Tampe R, Neefjes JJ,<br />
Chowdhury SI, Ressing ME, Rijsewijk FA,<br />
Wiertz EJ. Varicellovirus UL 49.5 proteins<br />
differentially affect the function of the<br />
transporter associated with antigen<br />
processing, TAP. PLoS Pathog<br />
<strong>2008</strong>;4:e1000080<br />
Neijssen J. The Fate of Intracellular<br />
Peptides and MHC Class I Antigen<br />
Presentation. Leiden University, <strong>2008</strong><br />
Rocha N, Neefjes J. MHC class II<br />
molecules on the move for successful antigen<br />
presentation. EMBO J <strong>2008</strong>;27:1-5<br />
van den Hoorn T, Neefjes J. Activated<br />
pDCs: open to new antigen-presentation<br />
possibilities. Nat Immunol <strong>2008</strong>;9:1208-10<br />
endosomes and explains why high levels of endosomal cholesterol induce endosome<br />
clustering as the result of dynein motor activity. In effect, our model is the first<br />
explanation of control of motor protein activities on intracellular vesicles.<br />
We are currently testing whether fusion and transport of endosomes could be<br />
coupled processes and how these are controlled.<br />
Genome-wide cell biology of new factors in MHC class II antigen presentation<br />
To get more insight into the cell biological processes controlling MHC class II<br />
antigen presentation, a FACS based genome-wide siRNA screen has been performed<br />
using two antibodies labeled with Cy3 and Cy5 respectively. One antibody monitored<br />
MHC class II expression levels whereas the other monitored the efficiency of antigen<br />
loading. After some 100.000 2-color FACS analyses and cross-correlation with<br />
expression profiles of primary human B cells, monocytes and dendritic cells<br />
(either resting or activated), 255 hits were identified as being involved in MHC<br />
class II expression or peptide loading.<br />
To understand these hits (and as an additional way to validate our data), we have<br />
established high-throughput techniques to arrive at pathways. To identify the hits<br />
involved in controlling transcription of MHC class II molecules, they were again<br />
silenced by siRNA and expression of the transcription master regulator CITA, its<br />
substrate MHC class II and the invariant chain were determined by qPCR. To further<br />
subdivide potential hits, transcription of MHC class I molecules was also determined<br />
as hits may affect the MHC locus rather than MHC class II expression only. This<br />
analyses yielded some 10 factors and cross-qPCR is currently been performed to test<br />
whether pathways controlling the expression of transcription regulators (CIITA) can<br />
be identified.<br />
Along the same token, we have silenced the hits in cells expressing Golgi markers<br />
and MHC class II-GFP molecules and subsequently fixed and stained early<br />
endosomes. For color confocal images are made and analyzed by ‘CellProfiler’ and<br />
other software programs to identify similar phenotypes. This yielded additional<br />
clusters and first hints on novel signaling pathways controlling MHC class II<br />
expression. In total we can explain the function of some 140 out of 255 hits most of<br />
which cluster in only 4 networks. How these pathways work is currently studied<br />
using yeast-2 hybrid, proteomics and other techniques.<br />
As a spin-off, we identified some 30 secreted factors not expressed in human primary<br />
immune cells. These factors may locally control MHC class II immune responses<br />
and are currently made to test this on primary human immune cells.<br />
Expanding the PKB/Akt1 pathway using bacterial infection systems We have<br />
shown that Salmonella activates the PKB/Akt1 pathway to survive intracellularly.<br />
In fact, a series of kinases in this pathway were activated for this purpose which are<br />
almost all involved in cancer as well. We argued that testing the effects of<br />
phosphatases on Salmonalla infection by siRNA would yield proteins that probably<br />
target the kinases already identified in our previous study. We first identified the<br />
phosphatases present in the human genome and then selected the corresponding<br />
siRNAs. Using these we identified a series of phosphatases, some of which were<br />
already defined as controlling PKB/Akt. Since esterases are homologous to<br />
phosphatases, we also identified two esterases. Simultaneously, Huib Ovaa’s group<br />
constructed a chemical phosphate-inhibitor library which was used in the same<br />
model screening system. This yielded two compounds inhibiting Salmonella<br />
infection. We are currently combining the siRNA data with the chemical library data<br />
to identify new target-lead combinations that may affect the PKB/Akt pathway.
ESTROGEN RECEPTOR AND BREAST CANCER<br />
Estrogen Receptor a (ERa)-positive breast cancer patients are commonly treated with<br />
tamoxifen. Resistance to tamoxifen treatment is observed in about half of the<br />
recurrences in breast cancer where the anti-estrogen tamoxifen acquires agonistic<br />
properties for transactivation of ERa. Tamoxifen resistant breast cancer is still<br />
sensitive to other anti-estrogens, all with compound specific properties and behavior.<br />
The clinical benefits of clarifying pathways underlying resistance can therefore be<br />
profound. We aim to get molecular understanding of tamoxifen and other antiestrogen<br />
resistance mechanisms and to develop tools to use as clinical decision<br />
markers in anti-estrogen therapy of breast cancer patients.<br />
Structural requirements in ERa for tamoxifen resistance We found that the two<br />
variants of the human Estrogen Receptor, ERa and ERb, differ in their response to<br />
estradiol and tamoxifen, which is determined by the ability of their N-terminal AF-1<br />
domains to interact with co-factors and by their hinge regions. Through a<br />
collaborative action of both AF-1 and hinge, ERa can induce transcription in the<br />
presence of its antagonist tamoxifen, a capacity which is lacking in ERb.<br />
Modifications in ER associated with resistance to anti-estrogens We have<br />
identified different combinations of ERa modulation by phosphorylation and/or<br />
overexpression of its cofactors, which affect the response to specific anti-estrogens.<br />
Resistance to Fulvestrant requires phosphorylation of Serine 305 by PKA in<br />
combination with phosphorylation of Serine 118 by MAPkinase or overexpression of<br />
SRC-1 and cyclin D1, whereas resistance to arzoxifene is specifically influenced by<br />
overexpression of cyclin D1 as co-factor of ER. This is highly relevant, since these<br />
anti-estrogens are currently in use in the clinic as alternatives for endocrine<br />
treatment.<br />
Overexpression of one of the three SRC-cofactors, SRC-2, predicted a poor response<br />
in a randomized trial that evaluated adjuvant tamoxifen treatment versus nil and was<br />
the cause of tamoxifen resistance in experimental studies.<br />
A total profile of modifications of ER that lead to resistance to anti-estrogens is<br />
presented in figure 1.<br />
In contrast to ERa, phosphorylation of ERb by either Protein Kinase A or<br />
MAPkinase, did not result in resistance to tamoxifen.<br />
Gene signature of ERa phosphorylated at Serine 305 by Protein kinase A Since<br />
phosphorylation of Serine 305 in ERa is causal for tamoxifen resistance, a gene<br />
profile was determined under these conditions. Microarray experiments on U2OS<br />
cell lines expressing ERa wt or a 305A mutant that does not become phosphorylated<br />
at that site, show specific gene targets of a tamoxifen bound ERa phosphorylated at<br />
Ser305 by Protein Kinase A. The results suggest an association between tamoxifen<br />
resistance by 305P and cell movement. Furthermore, phosphorylation at Serine 305<br />
in the presence of tamoxifen seems to affect the non-classical ER targets, rather than<br />
ERE regulated genes. Specific targets of a tamoxifen bound ERa phosphorylated at<br />
Serine 305 by Protein Kinase A were confirmed by both qPCR and an independent<br />
microarray experiment.<br />
Clinical relevance of phosphorylation of Serine 305 of ERa In collaboration with<br />
Marleen Kok and Sabine Linn (Division of Molecular Biology) and Goran Landberg<br />
and colleagues (University of Lund), we found that specific phosphorylation of the<br />
Estrogen Receptor at Serine 305 predicted a poor response in a randomized trial that<br />
evaluated adjuvant tamoxifen treatment as well as in metastatic breast cancer<br />
patients. In addition, we found that a combined measurement of two independent<br />
markers, phosphorylation of the Estrogen Receptor at Serine 305 by Protein kinase A<br />
and PAK-1, identified a substantial fraction of ER-positive breast cancer that had poor<br />
outcome after tamoxifen treatment.<br />
Application of the laboratory findings of how modulation of ER and its cofactors<br />
affects sensitivity towards anti-estrogens will lead to a tailoring of endocrine<br />
treatment of breast cancer patients.<br />
Group leader Rob Michalides<br />
Rob Michalides PhD Group leader<br />
Mariska Rondaij PhD Post-doc<br />
Renée De Leeuw MSc PhD student<br />
Wilbert Zwart MSc PhD student<br />
Cristiane Bentin Toaldo BSc Technical staff<br />
Désirée Verwoerd BSc Technical staff<br />
Publication<br />
33<br />
CELL BIOLOGY II<br />
Holm C, Kok M, Michalides R, Fles R,<br />
Koornstra R, Wesseling J, Hauptmann M,<br />
Neefjes J, Peterse J, Stal O, Landberg G,<br />
Linn S. Phosphorylation of the oestrogen<br />
receptor alpha at serine 305 and prediction of<br />
tamoxifen resistance in breast cancer.<br />
J Pathol <strong>2008</strong> (Epub ahead of print)<br />
Figure 1: ERa resistance profile by<br />
phosphorylation and cofactor overexpression.<br />
Graphic representation of antiestrogen<br />
sensitivity towards phosphorylation events on<br />
ERa (A) in possible conjunction with SRC-1<br />
and/or Cyclin D1 overexpression (B). The<br />
antiestrogen inhibits the ERa (black) unless<br />
the correct requirements are met to induce<br />
resistance against the compound (white).
34<br />
CELL BIOLOGY II<br />
Group leader Huib Ovaa<br />
Huib Ovaa PhD Group leader<br />
Boris Rodenko PhD Post-doc<br />
Silvia Cavalli PhD Post-doc<br />
Farid El Oualid PhD Post-doc<br />
Anitha Shanmugham PhD Post-doc<br />
Celia Berkers MSc PhD student<br />
Rieuwert Hoppes MSc PhD student<br />
Harald Albers MSc PhD student<br />
Henk Hilkmann Ing Technical staff<br />
Karianne Schuurman Msc Research assistant<br />
Annemieke de Jong MSc Research assistant<br />
Publications<br />
Bakker AH, Hoppes R, Linnemann C,<br />
Toebes M, Rodenko B, Berkers CR,<br />
Hadrup SR, van Esch WJ, Heemskerk<br />
MH, Ovaa H, Schumacher TN.<br />
Conditional MHC class I ligands and<br />
peptide exchange technology for the human<br />
MHC gene products HLA-A1, -A3, -A11,<br />
and -B7. Proc Natl Acad Sci U S A<br />
<strong>2008</strong>;105:3825-30<br />
Berkers CR, de Jong A, Ovaa H, Rodenko B.<br />
Transpeptidation and reverse proteolysis<br />
and their consequences for immunity.<br />
Int J Biochem Cell Biol 2009;41:66-71.<br />
Kraus M, Malenke E, Gogel J, Muller H,<br />
Ruckrich T, Overkleeft H, Ovaa H,<br />
Koscielniak E, Hartmann JT, Driessen C.<br />
Ritonavir induces endoplasmic reticulum<br />
stress and sensitizes sarcoma cells toward<br />
bortezomib-induced apoptosis. Mol Cancer<br />
Ther <strong>2008</strong>;7:1940-8<br />
Ovaa H, van Leeuwen F. Chemical<br />
Biology Approaches to Probe the Proteome.<br />
Chembiochem <strong>2008</strong>;9:2913-2919<br />
CHEMISTRY AND BIOLOGY OF PROTEOLYSIS AND<br />
ANTIGEN PRESENTATION<br />
The ubiquitin proteasome system and MHC class I antigen presentation<br />
T lymphocytes serve to recognize and eliminate cells that express foreign (e.g. virusderived<br />
or tumor-specific) proteins. The recognition of antigen presenting cells<br />
(APCs) is based on the binding of the T cell receptor to Major Histocompatibility<br />
Complexes (MHCs) that have bound a peptide derived from a foreign protein. The<br />
MHC class I pathway presents endogenously antigens, e.g. viral protein fragments,<br />
produced by cytosolic proteolysis, sequestered within the infected cell. Recognition of<br />
pMHC class I complexes by cytotoxic T cells leads to death of the antigen-expressing<br />
cell. The proteolytic cascades that produce MHC class I epitopes start with<br />
proteasomal degradation.<br />
The proteasome is a multi-catalytic proteolytic machine that is abundant and<br />
responsible for the turnover of many critical regulatory proteins including tumor<br />
suppressor proteins and cell cycle regulators. The destructive force of the proteasome<br />
as an important determinant of protein half-life is regulated by ubiquitination.<br />
Substrates are tagged with multiple ubiquitin (Ub) molecules for destruction by the<br />
proteasome. Ubiquitin is a 76 amino acid protein that can be conjugated onto<br />
substrates to guide protein destruction. The majority of proteins are targeted for<br />
proteasomal proteolysis by Ub polymers. Despite a wealth of literature on<br />
ubiquitination of proteasome substrates, little is known about the degradation<br />
process at a more detailed molecular level; ubiquitination status and protein stability<br />
currently cannot be predicted. It is clear however, that a ubiquitin code exists and that<br />
protein turnover by the proteasome is a tightly regulated and complex process that<br />
includes not only the complexity of the proteasome but also Ub polymer formation<br />
and remodelling and Ub recycling. Because of this complexity, tools that allow<br />
detailed studies of the effects of ubiquitination status on protein turnover are<br />
urgently needed.<br />
Figure 2: MHC class I-mediated immunity: antigen processing, -loading -presentation and the T cell kill.<br />
The proteasome is responsible for protein degradation assisted by other (endo)-proteolytic activities<br />
affording eptitopes that can be loaded into MHC class I complexes which may result in a T cell-mediated<br />
kill of the antigen presenting cell.<br />
Activity profiling with chemical probes Major progress has been made in<br />
understanding cancer on a genetic level but this knowledge is not easily translated<br />
into new drugs. Genetic methods generally do not assign protein function or enzyme<br />
activity, which are often in turn regulated by protein-protein interactions and posttranslational<br />
mechanisms. Post translational modifications and proteolytic<br />
processing modulate protein function, localization, half-life, and protein expression.<br />
The study of protein function is limited by current techniques. Commonly used<br />
techniques to study proteins include radiolabelling of newly synthesized<br />
polypeptides, use of antibodies that may not be specific for the full repertoire of posttranslational<br />
modifications, and determination of transcript levels with similar<br />
limitations. No single ideal technique currently exists and methods that determine<br />
protein function, enzyme activity and receptor function directly have to be developed.<br />
This is the primary aim of our lab, while we focus on targeted cytosolic proteolysis<br />
and antigen presentation.<br />
Our lab aims to develop tools to profile cellular enzymatic activities associated with<br />
post-translational modification of proteins with ubiquitin and we study proteasome<br />
activity, antigen production and antigen presentation by designing tools that should<br />
inferfere with individual components of these systems. We search for inhibitors of
enzymatic activities and ligands of receptors both by high throughput screening of<br />
small molecule compound collections using in vitro biochemical screens and cellbased<br />
assays and by rational chemical design leading to chemical optimization and<br />
all biochemistry further required. Ligands and inhibitors form the basis for the<br />
development of research probes that we use to achieve our goals.<br />
Research is centred around one central theme: chemistry and biology of targeted cytosolic<br />
proteolysis and antigen presentation, and currently divided into three main topics:<br />
(1) ubiquitin chemistry and biochemistry<br />
(2) proteasome action<br />
(3) phosphate ester hydrolysis<br />
(4) MHC class I antigen presentation<br />
Figure 3: Docking molecules into the MHC class I pocket<br />
Chemical reporters of UPS activity Pharmacological interference with UPSmediated<br />
protein degradation holds much promise. However, the only example of<br />
pharmacological modulation of the UPS approved for use in the clinic so far is the<br />
proteasome inhibitor bortezomib and tools to study proteasome action are in<br />
demand. A chemical approach using irreversible covalent inhibitors equipped with<br />
reporter groups offers several advantages over traditional approaches, including their<br />
applicability to any cell line or tissue. We recently developed such probes which have<br />
been shown to provide information that correlates directly with the functional state of<br />
enzyme active sites: active forms only and not latent or (auto)inhibited activities are<br />
reported. Using fluorescent proteasome activity reporters we have analyzed<br />
proteasome activity in mouse, we were able to monitor pharmacological inhibition in<br />
vivo and we were able to visualize active proteasomes in (primary) human cells both<br />
by confocal microscopy and flow cytometry.<br />
Conditional MHC class I ligands for epitope mapping MHC-bound peptides are<br />
essential for the stability of the MHC class I complex. Hence, standard strategies for<br />
the production of recombinant MHC complexes are based on in vitro refolding<br />
reactions with specific peptides and this severely limits the ability to produce large<br />
collections of peptide-MHC complexes. To address this issue, we developed in<br />
collaboration with the Schumacher lab conditional MHC ligands that can be cleaved<br />
in the MHC bound state under conditions that do not affect the integrity of the MHC<br />
structure. MHC class I molecules can efficiently be produced with these conditionally<br />
cleavable peptide ligands. These UV-labile ligands have been shown to disintegrate in<br />
the MHC-bound state upon exposure to UV light under mild conditions (neutral pH,<br />
4-37oC). Importantly, when UV-mediated cleavage is performed in the presence of<br />
another MHC binding peptide, an efficient ligand exchange reaction results in a class<br />
I molecule complexed with the epitope of choice. Ligands that disintegrate on<br />
command have now been identified for various different human MHC alleles (i.e.<br />
HLA-A2 and -A1, -A3, -A11 and –B7), indicating that it is straightforward to identify<br />
conditional ligands for other MHC class I alleles. Furthermore, this MHC exchange<br />
technology has been adapted to high-throughput applications in automated ELISA<br />
and fluorescence polarization assays.<br />
Future directions Improvements and development of new technologies to profile<br />
enzymatic UPS-related activities and MHC loading will allow new approaches to<br />
understanding the ubiquitin proteasome system and antigen presentation,<br />
unravelling novel phenomena. Despite major achievements in chemical probe<br />
development and in chemical investigations into the ubiquitin proteasome system<br />
and MHC class I antigen presentation, many challenges remain.<br />
Publication (continued)<br />
35<br />
CELL BIOLOGY II<br />
Piva R, Ruggeri B, Williams M, Costa G,<br />
Tamagno I, Ferrero D, Giai V, Coscia M,<br />
Peola S, Massaia M, Pezzoni G, Allievi C,<br />
Pescalli N, Cassin M, di Giovine S,<br />
Nicoli P, de Feudis P, Strepponi I,<br />
Roato I, Ferracini R, Bussolati B,<br />
Camussi G, Jones-Bolin S, Hunter K,<br />
Zhao H, Neri A, Palumbo A, Berkers C,<br />
Ovaa H, Bernareggi A, Inghirami G. CEP-<br />
18770: A novel, orally active proteasome<br />
inhibitor with a tumor-selective<br />
pharmacologic profile competitive with<br />
bortezomib. Blood <strong>2008</strong>;111:2765-75<br />
Raijmakers R, Berkers CR, de Jong A,<br />
Ovaa H, Heck AJ, Mohammed S.<br />
Automated online sequential isotope<br />
labeling for protein quantitation applied to<br />
proteasome tissue-specific diversity. Mol Cell<br />
Proteomics <strong>2008</strong>;7:1755-62<br />
Shanmugham A, Ovaa H. DUBs and<br />
disease: activity assays for inhibitor<br />
development. Curr Opin Drug Discov Devel<br />
<strong>2008</strong>;11:688-96<br />
Figure 4: MHC exchange. A. Principle of<br />
MHC class I-exchange and exchange-based<br />
screening assay based on fluorescence<br />
polarization (FP). B. A modified influenza<br />
A epitope is shown, designed to disintegrate<br />
upon UV exposure. The UV-cleavable<br />
2-nitrophenyl based b-peptide and cleavage<br />
products are depicted in red.
36<br />
CELL BIOLOGY II<br />
Group leader Peter Peters<br />
Peter Peters PhD Group leader<br />
Nicole Van der Wel PhD Associate staff<br />
scientist<br />
Sue Godsave PhD Post-doc -<br />
EU project-manager<br />
Pekka Kujala PhD Post-doc<br />
Musa Sani PhD Post-doc<br />
Matthijn Vos PhD Post-doc<br />
Diane Houben MSc PhD student<br />
Jason Pierson MSc PhD student<br />
Karin De Punder BSc Technical staff<br />
Hans Jansen BSc Technical staff<br />
Maaike Van Zon BSc Technical staff<br />
Nico Ong Research assistant<br />
Collaborators<br />
Jose Jesus Fernandez: Centro Nacional de<br />
Biotechnologia-Conejo Superior de<br />
Investigaciones Cientificas, Madrid, Spain<br />
Bram Koster: Department of Molecular<br />
Cell Biology, Leiden University Medical<br />
Center, Leiden<br />
Henny Zandbergen: Department of<br />
Physic, Technical University, Delft<br />
Helmut Gnaegi: Diatome Ltd, Biel,<br />
Switzerland<br />
NANOBIOLOGY<br />
One focal point of our structural biology group is to reveal and manipulate the<br />
macromolecular organisation of cells under normal and pathogenic conditions at the<br />
nano-scale level. We use cryo-electron tomography of vitreous sections, currently the<br />
only method that can obtain molecular resolution of molecular machines in cells in a<br />
near-native situation. The tomograms contain a 3D map of the cellular proteome at<br />
about 4 nm resolution and we are just beginning to explore its potential by placing<br />
high effort on developing methods for our nanotechnology. The other central point of<br />
our group is to visualise gene products in cells by electron microscopy at the highest<br />
resolution with gold probes on cryo-sections.<br />
Nanomachines in cells Cryo electron tomography of vitreous sections is becoming<br />
an established technique to study the three-dimensional structure of molecular<br />
machines in situ. Specifically we have been looking at the 80S eukaryotic ribosome<br />
within the cell. Using high-resolution tomography of unfixed frozen hydrated cryosections<br />
the structure and relative orientation of the ribosome, in a cellular context,<br />
has been established by semi-automatic particle selection, volumetric alignment and<br />
averaging. With this template a ‘molecular atlas’ was constructed, mapping the<br />
ribosome over the entire cellular volume within a thin vitreous section of a cell (see<br />
figure 5). This study illustrates the possibilities of this nanotechnology towards<br />
visualizing cellular machinery in situ at macromolecular resolutions in large<br />
eukaryotic cells. Future challenges include visualizing individual conformations<br />
(imposed by drugs) of GFP tagged ribosomes and using volumetric classification<br />
algorithms based on maximum likelihood statistical approach. Our initial attempt at<br />
understanding the spatial arrangements of the ribosome in situ is exciting and hints<br />
at the possibilities of imaging the vast majority of other biological machines in which<br />
the cellular spatial arrangement and molecular interactions that still remain elusive.<br />
One specific example that we now address is the transmembrane protein conduit<br />
machinery, the SecY/Sec61 system, which is attached to the ribosome and<br />
responsible for translocating nascent proteins into the lumen of the endoplasmic<br />
reticulum. With new technologies being developed by us and others a resolution of<br />
2.5 nm is approaching, allowing template matching the X-ray crystal structure with<br />
the ultimate goal to explain the structure of our averaged density maps.<br />
Figure 5: Constructing a<br />
Cellular Atlas of 80S<br />
Ribosomes A 10 nm slice<br />
through a reconstructed volume<br />
displaying cellular<br />
macromolecular machines,<br />
specifically the 80S Ribosome.<br />
A vacuole (V) and a<br />
Mitochondrion (M) can also be<br />
seen within the image. Upper<br />
Inset. High-resolution density<br />
map of the averaged 80S<br />
Ribosome. Lower Inset.<br />
A select area from the cellular<br />
Ribosomal atlas. Scale Bar,<br />
100 nm.<br />
Recombinant BCG and its translocation In collaboration with the group of<br />
Michael Brenner (Harvard, Boston) we published last year that after prolonged<br />
infection in macrophages and dendritic cells, M. tuberculosis translocates from<br />
phago-lysosomes to the cytosol (Cell. 2007 Jun 29;129(7):1287-98). The BCG<br />
vaccine strain failed to translocate from the phago-lysosome. The translocation into<br />
the cytosol was totally unexpected and against textbook knowledge. This difference<br />
in localization might give an explanation for the ineffectiveness of BCG as a MHC<br />
class I / CD8 triggered vaccine. Looking further into the difference in localization<br />
can bring us closer to a better vaccine against tuberculosis but perhaps also against
virus induced tumors such as HPV. The aim of our current work is to determine<br />
which regions of difference (RDs) between the M. tuberculosis and BCG genomes<br />
are important for translocation. In collaboration with the lab of Roland Brosch from<br />
Pasteur in Paris, we started with looking at BCG with a knock-in of the extended<br />
RD1 region (BCG::RD1), as this region is now known to encode the ESX-1 secretion<br />
system recently coined as a novel type VII secretion system (Abdallah AM et al., Type<br />
VII secretion. Nat Rev Microbiol. 2007;5(11):883-91). We found that this bacterium<br />
can be seen in the cytosol of the cell after 7 days of infection and conclude that the<br />
ESX-1 system (in a BCG background) is sufficient for translocation. In addition we<br />
are investigating ESX-5, another type VII secretion system from M. marinum (Bitter,<br />
VUmc Amsterdam). One of our next aims is the structure of ESX-1 secretion systems<br />
by cryo electron tomography. The proposed 3D-EM studies complement and expand<br />
upon on-going efforts to provide novel insights into the spatial organization of<br />
molecular machines within cells as outlined in the paragraph above. Currently we are<br />
also investigating by cryo-immunogold EM several different recombinant BCG based<br />
vaccine candidates. These are hypothesized to translocate from the phagosome to the<br />
cytosol using pore forming proteins from other bacteria, such as Listeriolysin from<br />
Listeria monocytogenes (Kaufmann, Berlin) and Perfringolysin O from Clostridium<br />
perfringens (Fulkerson, Aeras, Rockville USA).<br />
Prions Prion diseases are caused by accumulation of an abnormally folded isoform<br />
(PrP Sc ) of the cellular prion protein (PrP C ). The subcellular site where PrP Sc<br />
is formed is still unclear. In collaboration with the lab of Stan Prusiner (UCSF, San<br />
Francisco) we have used quantitative cryo-immunogold electron microscopy to localise<br />
different populations of PrP on hippocampal sections from prion-infected mice.<br />
At a late subclinical stage of prion infection we detected a 6 fold overall increase in<br />
PrP levels in the stratum oriens. The biggest increase (24 fold) occurred on vesicles<br />
resembling early endocytic or recycling vesicles in small neurites in the neuropil,<br />
suggesting that these vesicles are important in PrP Sc formation or pathology.<br />
We are also performing cryo-electron tomography on prion preparations, in order to<br />
solve the 3D structure of prions. In collaboration with Jesus Requena (University of<br />
Santiago de Compostela, Spain) we have examined purified samples of the proteinase<br />
K-resistant core of hamster PrP Sc , vitrified in the recently obtained FEI vitrobot. The<br />
preparations were found to contain 2 nm fibres, sometimes twisted together with<br />
other fibres. The data were consistent with a model in which individual fibres are<br />
comprised of ‘strings’ of monomers.<br />
Single intestinal stem cells The intestinal epithelium is the most rapidly selfrenewing<br />
tissue in adult mammals. In collaboration with the lab of Hans Clevers<br />
(Hubrecht Institute, Utrecht) we have recently demonstrated the presence of<br />
approximately six cycling Lgr5 +ve stem cells at the bottom of a small intestinal crypt<br />
(Barker N, et al., Nature. 2007;449(7165):1003-7). We have now established longterm<br />
culture conditions under which single crypts undergo multiple crypt fission<br />
events, whilst simultaneously generating villus-like epithelial domains in which all<br />
differentiated cell types are present. Single sorted Lgr5 +ve stem cells can also initiate<br />
these crypt-villus organoids. We conclude that intestinal crypt-villus units are selforganizing<br />
structures, which can be built from a single stem cell in the absence of a<br />
non-epithelial cellular niche.<br />
Publications<br />
37<br />
CELL BIOLOGY II<br />
Godsave SF, Wille H, Kujala P, Latawiec D,<br />
DeArmond SJ, Serban A, Prusiner SB,<br />
Peters PJ. Cryo-Immunogold Electron<br />
Microscopy for Prions: Toward<br />
Identification of a Conversion Site. J<br />
Neurosci. <strong>2008</strong>;28:12489-12499<br />
Hava DL, van der Wel N, Cohen N,<br />
Dascher CC, Houben D, León L, Agarwal S,<br />
Sugita M, van Zon M, Kent SC, Shams H,<br />
Peters PJ, Brenner MB. Evasion of peptide,<br />
but not lipid antigen presentation, through<br />
pathogen-induced dendritic cell maturation.<br />
Proc Natl Acad Sci U S A.<br />
<strong>2008</strong>;105:11281-6<br />
Peters PJ, Pierson J. Immunogold labeling<br />
of thawed cryosections. Methods Cell Biol.<br />
<strong>2008</strong>;88:131-49<br />
Abdallah AM, Savage ND, van Zon M,<br />
Wilson L, Vandenbroucke-Grauls CM,<br />
van der Wel NN, Ottenhoff TH, Bitter W.<br />
The ESX-5 secretion system of<br />
Mycobacterium marinum modulates the<br />
macrophage response. J Immunol.<br />
<strong>2008</strong>;181:7166-75<br />
Verbrugghe P, Kujala P, Waelput W,<br />
Peters PJ, Cuvelier CA. Clusterin in human<br />
gut-associated lymphoid tissue, tonsils, and<br />
adenoids: localization to M cells and<br />
follicular dendritic cells. Histochem Cell<br />
Biol. <strong>2008</strong>;129:311-20<br />
Zhang L, Lee SY, Beznoussenko GV,<br />
Peters PJ, Yang JS, Gilbert H, Brass AL,<br />
Elledge SJ, Isaacs SN, Moss B, Mironov A<br />
and Hsu VW. Coatomer and KDEL<br />
Receptor: Two host proteins implicated in<br />
early vaccinia biogenesis. PNAS (in press)<br />
Van der Flier L, Van Gijn M, Hatzis P,<br />
Kujala P, Haegebarth A, Stange D,<br />
Begthel H, Van den Born M, Guryev V,<br />
Oving I, Van Es JH, Barker N, Peters PJ,<br />
Van de Wetering M, Clevers H.<br />
Transcription factor Achaete scute-like 2<br />
(Ascl2) controls intestinal stem cell fate.<br />
Cell (in press)
38<br />
EXPERIMENTAL THERAPY<br />
Group leader Laura Van ’t Veer<br />
Laura Van ’t Veer PhD Group leader<br />
Petra Nederlof PhD PI and Academic staff<br />
Frans Hogervorst PhD Academic staff<br />
Flora Van Leeuwen PhD Academic staff<br />
Sabine Linn MD PhD Academic staff<br />
Senno Verhoef MD PhD Academic staff<br />
Annegien Broeks PhD Academic staff<br />
Marjanka Schmidt PhD Senior post-doc<br />
Youji He MD PhD Post-doc<br />
Tim Molloy PhD Post-doc<br />
Simon Joosse PhD student<br />
Marleen Kok MD PhD student<br />
Stella Mook MD PhD student<br />
Sjoerd Bruin MD Clinical fellow<br />
Astrid Bosma Technical staff<br />
Linde Braaf Technical staff<br />
Renske Fles Technical staff<br />
Richard Van Hien Technical staff<br />
Hasibe Iri-Kiraz Technical staff<br />
Sten Cornelissen Technical staff<br />
Izabela Mikolajewska Technical staff<br />
Kim Brandwijk Technical staff<br />
Lennart Mulder Technical staff<br />
Publications<br />
Broeks A, Braaf LM, Huseinovic A,<br />
Schmidt MK, Russell NS, Van Leeuwen FE,<br />
Hogervorst FB, Van 't Veer LJ. The<br />
spectrum of ATM missense variants and<br />
their contribution to contralateral breast<br />
cancer. Breast Cancer Res Treat.<br />
<strong>2008</strong>;107:243-8<br />
Horlings HM, van Laar RK, Kerst JM,<br />
Helgason HH, Wesseling J,<br />
van der Hoeven JJ, Warmoes MO,<br />
Floore A, Witteveen A, Lahti-Domenici J,<br />
Glas AM, Van 't Veer LJ, de Jong D. J Clin<br />
Gene expression profiling to identify the<br />
histogenetic origin of metastatic<br />
adenocarcinomas of unknown primary.<br />
Oncol. <strong>2008</strong>;26:4435-41<br />
EXPERIMENTAL THERAPY<br />
MOLECULAR PATHOLOGY AND MOLECULAR EPIDEMIOLOGY<br />
OF BREAST AND COLORECTAL CANCER<br />
Genome-wide genomic profiling in hereditary breast cancer (PI P Nederlof )<br />
We have shown that breast tumors from BRCA1 and BRCA2 mutation carriers show<br />
specific chromosomal changes, and that these genomic profiles can be used to<br />
classify individual tumors. We use a genome-wide array-CGH analysis containing<br />
~3500 1 Mb spaced BAC clones (www.sanger.ac.uk) with routinely formalin-fixed<br />
paraffin embedded (FFPE) tumor material. We showed that in a series of 48 breast<br />
tumors from BRCA1/2 negative breast and ovarian cancer (HBOC) families only two<br />
tumors showed a BRCA1-like profile, and we could confirm the inactivation of<br />
BRCA1 in one case (promoter methylation). Currently we apply the classification<br />
strategy to gain additional proof of significance for unclassified variants (UVs); gene<br />
alterations for which the pathogenicity is still unclear. In collaboration with Dr. P<br />
Devilee (Leiden University) we are collecting several breast cancer cases per family<br />
from BRCA1/2 negative HBC families (BRCAx). Preliminary results on 60 cases<br />
have shown that BRCAx tumors are heterogeneous but that within families the<br />
differences are smaller. This finding will be used in a larger set of tumors to<br />
determine different BRCAx subgroups that may allow linkage analyses within the<br />
subgroups, in order to find additional breast cancer genes.<br />
Molecular profiling for breast cancer classification and therapy response<br />
Half of the recurrences of ERa+ breast tumors respond to tamoxifen while the other<br />
half is resistant. Recently, an 81-gene tamoxifen response profile for advanced disease<br />
has been published (Jansen et al, JCO 2005). We validated this response profile on a<br />
more advanced whole genome platform and in an independent set of tumors,<br />
including stable disease (initially excluded). Additionally, an even more robust<br />
algorithm for tamoxifen response predicting is under development. Finally, these<br />
profiles are now being tested for clinical use in the adjuvant setting. In addition, we<br />
genotyped patients for SNPs in enzymes involved in the metabolism of tamoxifen,<br />
the cytochrome P450 (CYP) familie. In collaboration with Dr. Els Berns (Erasmus<br />
Medical Center) we showed that a SNP in CYP2C19 may result in a altered tamoxifen<br />
response. The ability to accurately predict tamoxifen treatment outcome would<br />
constitute a significant advance in the management of breast cancer.<br />
Genetic determinants of breast cancer risk and outcome<br />
In a retrospective nationwide cohort of Dutch breast cancer patients (BOSOM=<br />
Breast Cancer Outcome Of Mutation carriers) as well as within a large international<br />
collaboration (BCAC = breast cancer association consortium), we are investigating<br />
which germline variants (pathogenic mutations in e.g. BRCA1/2 as well as SNPs)<br />
influence breast cancer risk and outcome.<br />
Molecular profiling of bilateral breast tumor pairs<br />
With the increased incidence of breast cancer and the improved survival, studies on<br />
breast cancer outcome have become an important issue. This is especially true for<br />
young women who potentially have a long life expectancy after breast cancer, but also<br />
a high risk of developing a contralateral breast cancer (CBC) compared to older<br />
patients. Certain SNPs have been shown to increase the risk for the development of<br />
breast cancer and to predispose to a certain tumor subtype (e.g basal-like), as is seen<br />
in BRCA1 carriers. We are investigating which SNPs predispose to an increased risk<br />
for the development of a CBC and whether part of this risk can be predicted by the<br />
tumor subtype of the first cancer.<br />
Validation of Adjuvant! Online<br />
Nowadays, adjuvant treatment recommendations for early stage breast cancer<br />
patients are based on the estimated risk of breast cancer relapse and death and the<br />
expected benefit of adjuvant treatment. Several tools have been developed to make
estimates for the individual patient. One of these tools is Adjuvant! Online<br />
(www.adjuvantonline.com), which calculates a 10-year survival probability based on<br />
the patient’s age, tumor size, grade and estrogen receptor. In addition, the computer<br />
program also calculates the likely benefit to be expected from adjuvant systemic<br />
treatment. Adjuvant! Online is already being used by physicians. In addition, in the<br />
MINDACT trial the clinical risk is calculated by Adjuvant! Online. To assess the<br />
validity of Adjuvant! Online for Dutch breast cancer patients we are comparing<br />
predicted and observed disease outcome in 2 Dutch cohorts of approximately 10000<br />
patients in total.<br />
Validation of the 70-gene prognosis-signature in breast cancer<br />
subpopulations<br />
This validation study provides evidence that the 70-gene prognosis-signature<br />
(MammaPrint), is also a predictor of disease outcome in postmenopausal breast<br />
cancer patients and in women with 1-3 involved lymph nodes.<br />
The detection and prediction of circulating tumor cells in breast cancer<br />
patients<br />
The detection of circulating tumor cells in the blood of cancer patients is a promising<br />
tool for risk stratification. We previously developed a multi-marker mRNA QPCRbased<br />
detection platform for the semi-quantitation of tumor cell load in the peripheral<br />
blood of breast cancer patients. Recently we validated this assay in collaboration with<br />
the Radiumhospitalet in Oslo, Norway (B Naume), assaying a retrospective series of<br />
107 early-stage breast cancer patients from which blood was collected at time of<br />
diagnosis and stored frozen (median follow-up 7.4 years). The assay proved to be a<br />
powerful outcome predictor, with patients in which circulating tumor cells were<br />
detected having a significantly poorer relapse-free and overall survival. The assay was<br />
particularly powerful in lymph node negative patients (hazard ratio = 13.0 for relapsefree<br />
survival and 6.3 for overall survival, p < 0.01) and in multivariate analyses circulating<br />
tumor cell positivity was shown to be a significant and independent risk factor with<br />
respect to overall survival. This assay represents a powerful tool that could be used<br />
for prognostication in the clinic, and is currently being adapted and validated in other<br />
tumor types and also as a predictor of treatment response in patients.<br />
Specific DNA aberrations associated with colorectal cancer metastasis<br />
The overall five year survival of colorectal cancer (CRC) is 57% and up to 50% of all<br />
patients will develop metastasis. We are using array-CGH to investigate genome-wide<br />
chromosomal aberrations in primary and liver metastasized colorectal tumors. We<br />
found that primary colorectal tumors that developed liver metastasis have a unique<br />
chromosomal signature. We are now working on a validation study to validate the<br />
liver metastasis classifier in combination with mutation analysis. Secondary research<br />
will be done in operated colorectal liver metastasis for understanding the clinical<br />
behaviour after surgery. The ability to predict liver metastasis and understanding of<br />
their clinical behaviour by genomic aberrations is important for understanding the<br />
biology of the tumor and may lead to more individualized disease management.<br />
Avoidance of overtreatment with radiotherapy in rectal cancer patients<br />
The development of local recurrences (LR) is a major problem in the treatment of<br />
rectal cancer and there is a high clinical need to identify those patients who are at<br />
increased risk. In this study 1. We aim to develop a gene expression profile for the<br />
risk of LR in rectal cancer patients, allowing for selection of patients for preradiotherapy<br />
(RT). And, by establishing additional profiles for nodal status and<br />
circumferential margin, further refinement of the neo-adjuvant treatment can be<br />
achieved. Non irradiated fresh frozen tumor samples from 240 stage I-III rectal<br />
cancer patients were collected from the Dutch total mesorectal excision (TME) trial,<br />
in which rectal cancer patients were randomized for short-term pre-RT followed by<br />
TME or TME alone. RNA isolation was prepared and gene expression array will be<br />
performed on 200 samples. 2. By using PCR/sequencing on parallel extracted DNA,<br />
we also determined PIK3CA (exons 9 and 20), K-Ras (exon 1) and BRAF V600E<br />
mutations in 19 (7.9%), 81 (33.9%) and 5 (2.1%) rectal cancers, respectively.<br />
Interestingly, PIK3CA mutations revealed a strong association with increased LR.<br />
39<br />
EXPERIMENTAL THERAPY<br />
Publications (continued)<br />
Joosse SA, van Beers EH, Tielen IH,<br />
Horlings H, Peterse JL, Hoogerbrugge N,<br />
Ligtenberg MJ, Wessels LF, Axwijk P,<br />
Verhoef S, Hogervorst FB, Nederlof PM.<br />
Prediction of BRCA1-association in<br />
hereditary non-BRCA1/2 breast carcinomas<br />
with array-CGH. Breast Cancer Res Treat.<br />
<strong>2008</strong> Aug 14. (Epub ahead of print)<br />
Kok M, Linn SC, Van Laar RK, Jansen MP,<br />
Van den Berg TM, Delahaye LJ et al.<br />
Comparison of gene expression profiles<br />
predicting progression in breast cancer<br />
patients treated with tamoxifen. Breast<br />
Cancer Res Treat <strong>2008</strong> Jul 27. (Epub ahead<br />
of print)<br />
Molloy TJ, Bosma AJ, Van 't Veer LJ.<br />
Towards an optimized platform for the<br />
detection, enrichment, and semiquantitation<br />
circulating tumor cells. Breast<br />
Cancer Res Treat. <strong>2008</strong>;112:297-307<br />
Mook S, Schmidt MK, Viale G, Pruneri G,<br />
Eekhout I, Floore A, Glas AM, Bogaerts J,<br />
Cardoso F, Piccart-Gebhart MJ,<br />
Rutgers ET, Van 't Veer LJ; On behalf of the<br />
TRANSBIG consortium. The 70-gene<br />
prognosis-signature predicts disease outcome<br />
in breast cancer patients with 1-3 positive<br />
lymph nodes in an independent validation<br />
study. Breast Cancer Res Treat <strong>2008</strong> Jul 27<br />
Reyal F, Van Vliet MH, Horlings HM,<br />
Armstrong N, De Visser K, Kok M,<br />
Meuleman W, Teschendorff A, Mook S,<br />
Caldas C, Salmon R, Van de Vijver MJ,<br />
Wessels LFA. A comprehensive analysis of<br />
nine prognostic signatures reveals the high<br />
predictive capacity of Proliferation, Immune<br />
response and RNA splicing modules in<br />
breast cancer. Breast Cancer Res.<br />
<strong>2008</strong>;10:R93<br />
Van 't Veer LJ, Bernards R. Enabling<br />
personalized cancer medicine through<br />
analysis of gene-expression patterns. Nature.<br />
<strong>2008</strong>;452:564-70 (review)<br />
Weigelt B, Horlings HM, Kreike B,<br />
Hayes MM, Hauptmann M, Wessels LF,<br />
de Jong D, Van de Vijver MJ,<br />
Van 't Veer LJ, Peterse JL. Refinement of<br />
breast cancer classification by molecular<br />
characterization of histological special types.<br />
J Pathol. <strong>2008</strong>;216:141-50
40<br />
EXPERIMENTAL THERAPY<br />
Group leader Adrian Begg<br />
Adrian Begg PhD Group leader<br />
Conchita Vens PhD Research Associate<br />
Sari Neijenhuis MSc PhD student<br />
Monique De Jong MD PhD student<br />
Caroline Verhagen MD PhD student<br />
Ben Floot Technical staff<br />
Ingrid Hofland Technical staff<br />
Manon Verwijs Technical staff<br />
MECHANISMS AND PREDICTION OF TUMOR RESPONSE TO<br />
RADIATION<br />
We are pursuing two research lines, one on fundamental aspects of the radiation<br />
response and the other aiming more directly at clinical translation. The first involves<br />
how the cell handles DNA base damage and single strand breaks, an aspect of<br />
radiation damage that has indicated some novel targets for radiotherapy for cancer.<br />
The second focuses on prediction of outcome of radiotherapy, particularly using<br />
genome wide screening methods, which has also indicated some interesting potential<br />
targets.<br />
Mechanisms and modulation of radiosensitivity<br />
Possible involvement of polbDN in DSB induction or repair<br />
We have shown that a polbeta dominant negative (polbDN) radiosensitized<br />
mammalian cells, of potential clinical relevance, since a significant proportion of<br />
human cancers have polbeta mutations resembling this polbDN. More residual<br />
double strand breaks (DSB) after irradiation were found in polbDN-expressing cells<br />
than in vector controls, and we therefore tested polbDN’s involvement in DSB<br />
induction and repair. Studies with a DNA-PK inhibitor indicated no involvement of<br />
non-homologous endjoining (NHEJ) in radiosensitization by the polbDN, supporting<br />
previous data on DNA repair mutant cell lines. Premature chromosome<br />
condensation (PCC) assays indicated secondary double strand break formation after<br />
the initial immediate radiation-induced breaks, which was independent of DNA-PK<br />
activity. Interestingly, these extra breaks were not accompanied by gH2AX foci.<br />
Irradiation induced more chromosome and chromatid type aberrations in polbDNexpressing<br />
cells than in wildtype cells, but an increase in only chromatid type<br />
aberrations was seen after H 2O 2, also an oxidative damage inducer. This indicates a<br />
role for clustered damage, unique to ionizing radiation, in polbDN-induced<br />
radiosensitization.<br />
We further showed a replication dependence of polbDN radiosensitization, since<br />
sensitization was less in confluent than in log phase cells, and could be modeled<br />
assuming only S/G2 cells were sensitized. This is in contrast to polb-deficient<br />
(knockout) cells, which were more radiosensitive only under confluent conditions or<br />
in G1 log-phase cells. This is presumed to be partly due to the extra inhibition of long<br />
patch repair by the polbDN, and the role and availability of the various backup<br />
pathways.<br />
Figure 1: Expressions of a DNA polymerase beta<br />
dominant negative (polbDN), known to inhibit<br />
bae excision repair, causes increased<br />
chromosome aberrations after irradiation<br />
(upper panel), indicating formation of<br />
additional double strand breaks. This is<br />
probably the cause of the observed<br />
radiosensitization. These extra double strand<br />
breaks do not appear to signal the formation of<br />
gH2AX foci (lower panel).
Testing the hypothesis that BER-deficient cells are sensitive to HR inhibition<br />
Synthetic lethality has been shown when both homologous recombination (HR) and<br />
base excision repair (BER) are inhibited, arising from BRCA mutations and PARP<br />
inhibition. We hypothesized that this synthetic lethality would also arise if cells with<br />
mutations in the BER pathway, frequent in human tumors, are treated with HR<br />
inhibitors. We are currently testing this hypothesis. 17AAG has been shown to reduce<br />
BRCA2 and RAD51, thus affecting HR. Indeed, polDN-expressing cells (BER<br />
deficient) were radiosensitized more by 17AAG than wildtype cells. Experiments with<br />
XRCC1-knockout and polb-knockout cells (BER deficient) are underway, including<br />
treatment with caffeine and other putative HR-associated inhibitors.<br />
To look for novel HR or related inhibitors, we have set up a drug screening assay<br />
using the in-house robotics facility, with the goal of finding drugs which<br />
radiosensitize BER deficient more than proficient cells. This would provide tumor<br />
specific radiosensitization for those tumors exhibiting BER defects. For this, human<br />
tumor cells with and without expression of the polbDN were plated and irradiated in<br />
384-well plates. To assess post-irradiation growth, a automated microscopy based cell<br />
count assay was found to be far more sensitive than the CellTiter blue viability assay.<br />
In order to determine survival accurately after irradiation, assay times longer than 6<br />
days were necessary. We have begun to screen chemical libraries using this assay.<br />
Prediction of outcome We want to understand and predict causes of failure after<br />
radiotherapy, allowing selection of patients for alternative more effective therapies.<br />
Last year we measured expression profiles of advanced (T3-4) head and neck cancers<br />
(4 sites) prior to treatment, to search for genes which predict outcome in patients<br />
treated with concurrent radiotherapy and cisplatin (collaboration with departments of<br />
Head and Neck Surgery, Radiotherapy and Pathology). We found that a previously<br />
published signature, reported to distinguish head and neck cancer patients at high<br />
risk of either local or distant recurrence (Chung C et al, Cancer Res 2006), proved to<br />
be a highly significant predictor of loco-regional recurrence after combined radiochemotherapy<br />
in our patient series. We have now assessed the performance of this<br />
signature in a multivariable analysis with other clinical factors on 75 tumors where<br />
we also measured tumor volume at the time of treatment. The Chung signature<br />
proved to be independent of tumor site, volume and T-stage, with the signature and<br />
site being the most significant, yielding hazard ratios of around 4. This represents an<br />
independent validation of a signature, which appears to be independent of known<br />
clinical factors affecting outcome, further indicating its potential clinical usefulness.<br />
We have also completed a study on a series of larynx tumors from patients treated<br />
with radiotherapy alone and with less advanced tumors, representing a more<br />
homogenous patient and treatment group. Each patient with a local recurrence was<br />
matched for subsite and T-stage with one or two cured patients. This matched series<br />
comprised 19 recurrences and 30 cures, all with T1 or T2 tumors, in which<br />
pretreatment biopsies were taken and expression profiles measured (Illumina<br />
platform). In an hypothesis-driven approach, we tested the predictive potential of<br />
signatures known or suspected to affect response to radiotherapy. The putative stem<br />
cell marker CD44 was the most significant predictor of outcome (local control), with<br />
an acute hypoxia signature showing a strong trend. CD44 also emerged as the most<br />
significant gene in a data-driven approach searching for any set of genes<br />
distinguishing cures from recurrences. Signatures for intrinsic radiosensitivity and<br />
proliferation were not significant. We are now comparing these mRNA expression<br />
data with CD44 positivity assessed by immunohistochemistry. Further, we are<br />
measuring expression profiles on 30 more advanced (T3) larynx tumors and some<br />
oropharynx tumors, both treated by radiotherapy alone.<br />
In parallel, in a search for an intrinsic radiosensitivity predictor, we measured<br />
expression profiles on a series of 33 head and neck carcinoma cell lines with a range<br />
of radiosensitivities both before and up to 6h after 4Gy in vitro (collaboration with R<br />
Grenman, Turku, Finland). No genes which changed expression in response to<br />
irradiation were found which correlated with radiosensitivity. In contrast, a set of 288<br />
genes, which did not change after irradiation (remained high or low), were found<br />
which showed a significant correlation with intrinsic radiosensitivity. This represents<br />
the largest such dataset on site-specific cell lines. We are further investigating and<br />
validating the genes in this set.<br />
Publications<br />
41<br />
EXPERIMENTAL THERAPY<br />
Bartelink H, Begg AC. Prognostic and<br />
predictive markers in oncology. Semin<br />
Radiat Oncol. <strong>2008</strong>;18:73-74<br />
Vermeulen C, Verwijs-Janssen M, Begg AC,<br />
Vens C. Cell cycle phase dependent role of<br />
DNA polymerase beta in DNA repair and<br />
survival after ionizing radiation. Radiother<br />
Oncol. <strong>2008</strong>;86:391-398<br />
Vermeulen C, Bertocci B, Begg AC, Vens C.<br />
Ionizing radiation sensitivity of DNA<br />
polymerase lambda-deficient cells. Radiat<br />
Res. 2007;168:683-688<br />
Neijenhuis S, Verwijs-Janssen M,<br />
Kasten-Pisula U, Rumping G, Borgmann K,<br />
Dikomey E, Begg AC, Vens C. Mechanism<br />
of cell killing after ionizing radiation by a<br />
dominant negative DNA polymerase beta.<br />
DNA Repair <strong>2008</strong> (in press)<br />
Van den Broek GB, Wildeman M,<br />
Rasch CR, Armstrong N, Schuuring E,<br />
Begg AC, Looijenga LH, Scheper R,<br />
Van der Wal JE, Menkema L, Van Diest PJ,<br />
Balm AJ, Van Velthuysen MF,<br />
Van den Brekel MW. Molecular markers<br />
predict outcome in squamous cell carcinoma<br />
of the head and neck after concomitant<br />
cisplatin-based chemoradiation.<br />
Int J Cancer <strong>2008</strong> (in press)<br />
Wouters BG, Begg AC. Irradiation induced<br />
damage and the DNA damage response. In:<br />
Basic Clinical Radiobiology, pp 18-35, 4th Edition <strong>2008</strong> (in press)<br />
Begg AC. Molecular targeting and patient<br />
individualization. In: Basic Clinical<br />
Radiobiology, 4th Edition, pp 364-381, <strong>2008</strong><br />
(in press)
42<br />
EXPERIMENTAL THERAPY<br />
Group leader Fiona Stewart<br />
Fiona Stewart PhD Group leader<br />
Nicola Russell MD PhD Academic staff<br />
Fijs Van Leeuwen PhD Academic staff<br />
Saske Hoving PhD Post-doc<br />
Marion Scharpfenecker PhD Post-doc<br />
Ingar Seemann MSc PhD student<br />
Ben Floot Technical staff<br />
Johannes Te Poele Technical staff<br />
Nils Visser Technical staff<br />
RADIATION-INDUCED VASCULAR DAMAGE<br />
Improvements in detection and treatment of cancer have lead to increased numbers<br />
of survivors at risk for developing treatment related morbidity. More than half of all<br />
long-term survivors of cancer will have received radiotherapy as part of their<br />
treatment. Radiotherapy has been shown to be an independent risk factor for<br />
cardiovascular and cerebrovascular disease. This manifests as atherosclerosis in large<br />
vessels and telangiectasia and perfusion defects in capillaries. In our studies we focus<br />
on the mechanisms of development of radiation-induced vascular damage, with the<br />
ultimate goal of designing appropriate intervention strategies to inhibit or prevent<br />
this.<br />
Radiation induced atherosclerosis in large vessels (in collaboration with Mat<br />
Daemen, Cardiovascular Research Institute, Maastricht) We established a mouse model<br />
for studying radiation-induced atherosclerosis in the carotid arteries of ApoE-/- mice.<br />
These mice have increased cholesterol levels and develop atherosclerosis<br />
spontaneously with age, unlike wild type mice. ApoE-/- mice given single doses (8-14<br />
Gy) or clinically relevant fractionated irradiation (20 x 2 Gy in 4 weeks) to the neck,<br />
developed increased atherosclerotic plaque in the carotid arteries compared with agematched<br />
controls. The majority of lesions seen at 22-34 weeks after irradiation had an<br />
inflammatory, thrombotic phenotype, with a thin fibrous cap. The inflammatory<br />
phenotype was rare in unirradiated mice and lesions were protected by a thick<br />
fibrous cap. Wild type C57Bl6 mice did not develop atherosclerotic plaque, although a<br />
few (20%) did develop fatty streak lesions at 30 weeks after irradiation. These studies<br />
showed that irradiation, in combination with elevated cholesterol, accelerated the<br />
development of atherosclerosis and predisposed to the formation of a vulnerable,<br />
inflammatory, thrombotic plaque phenotype with a thin fibrous cap.<br />
Based on these results, we set up intervention studies, to investigate the potential of<br />
anti-platelet, anti-inflammatory and anti-cholesterol drugs to inhibit development of<br />
radiation-induced atherosclerosis. Drugs were given in the chow from 1 week before<br />
irradiation until termination of the experiment. Aspirin (ASA) and nitric oxide<br />
releasing aspirin (NO-ASA) inhibited endothelial cell (EC) expression of the adhesion<br />
molecule V-CAM and inhibited the formation of fatty streaks in these irradiated<br />
arteries at 4 weeks after irradiation. NO-ASA, but not ASA, also inhibited the<br />
formation of atherosclerotic lesions in unirradiated arteries at longer times (30 weeks)<br />
but there was no significant reduction in the number or size of lesions in the irradiated<br />
arteries. Clopidogrel and atorvastatin did not modify the development or progression<br />
of atherosclerotic lesions in irradiated or inirradiated vessels.<br />
In parallel clinical studies, we characterized radiation-induced vascular damage in<br />
resection material collected from head and neck (H&N; n = 70) and breast cancer<br />
patients (BC; n = 56) undergoing free-flap reconstructive surgery after previous<br />
radiotherapy or surgery alone. Using imaging analysis software the ratio of the<br />
thickness of the intima to the thickness of the media (IMR), was determined and<br />
sections were scored for proteoglycan content and inflammatory cell content. For the<br />
H&N cancer patients there was a 1.5-fold increase in the IMR at 4 years after<br />
irradiation (mean 66 Gy). Inflammatory cell content was also increased in the intima<br />
of the irradiated H&N vessels. For BC patients there was a 1.4-fold increase in IMR at<br />
3 years after irradiation (mean 49 Gy), after correction for the IMR value of the free<br />
flap artery, but this was not significant (p=0.072). There was a significant increase in<br />
the proteoglycan content of the media in the irradiated mammary vessels. This study<br />
demonstrated accelerated intimal thickening, increased proteoglycan content and<br />
inflammatory cell content, in medium sized arteries at relatively short times after<br />
radiation. These patients are likely to have an increased risk for development of<br />
atherosclerosis and subsequent cerebrovascular or cardiovascular disease.<br />
Radiation-induced telangiectasia (in collaboration with Peter ten Dijke, Leiden)<br />
Radiation induces late vascular damage that manifests in capillaries as telangiectasia.<br />
These are dilated, thin-walled blood vessels that may rupture thereby impairing organ<br />
function and causing excessive bleedings. Radiation-induced telangiectasia strongly
esembles the symptoms of patients with hereditary hemorrhagic telangiectasia<br />
(HHT). This autosomal dominant vascular disorder has been linked to mutations in<br />
the genes encoding transforming growth factor beta (TGF-b) receptor ALK1 and the<br />
co-receptor endoglin. Receptor haplo-insufficiency probably causes an imbalance<br />
between endothelial cell proliferation and migration (mediated via endoglin and<br />
ALK1 and their downstream target ID1) and vessel maturation (mediated via the<br />
TGF-b receptor ALK5 and its downstream target PAI-1). In this study we therefore<br />
aimed at elucidating the role of TGF-b as well as Notch signaling pathways in the<br />
development of radiation-induced telangiectasia.<br />
Irradiation of human microvascular endothelial cells in vitro resulted in reduced<br />
TGF-b/ALK1 signaling, with decreased activation of the signal transducing molecules<br />
Smad1 and Smad5 and diminished expression of the target gene ID1. TGF-b/ALK5<br />
signalling was increased as evidenced by activation of Smad2 and Smad3 and<br />
enhanced PAI-1 expression. Irradiation also activated the Notch signalling pathway<br />
shown by increased expression of the Notch ligand Jagged-1 and the downstream<br />
target gene Hey1. Functional studies showed that enhanced signalling through the<br />
anti-angiogenic ALK5 and Notch pathway leads to decreased cell migration and tube<br />
formation. Mechanistic and functional studies were further extended by analyzing<br />
irradiated kidneys from mice sacrificed at different time points after irradiation.<br />
Irradiation of wild type animals resulted in the development of telangiectasia.<br />
Expression of PAI-1 increased progressively from 10-30 weeks, whereas Id-1<br />
expression levels rose only transiently at 1 week after irradiation. Expression levels of<br />
ALK1 and ALK5 steadily increased whereas activation of the signal transducing Smad<br />
molecules changed during the course of the experiment. Further experiments<br />
indicated that changes in activity of the respective signalling pathways might be<br />
accomplished by altered expression of negative regulators. Endoglin levels were<br />
found to be increased by irradiation, suggesting an attempt to repair the damaged<br />
vasculature. As endoglin is known to have an impact on both ALK1 and ALK5<br />
signalling, we studied its importance on irradiation-induced TGF-ß signalling and<br />
telangiectasia development using endoglin heterozygous (Eng +/- ) mice. Eng +/- mice<br />
developed less telangiectasia. Whether these vessels display functional differences<br />
compared to the irradiated vasculature in wild type animals needs to be determined.<br />
Both ALK1 and ALK5 signalling were affected in Eng +/- animals demonstrating the<br />
importance of endoglin for both pathways in vivo. Future studies will address the<br />
question of how endoglin influences TGF-b signalling after irradiation and whether<br />
interfering with or promoting endoglin signalling is beneficial for irradiated cancer<br />
patients.<br />
Radiation induced cardiac damage (in collaboration with Christine Mummery,<br />
Leiden) Microvascular damage and perfusion defects are major underlying causes of<br />
heart failure after irradiation. This reflects inflammatory and thrombotic changes in<br />
EC function, as well as apoptotic EC loss and reduced capillary density.<br />
Revascularization of damaged heart tissue is essential to restore organ function and<br />
prevent myocardial infarct. Endoglin is highly expressed in damaged ECs and it is<br />
crucial for EC proliferation and vascular repair. In this project we will focus on the<br />
role of endoglin in microvascular damage and repair in the irradiated hearts of both<br />
wild type and endoglin deficient (Eng +/-) mice. In the first few months of this new<br />
project we have set up the procedure for local irradiation of mouse hearts and made<br />
base-line measurements of the heart volume. The imaging agent 99m Tc-labeled<br />
albumin is confined to the venous system. MicroSPECT/CT imaging of its<br />
distribution allows for a quantitative assessment of the percentage blood volume in<br />
the heart (pre-treatment values of 11.25 ± 1.45%). Consequently, it provides an<br />
accurate measure to study longitudinal changes in stroke volume. Alternatively,<br />
ultrasound imaging of the blood vessel microstructure in the myocardial wall will be<br />
performed using ultrasound and 99m Tc-sestamibi microSPECT/CT.<br />
Publications<br />
43<br />
EXPERIMENTAL THERAPY<br />
De Vreeze RSA, De Jong D, Haas RL,<br />
Stewart F, Van Coevorden F. Effectiveness<br />
of radiotherapy in myxoid sarcomas is<br />
associated with a dense vascular pattern.<br />
Int J Radiat Oncol Biol Phys <strong>2008</strong> (in press)<br />
Hendry JH, Akahoshi M, Wang LS,<br />
Lipschultz SE, Stewart FA. Radiationinduced<br />
cardiovascular injury. Radiat<br />
Environ Biophys <strong>2008</strong>;47:189-93<br />
Hoving S, Heeneman S, Gijbels MJ,<br />
te Poele JA, Russell NS, Daemen MJ,<br />
Stewart FA. Single dose and fractionated<br />
irradiation promote initiation and<br />
progression of inflammatory atherosclerosis<br />
in ApoE-/- mice. Int J Radiat Oncol Biol<br />
Phys <strong>2008</strong>;71:848-57<br />
Kruse JJCM, Floot BGJ, Te Poele JAM,<br />
Russell NS, Stewart FA. Radiation-induced<br />
activation of TGF-beta signaling pathways<br />
in relation to vascular damage in mouse<br />
kidneys. Radiat Res <strong>2008</strong> (in press)<br />
Scharpfenecker M, Kruse JJCM, Sprong D,<br />
Russell NS, ten Dijke P, Stewart FA.<br />
Ionizing radiation shifts the PAI1/ID1<br />
balance and activates Notch signaling in<br />
endothelial cells. Int J Radiat Oncol Biol<br />
Phys <strong>2008</strong> (in press)<br />
Stewart FA, Hoving, S, Kruse JJ.<br />
Radiation-induced cardiovascular and<br />
cerebrovascular effects: animal studies.<br />
Radiat Res. 2007;167:350-2
44<br />
EXPERIMENTAL THERAPY<br />
Group leader Jan Schellens<br />
Jan Schellens MD PhD Group leader<br />
Jos Beijnen PhD Academic staff<br />
Irma Meijerman PhD External staff<br />
Serena Marchetti MD PhD Academic staff<br />
Sander Veltkamp MSc PhD student<br />
Roos Oostendorp MSc PhD student<br />
Maarten Deenen MSc PhD student<br />
Lot Devriese MD MSc PhD student<br />
Suzanne Leijen MD PhD student<br />
Dick Pluim Technical staff<br />
Valerie Doodeman Technical staff<br />
Publications<br />
Damen CWN, Rosing H, Tibben MM,<br />
Van Maanen MJ, Lagas JS, Schinkel AH,<br />
Schellens JHM, Beijnen JH. A sensitive<br />
assay for the quantitative analysis of<br />
vinorelbine in mouse and human EDTA<br />
plasma by high-performance liquid<br />
chromatography coupled with electrospray<br />
tandem mass spectrometry. J Chromatogr B<br />
<strong>2008</strong>;868:102-09<br />
Harmsen S, Koster A, Beijnen JH,<br />
Schellens JH, Meijerman I. Comparison of<br />
two immortalized human cell lines to study<br />
nuclear receptor mediated CYP3A4 induction.<br />
Drug Metab Dispos <strong>2008</strong>;36:1166-71<br />
Hoebers FJ, Pluim D, Hart AA, Verheij M,<br />
Balm AJ, Fons G, Rasch CR, Schellens JH,<br />
Stalpers LJ, Bartelink H, Begg AC.<br />
Cisplatin-DNA adduct formation in<br />
patients treated with cisplatin-based<br />
chemoradiation: lack of correlation between<br />
normal tissues and primary tumor. Cancer<br />
Chemother Pharmacol <strong>2008</strong>;61:1075-81<br />
Marchetti S, de Vries NA, Buckle T, Pluim D,<br />
Beijnen JH, Mazzanti R, Van Tellingen O,<br />
Schellens JHM. Impact of the ABC-drug<br />
transporters ABCB1, ABCG2 and ABCC2<br />
on erlotinib disposition (Tarceva®) in in<br />
vitro and in vivo pharmacokinetic studies<br />
employing employing Bcrp1-/-/Mdr1a/1b-/-<br />
(triple knockout) and wild-type mice. Mol<br />
Cancer Ther <strong>2008</strong>;7:2280-87<br />
PHARMACODYNAMICS OF ANTICANCER DRUGS<br />
Our pharmacology group has an interest in studies exploring anticancer drug<br />
disposition, development of methods to improve oral pharmacokinetics (PK) of<br />
anticancer drugs and development of novel pharmacodynamic (PD) methods to<br />
support early clinical trials with anticancer drugs.<br />
Role of ABC drug transporters in absorption and disposition of anticancer<br />
drugs We performed a comprehensive preclinical pharmacokinetic study in wildtype,<br />
P-gp and/or BCRP knockout mice. Our results reveal that P-gp and BCRP only<br />
have modest effects on the absorption, distribution, metabolism and elimination of<br />
imatinib in comparison to metabolic elimination in mice. Of these two drug<br />
transporters, P-gp is the most dominant factor for systemic clearance and the most<br />
important for limiting brain penetration of imatinib. Interestingly, co-administration<br />
of the P-gp/BCRP inhibitor, elacridar, in mice leads to a 3-fold significant increase in<br />
the systemic exposure to imatinib in the presence or absence of P-gp/BCRP. This<br />
suggests that elacridar also inhibits other elimination pathways than P-gp and BCRP.<br />
Previous studies have shown that the human Organic Cation Transporter 1 (OCT1) is<br />
involved in the cellular uptake of imatinib. We show that elacridar inhibits OCT1 in<br />
vitro. In addition, OCT1/2 has a significant effect on the pharmacokinetics of<br />
imatinib in vivo by using OCT1/2 knockout mice. The considerable drug-drug<br />
interaction observed with elacridar and imatinib is only partly mediated by inhibition<br />
of OCT1/2. These uptake/elimination pathways can be important to explain the<br />
observed variability in the clinical pharmacokinetics of imatinib.<br />
We tested whether erlotinib hydrochloride (Tarceva, OSI-774), an orally active<br />
epidermal growth factor receptor tyrosine kinase inhibitor, is a substrate for the ATPbinding<br />
cassette drug transporters P-glycoprotein (P-gp; MDR1, ABCB1), breast<br />
cancer resistance protein (BCRP; ABCG2), and multidrug resistance protein 2<br />
(MRP2; ABCC2) in vitro and whether P-gp and BCRP affect the oral<br />
pharmacokinetics of erlotinib hydrochloride in vivo. In vitro, erlotinib was actively<br />
transported by P-gp and BCRP/Bcrp1. No active transport of erlotinib by MRP2 was<br />
observed. In vivo, systemic exposure (p = 0.01) as well as bioavailability of erlotinib<br />
after oral administration (5 mg/kg) were significantly increased in Bcrp1/<br />
Mdr1a/1b(-/-) knockout mice (60.4%) compared with wildtype mice (40.0%; p =<br />
0.02) (figure 1). Thus, erlotinib is transported efficiently by P-gp and BCRP/Bcrp1 in<br />
vitro. In vivo, absence of P-gp and Bcrp1 significantly affected the oral bioavailability<br />
of erlotinib. Possible clinical consequences for drug-drug and drug-herb interactions<br />
in patients in the gut between P-gp/BCRP-inhibiting substrates and oral erlotinib<br />
need to be addressed.<br />
New insights into the pharmacology, biotransformation, and cytotoxicity of<br />
gemcitabine and its metabolite 2’,2’-difluorodeoxyuridine We investigated the<br />
in vitro cytotoxicity, cellular uptake, efflux, biotransformation, and nucleic acid<br />
incorporation of dFdC and dFdU. dFdU monophosphate, diphosphate, and<br />
triphosphate (dFdU-TP) were formed from dFdC and dFdU. dFdU-TP was<br />
incorporated into DNA and RNA. The area under the intracellular concentration-time<br />
curve of dFdC-TP and dFdU-TP and their extent of incorporation into DNA and RNA<br />
inversely correlated with the IC(50) of dFdC and dFdU, respectively. The cellular<br />
uptake and cytotoxicity of dFdU were significantly enhanced by hCNT1. dFdU<br />
inhibited cell cycle progression and its cytotoxicity significantly increased with longer<br />
duration of exposure. dFdU is taken up into cells with high affinity by hCNT1 and<br />
phosphorylated to its dFdU-TP metabolite. dFdU-TP was incorporated into DNA and<br />
RNA, which correlated with dFdU cytotoxicity. These data provide strong evidence<br />
that dFdU can significantly contribute to the cytotoxicity of dFdC.<br />
DPYD pharmacogenetics in patients with metastatic colorectal cancer treated<br />
with capecitabine We are exploring the extent to which polymorphisms in DPYD<br />
contribute to development of severe toxicity in patients treated with capecitabine.<br />
Germline DNA was available from 576 patients with metastatic colorectal treated<br />
with capecitabine-based first line chemotherapy participating in the CAIRO2 study.<br />
All 23 exons of the entire DPYD gene were sequenced in 50 patients who suffered
from severe (grade 3/4) toxicity during the first two courses of treatment. The control<br />
group consisted of 100 patients selected at random from our study population.<br />
Polymorphisms that significantly differed between cases and cohort were assessed in<br />
the total population of 576 patients.<br />
A total of 29 different SNPs were detected in DPYD. The allele frequencies of the<br />
polymorphisms DPYD*2A and A2846T differed significantly between cases and<br />
cohort (6.0% vs 0.5% and 3.0 vs 0.0%, respectively). Assessment of these SNPs in<br />
the total population revealed that DPYD*2A and A2846T were significantly<br />
associated with grade 3/4 diarrhea (71% and 57%, respectively) and overall grade 3/4<br />
toxicity (100% and 71%, respectively). Furthermore, the average cumulative<br />
capecitabine dose per course was significantly lower compared to patients who are<br />
wildtype for these SNPs, with the lowest average cumulative doses in course number<br />
three being 49% and 63%, respectively.<br />
Pharmacogenomic and pharmacokinetic safety and cost analysis in patients<br />
treated with 5-FU / capecitabine Retrospectively, we could identify the presence of<br />
the DPYD*2A polymorphism in 13 patients all suffering from severe toxicity with one<br />
toxic death. Prolonged hospitalization periods were needed to recover from this<br />
severe toxicity induced by 5-FU or capecitabine. In our prospective study 530 patients<br />
have been genotyped for DPYD*2A. We have identified 7 patients carrying the<br />
DPYD*2A allele, of which 4 were safely treated with capecitabine at reduced doses.<br />
Figure 2: Systemic exposure and bioavailability of Erlotinib after oral administration were significantly<br />
increased in Bcrp1/Mdr1a/1b(-/-) knockout mice compared with wildtype mice.<br />
Circulating tumor cell detection in advanced solid cancer: validation of<br />
QPCR-based detection In the development of metasases, tumor cells intravasate<br />
into the bloodstream giving rise to circulating tumor cells (CTCs). We are performing<br />
a pilot study to explore the potential of CTC detection as a pharmacodynamic and<br />
prognostic marker in various types of solid cancer. In carcinoma patients we are<br />
performing positive selection with anti-EpCam(CD326) (melanoma: anti-MSCP) and<br />
depletion with anti-CD45. Ten types of solid cancer are being investigated: colorectal,<br />
esophageal, gastric, pancreatic, breast, ovarian, prostate, NSCLC, ACUP and<br />
melanoma. After mRNA isolation and cDNA synthesis, selected potential marker<br />
genes are being assessed in QPCR. This project is carried out in collaboration with<br />
A.J. Bosma, T.J. Molloy, L.J. van ’t Veer.<br />
Phase 1 and pharmacological study in monotherapy and in combination with<br />
gemcitabine, cisplatin or carboplatin in advanced solid tumors Wee1 is a<br />
tyrosine kinase involved in regulation of cell cycle checkpoints, particularly the G2/M<br />
checkpoint. MK-1775 is a highly selective, small molecule and an inhibitor of Wee1<br />
kinase. It potentiates the activity of cytotoxic agents in tumor cells. The protein p53<br />
regulates cell cycle checkpoints different from those regulated by Wee1, including the<br />
G1 checkpoint. The efficacy of MK-1775 is therefore expected to be greater in p53<br />
deficient tumors. In vivo, MK-1775 was well tolerated and showed enhancement of<br />
anti-tumor efficacy by gemcitabine, carboplatin and cisplatin in a nude xenograft<br />
tumor model. Preliminary results in patients showed that MK-1775 with<br />
chemotherapy was well tolerated and a substantial number of patients showed stable<br />
disease. Pharmacokinetics are linear.<br />
45<br />
EXPERIMENTAL THERAPY<br />
Publications (continued)<br />
Meijerman I, Beijnen JH, Schellens JHM.<br />
Combined action and regulation of phase<br />
enzymes and multidrug resistance proteins<br />
in multidrug resistance in cancer. Cancer<br />
Treat Rev <strong>2008</strong>;34:505-20<br />
Sparidans RW, Lagas JS, Schinkel AH,<br />
Schellens JHM, Beijnen JH. Liquid<br />
chromatography-tandem mass spectrometric<br />
assay for diclofenac and three primary<br />
metabolites in mouse plasma. J Chromatogr<br />
B <strong>2008</strong>;872:77-82<br />
Veltkamp SA, Jansen RS, Callies S,<br />
Pluim D, Visseren-Grul CM, Rosing H,<br />
Kloekers-Rhoades S, Andre VAM,<br />
Beijnen JH, Slapak CA, Schellens JHM.<br />
Oral administration of gemcitabine in<br />
patients with refractory tumoers: a clinical<br />
and pharmacologic study. Clin Cancer Res<br />
<strong>2008</strong>;14:3477-86<br />
Veltkamp SA, Pluim D, Van Tellingen O,<br />
Beijnen JH, Schellens JHM. Extensive<br />
metabolism and liver accumulation of<br />
gemcitabine following multiple oral and<br />
intravenous administration in mice. Drug<br />
Met Disp <strong>2008</strong>;36:1606-15<br />
Veltkamp SA, Witteveen EO, Capriati A,<br />
Crea A, Animati F, Voogel-Fuchs M,<br />
Heuvel Van den IJ, Beijnen JH, Voest EE,<br />
Schellens JHM. Clinical and pharmacologic<br />
study of the novel prodrug delimotecan<br />
(MEN 4901/T-0128) in patients with solid<br />
tumors. Clin Cancer Res <strong>2008</strong>; 14: 7535-44<br />
Brouwers EE, Tibben MM, Pluim D,<br />
Rosing H, Boot H, Cats A, Schellens JHM,<br />
Beijnen JH. Inductively coupled plasma<br />
mass spectrometric analysis of the total<br />
amount of platinum in DNA extracts from<br />
peripheral blood mononuclear cells and<br />
tissue from patients treated with cisplatin.<br />
Anal Bioanal Chem <strong>2008</strong> (in press)<br />
Harmsen S, Meijerman I, Beijnen JH,<br />
Schellens JHM. Nuclear receptor mediated<br />
induction of cytochrome P450 3A4 by<br />
anticancer drugs: a key role for the pregnane<br />
X receptor. Cancer Chemother Pharmacol<br />
<strong>2008</strong> (in press)<br />
Oostendorp RL, Buckle T, Beijnen JH,<br />
Van Tellingen O, Schellens JHM. The effect<br />
of P-gp (Mdr1a/1b), BCRP (Bcrp1) and<br />
P-gp/BCRP inhibitors on the in vivo<br />
absorption, distribution, metabolism and<br />
excretion of imatinib. Invest New Drugs<br />
<strong>2008</strong> (in press)
46<br />
GENE REGULATION<br />
Division head, group leader, Reuven Agami<br />
Reuven Agami PhD Group leader<br />
Eitan Zlotorynski PhD Post-doc<br />
Rani Elkon PhD Post-doc<br />
Nicolas Léveillé PhD Post-doc<br />
Murat Koseoglu PhD Post-doc<br />
Gijs van Haaften PhD Post-doc<br />
Maria Gomez Benito PhD Post-doc<br />
Henning Schaefer MD PhD Post-doc<br />
Carlos le Sage MSc PhD student<br />
Martijn Kedde MSc PhD student<br />
Jarno Drost MSc PhD student<br />
Remco Nagel MSc PhD student<br />
Marieke van Kouwenhove MSc PhD student<br />
Arnold Bos MSc PhD student<br />
Mariette Schrier PhD Technical staff<br />
Joachim Oude Vrielink BSc Technical staff<br />
Figure 1: A. A flow-chart of the genetic<br />
screen. Cells transduced with the miR-Lib<br />
were grown for two to three weeks in the<br />
presence or absence of RASV12.<br />
Subsequently, the population of inserts in<br />
each condition was recovered and compared<br />
using a barcode miR-array. B. Three<br />
independent miR-Array experiments were<br />
performed. The position of the reproducibly<br />
upregulated miR-Vecs is indicated for each<br />
experiment.<br />
DIVISION OF GENE REGULATION<br />
MICRORNAS IN CANCER<br />
Our main research objective is to understand the cancerous process in humans and<br />
identify essential cancerous genes. The knowledge obtained on these genes will allow<br />
us to design in the future novel therapeutic approaches. Most human tumors harbor<br />
multiple genetic alterations that activate oncogenes, inhibit tumor suppressors and<br />
induce genomic instability. As each tumor contains many genetic alterations, the<br />
study of the contribution of each alteration to the cancerous phenotype was obscured.<br />
In the past, we developed and successfully used an RNA interference (RNAi) approach<br />
to inactivate genes in mammalian cells. We used this RNAi system to characterize<br />
tumor suppressors and novel components of DNA damage signaling components.<br />
In the past three years we initiated studies to identify cancerous microRNAs<br />
(miRNAs), a newly emerging gene family encoding for endogenous small RNAs.<br />
We developed novel and unique genetic approaches to screen for cancer-causing and<br />
cancer-preventing miRNAs. With these tools we discovered and characterized the role<br />
of the miR-372 family in tumor growth and metastasis as well as the oncogenic role<br />
of miR-221 in glioblastoma.<br />
Interestingly, we noticed that the regions surrounding some functional miRNA<br />
targets (identified by our genetic screens) are highly conserved throughout evolution.<br />
We postulated that these regions recruit RNA binding proteins (RBPs) that regulate<br />
miRNA function. We performed genetic screens and identified RBPs that can inhibit<br />
or potentiate the accessibility of miRNAs to their target mRNAs. We suggest that the<br />
genetic interaction between miRNAs and RBPs determines developmental processes<br />
and cellular proliferation. We are working now to establish these interactions and<br />
their role in cancer development and progression.<br />
Functional genetic approaches identify cancerous miRNAs MicroRNAs<br />
(miRNAs) are potent post-transcriptional regulators of protein coding genes. Patterns<br />
of mis-expression of miRNAs in cancer suggest key functions of miRNAs in<br />
tumorigenesis. However, current bioinformatics tools do not fully support the<br />
identification and characterization of the mode of action of such miRNAs. To<br />
perform genetic screens for novel functions of miRNAs we developed a library of<br />
vectors expressing the majority of cloned human miRNAs and created corresponding<br />
DNA barcode arrays. In a screen for miRNAs that cooperate with oncogenes in<br />
cellular transformation we identified miR-372 and miR-373, each permitting<br />
proliferation and tumorigenesis of primary human cells that harbor both oncogenic<br />
RAS and active wild type p53 (figure 1). We provide evidence that these miRNAs are<br />
novel oncogenes participating in the development of human testicular germ cell<br />
tumors. By numbing the p53 pathway they allow tumorigenic growth in the presence<br />
of wild type p53. Intriguingly, in a genetic screen for miRNA genes that promote<br />
cellular migration we identified the same miRNA family. We characterized the role of<br />
miR-373 in the induction of tumor metastasis of breast cancer (figure 2).<br />
Second, we have used a novel functional genetic approach and identified miR-221<br />
and miR-222 (miR-221&222) as potent regulators of p27Kip1, a cell cycle inhibitor<br />
and tumor suppressor. Interestingly, high miR-221 level appears in signatures of poor<br />
prognosis cancers. Using miRNA-inhibitors we demonstrated that certain cancer cell<br />
lines require high activity of miR-221 for the maintenance of low p27Kip1 levels and<br />
continuous proliferation. We show that this interaction plays a role in human<br />
glioblastoma development.
Figure 2: MCF7 cells stably expressing luciferase and miR-373 were transplanted into SCID mice via tail<br />
vein injection. Bone metastasis in skull and pulmonary metastasis were observed in these mice.<br />
Interplay between RNA-binding proteins and miRNAs on mRNAs regulate<br />
gene expression MicroRNAs (miRNAs) are inhibitors of gene expression capable of<br />
controlling processes in normal development and cancer. In mammals, miRNAs use<br />
a seed sequence of 6-8 nucleotides to associate with 3’ UnTranslated Regions<br />
(3’UTRs) of mRNAs and inhibit their expression. Intriguingly, occasionally not only<br />
the miRNA-targeting site but also sequences in its vicinity are highly conserved<br />
throughout evolution. We therefore hypothesized that conserved regions in mRNAs<br />
may serve as docking platforms for modulators of miRNA activity. Here we<br />
demonstrate that the expression of dead end 1 (DND1), an evolutionary conserved<br />
RNA-binding protein, counteracts the function of several miRNAs in human cells<br />
and in primordial germ cells of zebrafish by binding mRNAs and prohibiting<br />
miRNAs from associating with their target sites (figure 3). These effects of DND1 are<br />
mediated through uridine-rich regions present in the miRNA-targeted mRNAs. Thus,<br />
our data unravel a novel role of DND1 in protecting certain mRNAs from miRNAmediated<br />
repression. We are now performing large scale analysis to broaden our<br />
early observations and our molecular understanding as to what extent this<br />
phenomena affect cellular growth and cancer<br />
Figure 3: A schematic model depicting the mechanism of DND1 action. The miRNA-RISC loaded with<br />
miRNAs targeting a 3’UTR inhibits its translation. By binding to U-rich regions in the 3’UTR of nanos,<br />
DND1 block miRNAs from binding to and inhibiting translation, thereby prohibiting miRNA function.<br />
Publications<br />
47<br />
GENE REGULATION<br />
Kedde M, Strasser MJ, Boldajipour B,<br />
Oude Vrielink JA, Slanchev K, le Sage C,<br />
Nagel R, Voorhoeve PM, van Duijse J,<br />
Orom UA, Raz R, Agami R. RNA-binding<br />
protein Dnd1 inhibits microRNA access to<br />
target mRNA. Cell 2007;131:1273-1286<br />
Duursma AM, Kedde, M, Schrier M,<br />
le Sage C, Agami R. miR-148 targets<br />
human DNMT3b protein coding region.<br />
RNA <strong>2008</strong>;14: 872-877<br />
Elkon R, Agami R. Removal of AU bias<br />
from microarray mRNA expression data<br />
enhances computational identification of<br />
active microRNAs. PLoS Comput Biol<br />
<strong>2008</strong>;4: e1000189<br />
Huang Q, Gumireddy K, Schrier M,<br />
le Sage C, Nagel R, Nair S, Egan DA, Li A,<br />
Huang G, Klein-Szanto AJ, Agami R.<br />
The microRNAs miR-373 and miR-520c<br />
promote tumour invasion and metastasis.<br />
Nat Cell Biol <strong>2008</strong>;10: 202-210<br />
Kedde M, Agami R. Interplay between<br />
microRNAs and RNA-binding proteins<br />
determines developmental processes. Cell<br />
Cycle <strong>2008</strong>; 7, 899-903<br />
Nagel R, le Sage C, Diosdado B,<br />
van der Waal M, Oude Vrielink JA,<br />
Bolijn A, Meijer GA, Agami R. Regulation<br />
of the adenomatous polyposis coli gene by<br />
the miR-135 family in colorectal cancer.<br />
Cancer Res <strong>2008</strong>;68: 5795-5802<br />
Zlotorynski E, Agami R. A PASport to<br />
cellular proliferation. Cell <strong>2008</strong>:134: 208-210
48<br />
GENE REGULATION<br />
Group leader Bas van Steensel<br />
Bas van Steensel PhD Group leader<br />
Katy Kolodziej PhD Post-doc<br />
Alexey Pindyurin PhD Post-doc<br />
Jop Kind PhD Post-doc<br />
Guillaume Filion PhD Post-doc<br />
Ulrich Braunschweig MSc PhD student<br />
Daniel Peric-Hupkes MSc PhD student<br />
Joke van Bemmel MSc PhD student<br />
Maartje Vogel MSc PhD student<br />
Ludo Pagie PhD Bioinformatician<br />
Wendy Talhout BAS Technical staff<br />
Inês de Castro Genebra de Jesus MSc<br />
Technical staff<br />
Publications<br />
de Wit E, van Steensel B. Chromatin<br />
domains in higher eukaryotes: insights from<br />
genome-wide mapping studies. Chromosoma<br />
<strong>2008</strong>; Oct 14. [Epub ahead of print]<br />
de Wit E, Braunschweig U, Greil F,<br />
Bussemaker HJ, van Steensel B. Global<br />
chromatin domain organization of the<br />
Drosophila genome. PLoS Genetics <strong>2008</strong>;<br />
4:e1000045<br />
Guelen L, Pagie L, Brasset E, Meuleman<br />
W, Faza MB, Talhout W, Eussen BH,<br />
de Klein A, Wessels L, de Laat W,<br />
van Steensel B. Domain organization of<br />
human chromosomes revealed by mapping<br />
of nuclear lamina interactions. Nature<br />
<strong>2008</strong>; 453:948-951<br />
Peric-Hupkes D, van Steensel B. Linking<br />
cohesin to gene regulation. Cell <strong>2008</strong>;<br />
132:925-928<br />
Vogel MJ. Genomic mapping of<br />
heterochromatin components using<br />
DamID. Thesis, University of Amsterdam;<br />
<strong>2008</strong>.<br />
CHROMATIN GENOMICS<br />
In every eukaryotic cell, hundreds of chromatin proteins work together to control the<br />
expression of thousands of genes. Each chromatin protein interacts with many other<br />
proteins and regulates specific parts of the genome. This network of interactions is<br />
enormously complex. To gain insight into this network and the roles in gene<br />
regulation, we take a broad integrative genomics approach, using both fruit fly and<br />
mammalian cells as model systems. We conduct our studies in the living cell, in the<br />
context of the entire genome. Thereby, we aim to identify general principles that<br />
govern gene regulation by chromatin. We develop and apply new whole-genome<br />
approaches to study the structure and composition of chromatin and the<br />
mechanisms of gene regulation. One major focus of investigation is currently how<br />
the genome is organized into large, multi-gene chromatin domains that may play a<br />
role in the coordinated control of neighboring genes. In relation to this, we<br />
investigate the role of the nuclear envelope in the spatial organization of the genome<br />
inside the nucleus, and its effects on gene expression.<br />
Genomics technology for chromatin research Over the past years, we have<br />
developed a technique (named ‘DamID’) for the genome-wide mapping of in vivo<br />
target loci of chromatin proteins and transcription factors. This microarray-based<br />
method allows us to generate detailed in vivo genomic binding maps of a large variety<br />
of chromatin proteins and transcription factors. We now routinely use DamID to<br />
construct such maps in Drosophila and mammalian cell lines. These binding maps<br />
provide a wealth of new insights into the roles of each protein in determining<br />
chromatin structure and gene regulation, and form the basis for systematic<br />
functional analysis of gene regulation by chromatin components. We also actively<br />
pursue the development of additional novel genomics methods that will uncover new<br />
aspects of chromatin and gene regulation.<br />
Heterochromatin organization in Drosophila Chromosome rearrangements can<br />
disrupt chromatin domain organization and thereby lead to aberrant regulation of<br />
genes. A classic example (known for more than 70 years) are white-mottled<br />
X-chromosomal inversions in Drosophila. In these inversions, the white eye color<br />
gene becomes partially silenced due to its relocation next to pericentric<br />
heterochromatin. It has been suggested that in these models the spreading of<br />
heterochromatin across the rearrangement breakpoint causes the silencing of white.<br />
However, the extent of this spreading and the precise pattern of heterochromatin<br />
redistribution have remained unclear. We therefore constructed high-resolution<br />
binding maps of Heterochromatin Protein 1 (HP1) on white-mottled chromosomes.<br />
We find that HP1 invades euchromatin across the inversion breakpoints over ~175kb<br />
and ~30kb, causing de novo association of HP1 with 20 genes. However, HP1 binding<br />
levels in these regions show substantial local variation, and white is the most strongly<br />
bound gene. Remarkably, white is also the only gene that is detectably repressed by<br />
heterochromatin. Thus, heterochromatin can invade a normally euchromatic region,<br />
yet the strength of HP1 binding and the effects on gene expression are highly<br />
dependent on local context. Our data suggest that the white gene has an unusual<br />
intrinsic affinity for heterochromatin, which may cause this gene to be more sensitive<br />
to PEV than most other genes. These results reveal how a chromosomal<br />
rearrangement can affect chromatin domain organization and alter the expression of<br />
genes in the affected genomic regions.<br />
Chromatin domain organization of the Drosophila genome Besides<br />
heterochromatin domains, there are several other examples of multi-gene chromatin<br />
domains. However, a global view of the chromatin organization of eukaryotic<br />
genomes is lacking. To systematically identify multi-gene chromatin domains, we<br />
constructed a compendium of genome-scale binding maps for a broad panel of 29<br />
chromatin components in Drosophila melanogaster. Next, we computationally<br />
analyzed this compendium for evidence of multi-gene chromatin domains. For this<br />
we used a novel statistical segmentation algorithm, developed in close collaboration<br />
with bioinformatics expert Dr Harmen Bussemaker (Columbia University, New<br />
York). We find that at least 50% of the fly genome is organized into chromatin
domains, which often consist of dozens of genes. The domains are characterized by<br />
various known and novel combinations of chromatin proteins. The genes in many of<br />
the domains are coregulated during development and share biological functions.<br />
Furthermore, fewer chromosomal rearrangements occur inside chromatin domains<br />
than outside domains during evolution. Our results indicate that a substantial<br />
portion of the Drosophila genome is packaged into functionally coherent, multi-gene<br />
chromatin domains. Presently, we are extending and refining the identification of<br />
chromatin domains by increasing the diversity, coverage and resolution of our<br />
DamID maps. In addition, we are investigating the role of various candidate proteins<br />
in the demarcation of specific domain types.<br />
Chromatin Protein Discovery Project DamID maps can be used to predict the<br />
molecular interactions and functions of chromatin proteins. In <strong>2008</strong> we have started<br />
the Chromatin Protein Discovery Project, which aims to generate genome-wide<br />
binding maps for ~100 candidate novel chromatin proteins in Drosophila. The<br />
candidate proteins are selected by computational predictions that take into account<br />
protein domain structure, interactions with known chromatin proteins, and<br />
likelihood of nuclear localization. For each of these we will generate full-genome,<br />
high-resolution DamID maps, which are expected to reveal many new molecular<br />
interactions and functions of the hitherto uncharacterized proteins. A pilot run for<br />
20 novel proteins is under way; we expect that in 2009 the mapping will be scaled up<br />
to the full set of candidate proteins.<br />
Nuclear lamina – genome interactions in human cells The nuclear lamina (NL)<br />
has long been thought to be an anchoring site of chromatin and to participate in the<br />
regulation of gene expression, but genomic sequences that interact with the NL in<br />
vivo have remained unknown. Using DamID, we have constructed for the first time a<br />
~1kb resolution map of the interaction sites of the entire human genome with the NL<br />
in fibroblasts. This map shows that human genome-NL interactions occur via more<br />
than 1,300 sharply defined large domains of 0.1-10Mb in size. These laminaassociated<br />
domains (LADs) together contain about 7,000 genes; the vast majority of<br />
these genes are expressed at very low levels, indicating that the NL represents a<br />
repressive chromatin environment. Furthermore, we found that the borders of LADs<br />
are often demarcated by specific sequence elements, indicating that LAD<br />
organization is a least in part ‘hard-coded’ in the genome. The border elements<br />
include binding sites for the insulator protein CTCF, promoters that are oriented<br />
away from LADs, and CpG islands, suggesting possible mechanisms of LAD<br />
confinement. Taken together, these results demonstrate that the human genome is<br />
divided into large, discrete domains that are units of chromosome organization<br />
within the nucleus. We are now conducting extensive studies to reveal the functions,<br />
dynamics, and molecular mechanisms of genome-NL interactions. This will reveal<br />
how three-dimensional organization of DNA inside the nucleus contributes to the<br />
regulation of gene expression.<br />
Bioinformatics Bioinformatics tools are essential for the detailed understanding of<br />
our genome-wide datasets (now amounting to millions of datapoints). In<br />
collaboration with national and international experts in bioinformatics, we are<br />
actively developing new algorithms and approaches for the systematic analysis of the<br />
chromatin protein binding patterns that we obtain. Integration with external datasets<br />
(gene expression profiles, genome annotation, sequence motifs) allows us to make<br />
functional predictions that can be further tested in our laboratory. In addition, we<br />
design our own custom oligonucleotide microrarrays.<br />
49<br />
GENE REGULATION<br />
Figure 4. Model of the folding of human<br />
chromosomes inside the cell nucleus. Large<br />
chromosomal domains (LADs) contact the<br />
nuclear lamina. Genes located in LADs are<br />
typically repressed. LADs are often<br />
demarcated by specific genomic elements<br />
that are named border elements.
50<br />
GENE REGULATION<br />
Group leader Fred van Leeuwen<br />
Fred van Leeuwen PhD Group leader<br />
Iris Stulemeijer PhD Post-doc<br />
Floor Frederiks MSc PhD student<br />
Kitty Verzijlbergen MSc PhD student<br />
Tibor van Welsem Technical staff<br />
EPIGENETIC REGULATION OF GENE EXPRESSION<br />
In eukaryotic cells the DNA is packaged into chromatin by histone proteins. Posttranslational<br />
modifications of the histone proteins and methylation of DNA can affect<br />
chromatin structure and function. Indeed, chromatin modifications are involved in<br />
regulation of gene expression and DNA damage response. Changes in chromatin<br />
modification can also result in heritable changes in gene expression without changes<br />
in the actual genetic code and these epigenetic changes can lead to cancer. The<br />
mechanisms by which epigenetic imprints are established or prevented are still<br />
poorly understood. Many chromatin modifiers are conserved from yeast to humans.<br />
Our group uses the budding yeast Saccharomyces cerevisiae as a powerful model<br />
system to identify new epigenetic regulators and to unravel the molecular<br />
mechanisms by which chromatin-modifying enzymes affect chromatin structure,<br />
gene expression and DNA damage response.<br />
Figure 5: Telomeric silencing in budding yeast; red<br />
(dark) and white (light) sectors in a yeast colony<br />
indicate epigenetic inheritance of active and silent<br />
chromatin.<br />
Function and regulation of histone H3 lysine 79 (H3K79) methylation by Dot1<br />
We previously discovered a novel histone methyltransferase Dot1, which can add one,<br />
two, or three methyl groups to lysine 79 of histone H3 on the surface of the<br />
nucleosome core. Dot1, which is conserved from yeast to humans, influences<br />
heterochromatin structure and the DNA damage response, and has been implicated<br />
in oncogenic transformation in mammals. A major goal of our research is to<br />
understand how H3-Lys79 methylation affects chromatin structure and function. We<br />
have used a combination of genetics and biochemistry to study the mechanism of<br />
methylation of H3-Lys79 and the consequences thereof. We found that Dot1, in<br />
contrast to other known protein methyltransferases, acts by a non-processive<br />
mechanism. Interestingly, this unusual mechanism of synthesis affects the function<br />
of the methylated lysine. In general, different forms of lysine methylation have<br />
specific functions in chromatin. In contrast, the different methyl forms of histone<br />
H3K79 showed functional overlap, suggesting that the complex methylation pattern<br />
of H3K79 is read as a binary code of methylation or no methylation. In addition,<br />
whereas histone methylation typically acts as a binding signal, our results show that<br />
H3K79 methylation acts as an anti-binding signal. Since human and yeast Dot1 are<br />
structurally very similar, we expect that similar rules apply to human cells. We are<br />
currently performing new genome-wide screens to understand how H3K79<br />
methylation specifically affects heterochromatin function. Dot1 has also been<br />
implicated in the DNA damage response. However, the mechanism by which H3K79<br />
methylation affects DNA damage checkpoint activation is still unclear. Our recent<br />
studies indicate that the role of Dot1 is modulated by other DNA damage response<br />
pathways. We are currently investigating how Dot1 interacts with other factors to<br />
mount a cellular response upon induction of DNA damage.
Figure 6: A novel assay to determine protein turnover in<br />
vivo. The epitope tag on histone H3 can be switched at<br />
the genomic level from an old to a new tag in arrested<br />
yeast cells. Upon re-entry into the cell cycle old histone<br />
proteins disappear and new histone proteins accumulate.<br />
Histone One of the main goals of our group is to understand how chromatin<br />
modifications can have long-term effects on gene expression. Post-translational<br />
modifications of histones have been proposed to be involved in epigenetic memory.<br />
When a cell divides, parental histones (containing the epigenetic marks) and newly<br />
synthesized histones (unmodified or in a ground state) are somehow assembled onto<br />
the daughter DNA strands in a manner that faithfully reproduces the transcriptional<br />
states of chromatin that existed prior to chromosome duplication. It is thought that<br />
the post-translational modifications on histones can serve as epigenetic memory<br />
marks during cell division. However, the exact mode of histone inheritance is still<br />
unclear and recent studies have shown that chromatin can be dynamic. Since yeast<br />
has only two copies of each histone gene, it provides a unique system to manipulate<br />
histones and study their inheritance. We developed a novel assay in yeast to<br />
determine protein turnover in vivo. The assay involves differential labeling of existing<br />
and newly synthesized proteins. Using this assay we found that histones throughout<br />
the genome are subject to extensive replication-independent exchange, suggesting<br />
that histones and their post-translational marks are not permanent residents in<br />
chromatin. Methods will be developed to search for proteins responsible for this<br />
mode of histone deposition and eviction. We expect that our studies will provide<br />
more insight into the role of histones in maintaining established patterns of gene<br />
expression.<br />
Publications<br />
51<br />
GENE REGULATION<br />
van Welsem T, Frederiks F,<br />
Verzijlbergen KF, Faber AW, Nelson ZW,<br />
Egan DA, Gottschling DE, van Leeuwen F.<br />
Synthetic lethal screens identify gene<br />
silencing processes in yeast and implicate<br />
the acetylated amino terminus of Sir3 in<br />
recognition of the nucleosome core. Mol Cell<br />
Biol <strong>2008</strong>;28:3861-72<br />
Frederiks F, Tzouros M, Oudgenoeg G,<br />
van Welsem T, Fornerod M, Krijgsveld J,<br />
van Leeuwen F. Nonprocessive methylation<br />
by Dot1 leads to functional redundancy of<br />
histone H3K79 methylation states. Nat<br />
Struct Mol Biol <strong>2008</strong>;15:550-7<br />
Backer R, van Leeuwen F, Kraal G,<br />
den Haan JM. CD8- dendritic cells<br />
preferentially cross-present Saccharomyces<br />
cerevisiae antigens. Eur J Immunol<br />
<strong>2008</strong>;38:370-80<br />
Ovaa H, van Leeuwen F. Chemical Biology<br />
Approaches to Probe the Proteome.<br />
Chembiochem <strong>2008</strong> (in press)
52<br />
GENE REGULATION<br />
Group leader Maarten Fornerod<br />
Maarten Fornerod, PhD Group leader<br />
Nikos Xylourgidis PhD Post-doc<br />
Stijn Heessen PhD Post-doc<br />
Bernike Kalverda MSc PhD student<br />
Dieuwke Engelsma MSc PhD student<br />
Hella van der Velde Technical staff<br />
Publications<br />
De Keersmaecker K, Rocnik JL, Bernad R,<br />
Lee BH, Leeman D, Gielen O, Verachtert H,<br />
Folens C, Munck S, Marynen P, Fornerod M*,<br />
Gilliland DG*, Cools J*. Kinase activation<br />
and transformation by NUP214-ABL1 is<br />
dependent on the context of the nuclear<br />
pore. Mol Cell <strong>2008</strong>;31:134-42.<br />
*co-corresponding authors<br />
Engelsma D, Valle N, Fish A, Salome N,<br />
Almendral JM, Fornerod M. A<br />
supraphysiological nuclear export signal is<br />
required for parvovirus nuclear export.<br />
Mol Biol Cell <strong>2008</strong>;19:2544-52<br />
Fornerod M, Clarke PR. To the centre of<br />
the volcano. Workshop on the mechanisms<br />
of nucleocytoplasmic transport. EMBO Rep<br />
<strong>2008</strong>;9:419-24<br />
Frederiks F, et al. See page 51<br />
Hendriksen J, Jansen M, Brown CM,<br />
van der Velde H, van Ham M, Galjart N,<br />
Offerhaus GJ, Fagotto F, Fornerod M.<br />
Plasma membrane recruitment of<br />
dephosphorylated beta-catenin upon<br />
activation of the Wnt pathway. J Cell Sci<br />
<strong>2008</strong>;121:1793-802<br />
Kalverda B, Roling MD, Fornerod M.<br />
Chromatin organization in relation to the<br />
nuclear periphery. FEBS Lett<br />
<strong>2008</strong>;582:2017-22<br />
Van Vlierberghe P, van Grotel M, Tchinda J,<br />
Lee C, Beverloo HB, van der Spek PJ,<br />
Stubbs A, Cools J, Nagata K, Fornerod M,<br />
et al. The recurrent SET-NUP214 fusion as<br />
a new HOXA activation mechanism in<br />
pediatric T-cell acute lymphoblastic<br />
leukemia. Blood <strong>2008</strong>;111:4668-80<br />
DYNAMIC INTERACTIONS AT THE NUCLEAR ENVELOPE<br />
The nuclear envelope arguably is the most important border within the eukaryotic<br />
cell. Borders in general are stable, but can reflect the dynamics of an entire<br />
organization. This makes them an attractive platform from which to study a complex<br />
system – such as the eukaryotic cell. The current focus of the lab is nuclear transport,<br />
chromatin dynamics at the nuclear envelope and connections between these two<br />
processes.<br />
Nuclear transport Nucleocytoplasmic transport is accomplished by distinct classes<br />
of soluble transport receptors that interact with transport signals and the nuclear<br />
pore complex. We investigate how this fundamental process is used in the regulatory<br />
circuits of the cell.<br />
CRM1-mediated nuclear export Exportins are nuclear export receptors that bind<br />
their cargoes cooperatively with RanGTP. They are responsible for multiple protein<br />
and ribonucleoprotein export pathways. Present evidence suggests that most<br />
exportins export one or a few structurally related groups of substrates. The main<br />
exception is CRM1/Exportin1, that exports multiple functionally unrelated protein<br />
and ribonucleoprotein cargoes, and can be thought of as the only ‘multi-purpose’<br />
exportin identified to date.<br />
A supraNES in parvovirus NS2 required for viral nuclear export and infectivity<br />
We have previously identified nuclear export signals from a 15-mer random peptide<br />
library with unusually high affinity to its receptor CRM1, which we termed<br />
supraNESs. Characterization of these supraNESs lead to the insight that regular<br />
NESs must have low affinity for CRM1 in order to function properly. However,<br />
supraNESs have so far not been identified in endogenous proteins. We now identify a<br />
supraNES in a viral protein, the NS2 protein of mouse parvovirus MVM. NS2<br />
strongly interacts with CRM1 without the requirement of RanGTP, while addition of<br />
RanGTP renders the complex very stable. A single mutation of a hydrophobic residue<br />
that inactivates regular NESs, reduces the affinity of the NS2 NES for CRM1 from<br />
supraphysiological to regular. Interestingly, an MVM mutant harbouring such<br />
regular NES is severely hampered in capsid nuclear export and viral productivity. In<br />
virus-infected cells, we observed a co-localization of NS2, CRM1 and mature virions,<br />
dependent on the supraphysiological NS2 NES. These data suggest a critical role for<br />
the NS2 supraNES in capsid nuclear export and parvovirus pathogenicity.<br />
Kinase activation and transformation by NUP214-ABL1 is dependent on the<br />
context of the nuclear pore Genetic alterations causing constitutive tyrosine kinase<br />
activation are observed in a broad spectrum of cancers. Thus far these mutant<br />
kinases have been localized to the plasma membrane or cytoplasm where they<br />
engage proliferation and survival pathways. In collaboration with the lab of Dr. J.<br />
Cools (Univerrsity of Leuven, Belgium, and the lab of Gary Gilliland (Harvard<br />
University, USA), we have found that the NUP214-ABL1 fusion is unique among<br />
these because of its requisite localization to the nuclear pore complex for its<br />
transforming potential. We show that NUP214-ABL1 displays attenuated<br />
transforming capacity as compared to BCR-ABL1 and that NUP214-ABL1<br />
preferentially transforms T-cells, which is in agreement with its unique occurrence in<br />
T-cell acute lymphoblastic leukemia. Furthermore, NUP214-ABL1 differs from BCR-<br />
ABL1 in subcellular localization, initiation of kinase activity and signaling and lacks<br />
phosphorylation on its activation loop. In addition to delineating a novel mechanism<br />
for kinase activation, this study provides new insights into the spectrum of<br />
chromosomal translocations involving nucleoporins by indicating that the nuclear<br />
pore context itself may play a central role in transformation.
DIVISION OF IMMUNOLOGY<br />
LYMPHOCYTE ACTIVATION AND SURVIVAL<br />
Our interest is to determine how cells decide between living and dying. We focus on<br />
the mechanism of action of TNF receptor family members, since these govern such<br />
decisions. Lymphocytes are our main cell type of interest, since throughout their<br />
existence, they mostly live ‘on the edge’ between life and death. Our work is inspired<br />
by the desire to improve cancer immunotherapy. The second aim of our work is to<br />
contribute to the design of novel therapies aimed at killing cancer cells by activating<br />
apoptotic pathways.<br />
TNF receptor family members and control of the immune response From our<br />
work, TNF receptor family member CD27 and its ligand CD70 have emerged as<br />
interesting targets to improve anti-tumor immunity. We have demonstrated that this<br />
costimulatory receptor/ligand pair promotes the generation and maintenance of<br />
effector CD8 T cells, the formation of memory CD8 T cells and their secondary<br />
expansion. CD27 rescues primed T cells from apoptosis, which at least in part<br />
explains its impact on the magnitude of the CD8 T cell response.<br />
To determine by which molecular mechanisms CD27 directs the T cell response, we<br />
have used genome-wide expression profiling comparing CD8 T cells that did or did<br />
not receive CD27 input in vitro or in vivo. In this way, we have identified a cytokine as<br />
CD27 target gene that is solely responsible for the anti-apoptotic effect of CD27<br />
during TCR-driven CD8 T cell expansion in vitro. Primary influenza virus-specific<br />
CD8 T cells were reconstituted to express the cytokine and examined side by side<br />
with mock-transduced cells for their accumulation after virus infection. These<br />
experiments showed complementation of the survival defect of CD27 -/- cells in vivo,<br />
while wild-type CD8 T cells did not benefit from provision of the cytokine gene.<br />
Similar experiments were performed to examine the role of the anti-apoptotic<br />
regulator Bcl-x L. Whereas Bcl-x L clearly afforded protection against apoptosis, this<br />
effect was equal in magnitude for wild-type and CD27-deficient cells. These data tie<br />
in with earlier studies using Bcl-x L transgenic T cells, which also pointed out that<br />
Bcl-x L, despite being upregulated at the protein level upon CD27 signaling, is not the<br />
key effector of its anti-apoptotic function. Gene expression profiling also identified a<br />
chemokine as CD27 target gene in CD8 T cells which supports effector T cell<br />
accumulation in vivo according to recent reconstitution experiments.<br />
In a protein immunization model, we have found that CD4 T cells require CD27 to<br />
support their own survival, but also to enable them to help CD8 T cells, in particular<br />
to imprint memory characteristics into them. By genome-wide expression analysis of<br />
CD4 T cells undergoing an immune response in vivo, we have found the membrane<br />
protein MS4A4B as CD27 target gene. Strikingly, the same molecule was identified<br />
as a unique marker for CD8 memory T cells that had received CD27-proficient CD4<br />
T cell help. Our findings suggest that MS4A4B is instrumental in programming<br />
memory CD8 T cells for secondary expansion. We have recently raised monoclonal<br />
antibodies to this molecule in Armenian hamsters. These unique reagents will allow<br />
us to examine the value of the MS4A4B as marker of helped versus helpless CD8 T<br />
cells. Genetic interventions will be used to determine its functional contribution.<br />
Recent findings suggest that the functional capabilities of T cells are to a large extent<br />
imprinted in the priming phase, during T cell-dendritic cell (DC) contact. We have<br />
made transgenic mice constitutively expressing CD70 on immature conventional DC<br />
(CD11c-Cd70tg). Such DCs normally acquire CD70 upon maturation. In two in vivo<br />
models, transgenic CD70 expression switched the system from tolerance induction<br />
to induction of full CD8 T cell responsiveness, allowing the elimination of tumors<br />
and virus, respectively (figure 1). The transgenic DC could also induce an effective<br />
anti-tumor immune response in an adoptive transfer setting, which mimics a clinical<br />
situation of cancer immunotherapy. We have also generated CD70-deficient mice,<br />
which will be used as recipients to further address the potential of CD70 transgenic DC.<br />
In DCs, transport of CD70 to the cell surface is tightly controlled. We have found that<br />
CD70 is sorted exactly like MHC class II to the inner vesicles of late endocytic<br />
53<br />
IMMUNOLOGY<br />
Division head, group leader Jannie Borst<br />
Jannie Borst PhD Group leader<br />
Jonathan Coquet PhD Post-doc<br />
Sabine Middendorp PhD Post-doc<br />
Yanling Xiao MD PhD Post-doc<br />
Anna Keller MSc PhD student<br />
Chiel Maas MSc PhD student<br />
Victor Peperzak MSc PhD student<br />
Rogier Rooswinkel MSc PhD student<br />
Elise Veraar MSc PhD student<br />
Inge Verbrugge MSc PhD student<br />
Evert De Vries Technical staff<br />
Gerda Van Der Horst Technical staff<br />
Figure 1: Transgenic CD70 expression<br />
converts tolerance to anti-tumor immunity.<br />
CD11c-Cd70tgCd27 -/- or control Cd27 -/mice<br />
were injected subcutaneously with B16<br />
tumor cells expressing an ovalbumin epitope<br />
(OVA). Three days later, 10 5 WT or CD27 -/-<br />
OVA-specific OT-I T cells were transferred<br />
and one day later (day 0), mice were<br />
challenged with OVA peptide in PBS, as<br />
indicated. Percentages of MHC tetramer +<br />
cells among CD8 + T cells in blood and<br />
tumor size measured by caliper are<br />
indicated (n=4).
54<br />
IMMUNOLOGY<br />
Publications<br />
Xiao Y*, Peperzak V*, Keller AM, Borst J.<br />
CD27 instructs CD4+ T cells to provide<br />
help for the memory CD8+ T cell response<br />
after protein immunization. * equal first<br />
author J Immunol <strong>2008</strong>;181:1071-1082.<br />
Mousavi SF, Soroosh P, Takahashi T,<br />
Yoshikai Y, Shen H, Lefrancois L, Borst J,<br />
Sugamura K, Ishii N. OX40 costimulatory<br />
signals potentiate the memory commitment<br />
of effector CD8+ T cells. J Immunol<br />
<strong>2008</strong>;181:5990-6001.<br />
Keller AM, Schildknecht A, Xiao Y,<br />
van den Broek M, Borst J. Deliberate<br />
expression of CD70 on steady state<br />
dendritic cells breaks CD8+ T cell tolerance<br />
and permits effective immunity.<br />
Immunity <strong>2008</strong>; Dec 15.<br />
Verbrugge I, Wissink EHJ, Rooswinkel RW,<br />
Jongsma J, Beltraminelli N, Dupuis M,<br />
Borst J, Verheij, M. Combining<br />
radiotherapy with APO010 (MegaFas<br />
Ligand) in cancer treatment, a preclinical<br />
evaluation of feasibility. Clin Cancer Res<br />
<strong>2008</strong> (in press)<br />
Verbrugge T, De Vries E, Tait SW,<br />
Wissink EH, Walczak H, Verheij M, Borst<br />
J. Ionizing radiation modulates the TRAIL<br />
death inducing signaling complex, allowing<br />
bypass of the mitochondrial apoptosis<br />
pathway. Oncogene <strong>2008</strong>;27:574-584<br />
compartments, known as MIIC. We have shown that this CD70 routing serves in the<br />
first place to coordinate CD70 cell surface deposition temporally and spatially with<br />
antigen presentation by MHC class II. CD70 transport also impacts on the CD8 T<br />
cell response. We found that routing of CD70 to MIIC is controlled by a chaperone<br />
protein. In DC, genetically deficient for this chaperone, cell surface deposition of<br />
CD70 is enhanced. This has a strong impact on CD8 T cell priming, as shown in an<br />
in vitro cross-presentation setting with ovalbumin protein antigen. These findings<br />
demonstrate a novel role of the chaperone in determining the threshold for CD8 T<br />
cell priming by controlling the level of CD70 cell surface expression. This work is<br />
carried out in close collaboration with the group of J Neefjes (Division of Cell Biology II).<br />
Apoptosis signaling and cancer therapy Death receptors, such as the TRAIL<br />
receptors and Fas/CD95 are TNF receptor family members that share the ability to<br />
induce apoptosis. We aim to further unravel pro-apoptotic signaling by death<br />
receptors and to exploit this knowledge for cancer therapy.<br />
In collaboration with M Verheij of the Division of Radiotherapy we are carrying out a<br />
project aimed at exploiting death ligands to enhance the efficacy of radiotherapy. As<br />
proof of principle, we have used human lymphoid tumor cells. In these cells,<br />
radiation (and DNA damaging drugs) can sensitize for TRAIL receptor- and Fas/<br />
CD95 induced apoptosis when the mitochondrial pathway is blocked and p53 is<br />
lacking. APO010, a novel recombinant form of Fas/CD95 ligand, is currently in a<br />
Phase I dose-escalation trial in patients with solid tumors. We have investigated the<br />
feasibility of combination therapy with APO010 and radiotherapy in leukemic and<br />
solid tumor cell types. This approach is attractive, since a potential combined<br />
treatment effect would be local and limit normal tissue toxicity. We found a<br />
significant combined effect of both treatment regimens on cell death induction, as<br />
read out by apoptosis and clonogenic survival in vitro. In vivo, however, the<br />
combination of systemically administered APO010 and radiotherapy did not<br />
significantly enhance tumor growth delay, as compared to the single modality<br />
treatments. Toxicity data indicated that further dose-escalation of APO010 was not<br />
achievable. We suggest therefore that future studies should address the efficacy of<br />
locally applied APO010 as radiation-enhancing agent.<br />
After death receptor stimulation, the Bcl-2 family member Bid is cleaved by<br />
Caspase-8 and its carboxy-terminal fragment translocates to mitochondria where it<br />
induces release of pro-apoptotic mediators. We have discovered that Bid function is<br />
regulated by a novel form of serine/threonine/cysteine ubiquitination. This serves to<br />
target Bid’s amino-terminal fragment for degradation, thus liberating its BH3 domain<br />
for apoptosis-induction. Although Bid was seen as a unique mediator of death<br />
receptor-induced apoptosis, we have found that it can also be essential for apoptosisinduction<br />
by DNA damaging stimuli. However, its mode of regulation is distinct in<br />
this scenario, since it is not cleaved at the conventional sites by upstream caspases.<br />
We have developed a novel FRET-based read-out for Bid cleavage to map the relevant<br />
sites for proteolytic activation of Bid after DNA damage and to identify the protease<br />
involved.<br />
In a functional genetic screen for mediators of TRAIL-induced apoptosis in MCF-7<br />
breast cancer cells, we have identified a molecule that determines the extent of cell<br />
surface expression of TRAIL receptor-1, but not TRAIL receptor-2. Both receptors are<br />
partially retained in intracellular vesicles. We find that the plasma membrane pool –<br />
rather than the intracellular pool – engages in pro-apoptotic signaling. Our data<br />
implicate vesicular trafficking in regulating plasma membrane levels of TRAIL<br />
receptors both before and after ligand binding, and thereby sensitivity to TRAILinduced<br />
apoptosis.
DISSECTING VIRUS- AND TUMOR-SPECIFIC T CELL IMMUNITY<br />
The aim of our research is straightforward 1) to design novel technologies that can be<br />
used to examine and modify antigen-specific T cell immunity 2). To use these tools to<br />
unravel and manipulate the molecular processes underlying immune recognition by<br />
T lymphocytes. Within these projects, a main focus is on the design and testing of<br />
novel concepts for adoptive immunotherapy.<br />
Dissecting antigen-specific T cell immunity (collaboration with Ovaa and Perrakis<br />
labs) Peptide-(p)MHC tetramers and other types of pMHC multimers have become<br />
an essential tool for the analysis of T cell immunity. However, the inefficiency of the<br />
classical strategy for pMHC generation has precluded the development of large<br />
libraries of MHC multimers for high-throughput screening, or of a collection of<br />
clinical grade pMHC multimer reagents for cellular therapy. In collaboration with<br />
H Ovaa (Division of Cell Biology II) we have in the past years addressed this issue, by<br />
designing MHC class I molecules occupied with UV-sensitive ‘conditional’ peptide<br />
ligands.<br />
While the UV-induced ligand exchange technology has successfully been utilized to<br />
identify T cell antigens in animal model systems by us and others, limitations on<br />
patient sample size preclude an equally comprehensive analysis of T cell immunity<br />
for most human diseases. To tackle this issue, we have developed a combinatorial<br />
coding strategy that would allow the parallel detection of a large number of different<br />
T cell populations within a single sample. This technology is based on the concept<br />
that a given T cell specificity is not encoded by a single identifier, but is encoded by a<br />
defined combination of two or more identifiers. Using such ‘combinatorial coding’,<br />
the number of specificities that can be encoded increases dramatically (figure 2). In a<br />
series of proof-of principle experiments, in which up to 25 different antigen specificities<br />
were encoded per sample, we have demonstrated that the detection of antigen-specific T<br />
cells from peripheral blood by combinatorial coding is highly efficient. Furthermore, to<br />
exemplify the value of the large-scale screening of patient material made possible by<br />
combinatorial coding, we have analyzed T cell responses against known and potential<br />
melanoma-associated antigens in peripheral blood from melanoma patients. In parallel<br />
work, this technology is being utilized to identify leukemia-reactive T cell responses in a<br />
joint effort with the Heemskerk/Falkenburg group (LUMC).<br />
Dissection of T cell immunity through retroviral barcoding The ability to<br />
visualize antigen-specific T cell responses and to determine the differentiation<br />
pathways of different subsets of T cells is essential for our understanding of<br />
pathogen- and vaccine-induced immunity. While MHC tetramer technology makes it<br />
possible to follow the development of immunity at the T cell population level, it<br />
Figure 2: Combinatorial coding with MHC-qDots. To allow the simultaneous detection of multiple<br />
antigen-specific T cell populations in patient samples, individual antigen-specificities are encoded by<br />
pMHC multimers that are coupled to unique combinations of fluorochromes. Left plot depicts the binding<br />
of pMHC multimers conjugated to two different fluorochromes to an antigen-specific T cell. Right plot<br />
shows the maximal number of antigen-specificities that can be encoded by classical single fluorochrome<br />
encoding and by two-dimensional encoding as a function of the number of available detection channels.<br />
Group leader Ton Schumacher<br />
Ton Schumacher PhD Group leader<br />
Gavin Bendle PhD Post-doc<br />
Bianca Heemskerk PhD Post-doc<br />
Andrew Kaiser PhD Post-doc<br />
Shalin Naik PhD Post-doc<br />
Sine Reker Hadrup PhD Post-doc<br />
Remko Schotte PhD Post-doc<br />
Jenny Shu PhD Post-doc<br />
Carmen Gerlach MSc PhD student<br />
Jeroen Van Heijst MSc PhD student<br />
Carsten Linnemann MSc PhD student<br />
Erwin Swart Technical staff<br />
Mireille Toebes Technical staff<br />
Jos Urbanus Technical staff<br />
Publications<br />
55<br />
IMMUNOLOGY<br />
Bakker AH, Hoppes R, Linnemann C,<br />
Toebes M, Rodenko B, Berkers CR,<br />
Reker-Hadrup S, van Esch WJE,<br />
Heemskerk MHM, Ovaa H,<br />
Schumacher TNM. Conditional MHC<br />
class I ligands and MHC exchange<br />
technology for the human MHC alleles<br />
HLA-A1, -A3, -A11 and -B7. Proc Natl Acad<br />
Sci USA. <strong>2008</strong>;105:3825-30<br />
Bos R, van Duikeren S, Morreau H,<br />
Schumacher TN, Haanen JB,<br />
van der Burg SH, Melief CJM, Offringa R.<br />
Balancing between anti-tumor efficacy and<br />
auto-immune pathology in T-cell mediated<br />
targeting of carcinoembryonic antigen.<br />
Cancer Res. <strong>2008</strong> ( in press)<br />
Coccoris M, Swart E, van Heijst JW,<br />
de Witte MA, Haanen JBAG, Schepers K,<br />
Schumacher TNM. Long-term functionality<br />
of TCR transduced T cells in vivo.<br />
J Immunol. <strong>2008</strong>;180:6536-43<br />
De Witte MA, Bendle GM,<br />
van den Boom MD, Coccoris M,<br />
Schell TD, Tevethia SS, Mesman EM,<br />
Song JY, Schumacher TNM. TCR gene<br />
therapy of spontaneous prostate carcinoma<br />
requires in vivo T cell activation. J<br />
Immunol. <strong>2008</strong>;181:2563-71
56<br />
IMMUNOLOGY<br />
Publications (continued)<br />
De Witte MA, Jorritsma A, Kaiser A,<br />
van den Boom MD, Dokter M, Bendle G,<br />
Haanen JBAG, Schumacher TNM.<br />
Requirements for effective anti-tumor<br />
responses of TCR transduced T cells.<br />
J Immunol. <strong>2008</strong>;181:5128-36<br />
De Witte MA, Jorritsma A, Swart E,<br />
Straathof KC, de Punder K, Haanen JB,<br />
Rooney C, Schumacher TNM. An<br />
inducible caspase 9 safety switch can halt<br />
cell therapy-induced autoimmune disease.<br />
J Immunol. <strong>2008</strong>;180:6365-73<br />
Jorritsma A, Bins AD, Schumacher TN,<br />
Haanen JB. Skewing the T-cell repertoire by<br />
combined DNA vaccination, host<br />
conditioning, and adoptive transfer. Cancer<br />
Res. <strong>2008</strong>;68:2455-62<br />
Reker Hadrup S, Toebes M, Rodenko B,<br />
Bakker AH, Egan DA, Ovaa H,<br />
Schumacher TNM. High-Throughput<br />
T Cell Epitope Discovery Through MHC<br />
Peptide Exchange. In Methods in<br />
Molecular Biology. Epitope Mapping<br />
Protocols: Second Edition. <strong>2008</strong> (in press)<br />
Schepers K, Swart E, van Heijst JW,<br />
Gerlach C, Castrucci M, Heimerikx M,<br />
Velds A, Kerkhoven RM, Arens R,<br />
Schumacher TN. Dissecting T cell lineage<br />
relationships by cellular barcoding. J Exp<br />
Med. <strong>2008</strong> (in press)<br />
Verstrepen BE, Bins AD, Rollier CS,<br />
Mooij P, Koopman G, Sheppard NC, et<br />
al. Improved HIV-1 specific T-cell responses<br />
by short-interval DNA tattooing as<br />
compared to intramuscular immunization<br />
in non-human primates. Vaccine.<br />
<strong>2008</strong>;26:3346-51<br />
Van den Berg JH, Nuijen B, Beijnen JH,<br />
Vincent A, van Tinteren H, Kluge J,<br />
Woerdeman LAE, Hennink WE, Storm G,<br />
Schumacher TN, Haanen JBAG.<br />
Optimization of intradermal vaccination<br />
by DNA tattooing in human skin. Hum<br />
Gene Ther. <strong>2008</strong> (in press)<br />
doesn’t allow the analysis of cell fate and cellular differentiation pathways.<br />
To allow such lineage tracking we have developed an approach in which individual<br />
T cells are tagged with a genetic barcode. For this purpose we have created an<br />
oncoretroviral and lentiviral library containing some 5,000 different barcodes, and<br />
the same set of barcodes has been used to generate a barcode micro-array. Infection<br />
of cell populations by these libraries of viral vectors and subsequent micro-array<br />
analysis can then be used to trace the progeny of individual cells. Following earlier<br />
work in which we demonstrated how T cell populations primed at distinct sites in the<br />
body intermingle, we have more recently used this technology to trace hematopoietic<br />
differentiation and T cell differentiation. With respect to the latter, using a strategy<br />
for the retroviral labelling of naïve T lymphocytes we have analyzed to what extent<br />
effector T cells and memory T cells are derived from the same naïve T cell clones.<br />
Contrary to models that assume that T cell fate is to a large extent determined during<br />
the initial priming event, these data show that effector and memory T cell subsets are<br />
more or less completely derived from the same set of naïve T cell precursors. In<br />
addition, we have utilized retroviral barcoding to determine to what extent the<br />
magnitude of T cell responses is determined by the recruitment of antigen-specific<br />
T cells from the naive T cell pool. These data demonstrate that under conditions in<br />
which the magnitude of antigen-specific T cell responses varies widely, the<br />
recruitment of antigen-specific T cell precursors remains largely unchanged and is in<br />
fact near complete. These data demonstrate that regulation of the magnitude of T cell<br />
responses occurs primarily at the level of T cell expansion.<br />
TCR gene therapy (collaboration with Haanen lab) In the past years our group has<br />
developed the retroviral introduction of antigen-specific T cell receptors into<br />
peripheral T cells as a means to induce tumor-specific T cell immunity in vivo. In this<br />
strategy, autologous or donor-derived T cell populations are equipped with a TCR of<br />
defined reactivity in short-term ex vivo procedures, and re-infusion of the redirected<br />
cells is used to supply T cell reactivity against defined antigens.<br />
An important development in this line of research in the past year has been the<br />
observation of acute Graft-versus-Host-Disease (GVHD) in mouse models for TCR<br />
gene transfer. This pathology occurs under conditions in which the in vivo function of<br />
TCR modified T cells is strongly promoted. Further experiments have shown that this<br />
pathology can be explained by the formation of so-called mixed dimers that are<br />
formed by the joint assembly of endogenous and exogenous TCR chains (figure 3).<br />
These data indicate that it will be essential to restrict the formation of mixed TCR<br />
dimers in clinical trials of TCR gene transfer. With respect to such trials, in the past<br />
year we have initiated the production of clinical grade retrovirus encoding a<br />
melanoma-reactive TCR for the gene modification of human T cells. Importantly, in<br />
view of the safety issues seen in mouse models, this TCR has been modified such<br />
that the formation of mixed dimers is to a large extent suppressed. Finally, in a joint<br />
effort with the Haanen lab and <strong>NKI</strong>-AVL pharmacy, a large effort is currently made to<br />
develop the pharmaceutical process that will be utilized in this trial.<br />
Figure 3: Off-target toxicity through TCR gene therapy. Introduction of an unmodified TCR into<br />
peripheral T cells leads to the formation of mixed TCR dimers, consisting of endogenous and exogenous<br />
TCR chains. As these TCR heterodimers have not been selected against reactivity with self antigens,<br />
autoreactivity may ensue.
IMMUNOTHERAPY<br />
The main objective of this line of research is the development of novel T cell<br />
immunity-based strategies that can be translated to clinical application. This work is<br />
performed in close collaboration with the Schumacher group. The focus is on<br />
treatment of patients with solid tumors, especially melanoma and renal cell<br />
carcinoma. A second line of research is focusing on immunomonitoring. The<br />
primary aim of this line of research is to evaluate specific and cytokine-based<br />
immunotherapies, using advanced technologies for characterization of immune<br />
responses in peripheral blood and at the tumor site. These studies include<br />
immunomonitoring of clinical trials of cancer patients in our hospital and are<br />
conducted in close collaboration with Christian Blank (division of Immunology and<br />
Medical Oncology), Axel Bex (division of Surgical Oncology), Michel van den Heuvel<br />
(division of Medical Oncology) and the clinical chemistry laboratory headed by<br />
Willem Nooijen (division of Diagnostic Oncology).<br />
DNA vaccination for the treatment of cancer<br />
Phase I study in melanoma patients Induction of immunity following DNA vaccination<br />
is generally considered a slow process. We have shown that DNA delivery to the skin<br />
results in a highly transient pulse of antigen expression. Based on this information<br />
we developed a novel, rapid and potent intradermal DNA vaccination method. By<br />
short-interval intradermal DNA delivery, robust T cell responses, of a magnitude<br />
sufficient to reject established subcutaneous tumors, are generated within 12 days.<br />
These results were confirmed in a non-human primate model (in collaboration with<br />
the BPRC, Rijswijk). We showed that DNA tattooing results in a 10-100-fold increase<br />
in vaccine-specific T cells compared to intramuscular vaccination.<br />
Together with B Nuijen (Pharmacy, Slotervaart Hospital), W Hennink and G Storm<br />
(Department of Biopharmacy, Utrecht University) we have developed an ex vivo<br />
human skin model to optimize DNA tattoo vaccination and to create a platform for<br />
testing novel DNA formulations for transfection and targeting of human skin cells<br />
(figure 4). Results from these studies have been crucial for approval by the Central<br />
Committee on Research involving Human Subjects (CCMO) and the Dutch Ministry<br />
for Environmental Affairs to start a first gene therapy phase I trial with DNA tattoo<br />
vaccination. For this purpose a clinical grade DNA vaccine has been produced in the<br />
recently built GMP DNA plasmid production unit (Amsterdam-Biotherapeutics Unit)<br />
at the <strong>NKI</strong>-AVL/Slotervaart Hospital pharmacy department.<br />
Figure 4: Tattooing procedure of the human skin (A) and typical expression of luciferase (B), visualized<br />
with a light sensitive camera, 18 hours after tattooing. Each area of 50mm2 was tattooed with a different<br />
tattoo setting. Note the marked variation in luciferase signal obtained with different vaccination<br />
conditions.<br />
DNA vaccination for the treatment of high risk human papilloma virus (HPV) associated<br />
cancers Human papilloma virus infection (serotypes 16 and 18) is strongly associated<br />
with the development of squamous cell cancer of the cervix, but also penis, vulva,<br />
anus and oropharynx. Because persistence of oncogenic HPV proteins E6 and E7 is<br />
required for carcinogenesis, these viral antigens are exquisite targets for<br />
immunotherapeutic intervention. Indeed, therapeutic vaccinations targeting these<br />
viral antigens have shown some promise in woman suffering from cervical cancer. In<br />
the next years we will perform a phase I/II study in patients with HPV 16-positive<br />
squamous cell cancer of the penis and cervix using our novel and potent intradermal<br />
DNA vaccination strategy. We have developed highly immunogenic and safe HPV 16<br />
E6 and E7 containing DNA vaccines that we will start producing in <strong>2008</strong>-2009 for a<br />
first clinical trial.<br />
Group leader John Haanen<br />
John Haanen MD PhD Group leader<br />
Florry Vyth-Dreese PhD Senior staff<br />
scientist<br />
Bianca Heemskerk PhD Post-doc<br />
Annelies Jorritsma PhD Post-doc<br />
Esther Tjin PhD Post-doc<br />
Silvia Ariotti MSc PhD student<br />
Koen Oosterhuis MSc PhD student<br />
Joost Van den Berg MSc PhD student<br />
Yeung-Hyen Kim MSc PhD student<br />
Trees Dellemijn Technical staff<br />
Raquel Gomez MSc Technical staff<br />
Johan Sein Technical staff<br />
Willeke Van de Kasteele Technical staff<br />
Martin Van der Maas Technical staff<br />
Publications<br />
57<br />
IMMUNOLOGY<br />
Abad JD, Wrzensinski C, Overwijk W,<br />
De Witte MA, Jorritsma A, Hsu C,<br />
Gattinoni L, Cohen CJ, Paulos CM,<br />
Palmer DC, Haanen JB, Schumacher TN,<br />
Rosenberg SA, Restifo NP, Morgan RA.<br />
T-cell receptor gene therapy of established<br />
tumors in a murine melanoma model. J<br />
Immunother. <strong>2008</strong>;31:1-6<br />
Bos R, van Duikeren S, Morreau H,<br />
Franken K, Schumacher TN, Haanen JB,<br />
van der Burg SH, Melief CJ, Offringa R.<br />
Balancing between antitumor efficacy and<br />
autoimmune pathology in T-cell-mediated<br />
targeting of carcinoembryonic antigen.<br />
Cancer Res. <strong>2008</strong>;68:8446-55<br />
Coccoris M, Swart E, de Witte MA,<br />
van Heijst JW, Haanen JB, Schepers K,<br />
Schumacher TN. Long-term functionality<br />
of TCR-transduced T cells in vivo. J<br />
Immunol. <strong>2008</strong>;180:6536-43<br />
De Witte MA, Jorritsma A, Kaiser A,<br />
van den Boom MD, Dokter M, Bendle<br />
GM, Haanen JB, Schumacher TN.<br />
Requirements for effective antitumor<br />
responses of TCR transduced T cells. J<br />
Immunol. <strong>2008</strong>;181:5128-36
58<br />
IMMUNOLOGY<br />
Publications (continued)<br />
De Witte MA, Jorritsma A, Swart E,<br />
Straathof KC, de Punder K, Haanen JB,<br />
Rooney CM, Schumacher TN. An<br />
inducible caspase 9 safety switch can halt<br />
cell therapy-induced autoimmune disease. J<br />
Immunol. <strong>2008</strong>;180:6365-73<br />
Griffioen A, Vyth-Dreese FA. Angiostasis<br />
as a way to improve immunotherapy.<br />
Thrombosis and Hemostasis. <strong>2008</strong> (in<br />
press)<br />
Jorritsma A, Bins AD, Schumacher TN,<br />
Haanen JB. Skewing the T-cell repertoire by<br />
combined DNA vaccination, host<br />
conditioning, and adoptive transfer. Cancer<br />
Res. <strong>2008</strong>;68:2455-62<br />
Quaak SG, van den Berg JH, Toebes M,<br />
Schumacher TN, Haanen JB, Beijnen JH,<br />
Nuijen B. GMP production of pDERMATT<br />
for vaccination against melanoma in a<br />
phase I clinical trial. Eur J Pharm<br />
Biopharm. <strong>2008</strong>. [Epub ahead of print]<br />
Verstrepen BE, Bins AD, Rollier CS,<br />
Mooij P, Koopman G, Sheppard NC,<br />
Sattentau Q, Wagner R, Wolf H,<br />
Schumacher TN, Heeney JL, Haanen JB.<br />
Improved HIV-1 specific T-cell responses by<br />
short-interval DNA tattooing as compared<br />
to intramuscular immunization in nonhuman<br />
primates. Vaccine. <strong>2008</strong>;26:3346-51<br />
Figure 5: Multi parameter confocal<br />
microscopic analysis of renal cell carcinoma<br />
specimen. Merge graph of cryosection<br />
stained for CD31/34 on blood vessels<br />
(green), Ki67 proliferation marker (nuclear<br />
label, red) and CD3 T cells (blue). Arrows<br />
indicate T cell (1), proliferating T cell (2),<br />
blood vessel (3), proliferating blood vessel<br />
(4) and proliferating tumor cell (5).<br />
Adoptive immunotherapy program TIL therapy Recently, we started a new project<br />
concerning adoptive therapy with Tumor-infiltrating Lymphocytes (TIL). With this<br />
approach, the Surgery Branch, NIH, Bethesda, MD, USA finds a 50% objective<br />
response rate in heavily pretreated stage IV melanoma patients. Treatment combines<br />
ex vivo culture of melanoma-reactive T cells from resected metastases with nonmyeloablative<br />
chemotherapy and high dose bolus IL-2. Our goals are to show that<br />
this treatment can be given safely in an independent cancer centre, to show in an<br />
randomized controlled trial that this treatment improves progression-free survival<br />
compared to standard chemotherapy and to perform a comprehensive analysis of the<br />
T cell specificities of the melanoma-reactive TIL prior to and after adoptive transfer.<br />
We expect to start treatment of the first patients in 2009.<br />
T-cell receptor gene therapy In close collaboration with the Schumacher group and<br />
several international groups, we have selected a highly avid T cell receptor (TCR)<br />
specific for melanocyte differentiation antigen MART-1 26-35. This TCR, called 1D3,<br />
has been cloned into a retroviral vector (MP-71) and will be produced by a German<br />
GMP manufacturer. The TCR has been equipped with safeguards to prevent pairing<br />
with endogenous TCR chains of transduced peripheral T cells to prevent potential<br />
side effects. The process of clinical grade culturing and T cell transduction with the<br />
1D3-MP-71 retrovirus is validated step-by-step in our laboratory and GMP facility. A<br />
large translational gene therapy grant from ZonMW will allow us to perform a phase<br />
I clinical study in melanoma patients in the next years.<br />
Immunotherapy monitoring (This work is performed under supervision of Florry<br />
Vyth-Dreese, staff scientist in the group.) The primary aim of this work is to evaluate<br />
specific and cytokine-based immunotherapies, using advanced technologies to<br />
characterize immune responses in peripheral blood and at the tumor site. The<br />
Immunomonitoring Facility is implementing a full spectrum of immunomonitoring<br />
tools and assays.<br />
Immunotherapy in patients with renal cell carcinoma (RCC) Since the past 3 years<br />
metastatic RCC patients have been treated by targeted therapy with tyrosine kinase-<br />
and mTOR kinase inhibitors and anti-VEGF mAb. These therapies aim at inhibition<br />
of angiogenesis as well as direct targeting of the tumor. In addition, they may<br />
potentiate anti-tumor immune responses. In collaboration with others, we investigate<br />
inhibition of angiogenesis at the peripheral blood and tumor tissue level. Clinical<br />
tumor specimens obtained from RCC patients treated with Sunitinib, Avastin or<br />
Interferon alpha, non small cell lung cancer (NSCLC) patients treated with Erlotinib,<br />
or untreated patients, are examined using immunohistological methods (figure 5).<br />
Preliminary data indicate that inhibition of angiogenesis results in enhanced tumor<br />
infiltration by immune cells and induces apoptosis of tumor cells and endothelial<br />
cells.<br />
Detection of antigen-specific T lymphocytes in experimental and human tissues We have<br />
successfully applied in situ tetramer technique for the detection of minor<br />
Histocompatibility Antigen-specific T cells in a human ex vivo in situ skin explant<br />
model of Graft versus Host reactivity (in collaboration with the group of E Goulmy,<br />
Leiden University Medical Center). Recently, we published a method to detect minor<br />
antigen specific T cells, not only in fresh, but also in cryopreserved tissues using socalled<br />
MHC-dextramers. This will enable analysis of patient material post-surgically.<br />
Development of a human ex vivo in situ skin model for vitiligo and melanoma In<br />
collaboration with R Luiten (SNIP and Dermatology, Academical Medical Center,<br />
University of Amsterdam, Amsterdam) and C Melief (Leiden University Medical<br />
Center) a human in situ skin model has been developed to study immune and<br />
environmental factors involved in the development of vitiligo and potential therapy of<br />
melanoma. Using melanocyte-specific T cell clones co-cultured with normal skin<br />
tissues, we were able to mimic the induction of vitiligo ex vivo. In addition, bulk<br />
T cells obtained from vitiligo lesions were shown to induce severe lesions in this skin<br />
model. Separate studies currently show that a similar model can be applied to<br />
visualize melanoma -T cell interactions ex vivo in situ.
PROGRAMMED MUTAGENESIS<br />
To improve immunity, B cells have the unique capacity to mutate their immunoglobulin<br />
genes by programmed mutagenesis. Our research-focus is three-fold: i) Unravel the<br />
molecular mechanism of programmed mutagenesis, ii) Determine the targeting<br />
specificity of the mutation process, and iii) Understand the decision making between<br />
repair and mutagenesis.<br />
Binding of the mutagen AID within the chromatin of B cells In B cells of the<br />
germinal center (GC) the antigen specific immunoglobulin (Ig) gene repertoire is<br />
diversified by somatic hypermutation (SHM) and class switch recombination (CSR).<br />
While SHM introduces point mutations into the Ig variable region to generate high<br />
affinity Ig variants, CSR is a recombination process enabling isotype switching<br />
within the Ig heavy chain constant region. Both SHM and CSR are initiated by the<br />
activation induced cytidine deaminase (AID), which deaminates cytosine in the<br />
variable- and switch-regions of Ig genes. Based on the translocation break points and<br />
somatic mutations in cancer genes, aberrant targeting of AID in B cells of the GC<br />
seems to boost the accumulation of oncogenic mutations in GC B cells. How<br />
specifically AID is targeted to Ig loci remains unknown. To determine genome-wide<br />
the binding specificity of AID, we applied DamID technology. We showed that<br />
aberrant targeting of AID can occur throughout the genome. Moreover, consistent<br />
with its single-stranded DNA binding activity, AID targets predominantly actively<br />
transcribed regions. Having established the DamID technology for B cells, future<br />
studies should allow us to provide a detailed, genome-wide binding profile of AID.<br />
This profile will be critical in identifying aberrant AID target sites (ATS), which will<br />
help to identify cancer-associated ATS causal to the development of GC- and post-GC<br />
derived B cell lymphomas. ATS are presently used to identify molecular predictors<br />
that favor AID binding.<br />
Role of PCNA and TLS in programmed mutagenesis During replication, DNA<br />
lesions can block high fidelity DNA polymerases, causing replication forks to arrest.<br />
To increase the fitness of replicating cells in the presence of DNA lesions, i.e. to<br />
continue replication without an a priori repair of the initial lesion, polymerase<br />
switching is required. Monoubiquitination of the highly conserved lysine residue 164<br />
(K164) of the proliferating cell nuclear antigen (PCNA-Ub) triggers switching from a<br />
damage sensitive, high fidelity DNA polymerase to a damage tolerant, low fidelity<br />
translesion synthesis (TLS) DNA polymerase. TLS polymerases can continue<br />
replication across non Watson Crick base pairs. B cells use TLS to introduce somatic<br />
mutations around genetic lesions caused by AID. Owing to the reduced accuracy of<br />
TLS polymerases, TLS is the major cause of DNA-damage-induced mutations. The<br />
finding that DNA damage-inducible ubiquitination of PCNA occurs at identical<br />
positions in yeast and humans suggested an evolutionary conservation of this<br />
molecular switch-board. To determine whether this concept applies to DNA damage<br />
tolerance in mammals, we have generated recombinant mouse strains and cell lines<br />
thereof carrying a conditional PCNA allele as well as a mutant K164R allele. While<br />
PCNA K164R mutant B cells proliferate normally, the mutation spectrum of<br />
hypermutated Ig genes alters dramatically. The failure to modify PCNA K164 in<br />
homozygous mutant B cells resulted in a 10-fold reduction of transitions and<br />
transversions at template A/T - normally accounting for 50% of all mutations<br />
generated (figure 6b). The selective failure to mutate template A/T is compensated by<br />
an increase in mutations at template G/C. This phenotype is similar to that found in<br />
polymerase eta (Polh) and mismatch recognition deficient B cells. Our data indicate<br />
the existence of two alternative mutator pathways, a major PCNA K164 dependent A/T<br />
mutator pathway and a major PCNA K164 independent G/C mutator pathway.<br />
PCNA-Ub, mismatch recognition (MSH2, MSH6) and Polh likely cooperate in<br />
establishing the vast majority of mutations at template A/T during replication of Ig<br />
genes. Consistent with the defined role of PCNA-Ub in yeast, B cells from PCNA K164R<br />
mutant mice are highly sensitive to ultraviolet light (UV), cisplatin (CisPt) and<br />
methyl-methanesulphonate (MMS). PCNAK164R mutant cells experience not just an<br />
S/G2 but also a G1 arrest. We show that the increased DNA damage sensitivity is<br />
caused by a defective recruitment of Polh.<br />
Group leader Heinz Jacobs<br />
59<br />
IMMUNOLOGY<br />
Heinz Jacobs PhD Group leader<br />
Petra Langerak MSc PhD student<br />
Marinus Heideman MSc PhD student<br />
Peter Krijger MSc PhD student<br />
Marc Hogenbirk MSc PhD student<br />
Niek Wit MSc PhD student<br />
Paul Van den Berk Technical Staff<br />
Figure 6a: DNA sliding clamp usage, DNA<br />
polymerases and mutagenesis: SHM is<br />
dominated by the introduction of point<br />
mutations (from: A, G, C, or T to: A, G, C,<br />
or T). We have shown previously, that most<br />
mutations at template A and T - which<br />
depend on mismatch repair (MMR)<br />
proteins Msh2 and Msh6 - require<br />
PCNA-Ub. Equally relevant, our data<br />
indicate that mutations at template G/C do<br />
not depend on PCNA-Ub. PCNA-Ub<br />
enables a polymerase switch between high<br />
fidelity DNA polymerases (Pold/e) and the<br />
A/T mutator Polh. Uracil, which is<br />
generated by AID cytidine deamination<br />
instructs a template T to generate G to A<br />
and C to T transitions. The TLS polymerase<br />
Rev1 contributes to the generation of C to G<br />
and G to C transversions. The<br />
polymerase(s) (Pol?) responsible for G to T<br />
and C to A transversions remain to be<br />
identified.
60<br />
IMMUNOLOGY<br />
Publications<br />
Langerak P, Krijger PH, Heideman MR,<br />
van den Berk PC, Jacobs H. Somatic<br />
hypermutation of immunoglobulin genes:<br />
Lessons from PCNAK164R mutant mice.<br />
Philos Trans R Soc Lond B Biol Sci. <strong>2008</strong><br />
Nov 12 (Epub ahead of print)<br />
Van Maldegem F, Scheeren FA,<br />
Jibodh RA, Bende RJ, Jacobs H,<br />
van Noesel CJM. AID splice variants are<br />
deaminase-activity deficient. Blood. <strong>2008</strong><br />
(in press)<br />
Our data identify a dual role for PCNA ubiquitination and Polh: Anti-mutagenic<br />
damage tolerance of UV-induced DNA damage and mutagenic processing of AIDinduced<br />
DNA damage in Ig genes. Polh requires PCNA-Ub to establish mutations at<br />
template A/T. Equally essential, the G/C mutator appears independent of PCNA-Ub.<br />
9-1-1 Complex: An alternative platform for PCNA-independent TLS? The<br />
RAD9/RAD1/Hus1 complex (also known as 9-1-1 complex) is a heterotrimeric DNA<br />
sliding clamp that is structurally very similar to PCNA. In yeast, 9-1-1 was shown to<br />
interact with TLS polymerases and induce TLS independent of PCNA K164<br />
modification. For various reasons, we consider that the 9-1-1 complex provides an<br />
additional platform capable of recruiting specific TLS polymerases in mammalian<br />
cells. At present, we are testing this hypothesis in mouse models carrying genetically<br />
predefined mutations in the 9-1-1 complex.<br />
Figure 6b: Model: A role of the 9-1-1 DNA sliding clamp in SHM Both, MMR and long patch base<br />
excision repair (BER) generate single strand gaps at sites of DNA damage (asterisk). The ubiquitin<br />
conjugase/ligase Rad6/Rad18 complex binds single stranded DNA and mediates ubiquitination of PCNA<br />
and - as shown so far only in yeast - ubiquitination of an alternative DNA sliding clamp, the 9-1-1<br />
complex. We speculate that 9-1-1 complex is required to generate most G/C transversions. This model is<br />
currently addressed in mice carrying specific mutations in 9-1-1.<br />
Allelic exclusion at the immunoglobulin heavy chain locus initiates at the<br />
level of transcription Developing lymphocytes generate their antigen receptor<br />
genes by DNA recombination. Once a gene has been productively assembled, the<br />
receptor is expressed and terminates rearrangement of the second allele to ensure<br />
mono-specificity. Using specific, non-functional IgH knock-in and transgenic mice,<br />
we showed in collaboration with J. Lutz and H-M. Jäck from the University of<br />
Erlangen, that stable expression of a non-coding µ heavy chain (HC) mRNA in B cells<br />
reduces DNA recombination at the IgH locus and impairs early B cell development.<br />
Functional µHC mRNA serves therefore not only as a classical messenger but also as<br />
a sensor for productive IgH rearrangements and as a temporary regulator of allelic<br />
exclusion during B cell development.
COMBATING TUMOR IMMUNE ESCAPE<br />
The aim of our research is to identify mechanisms that tumors use to escape from<br />
anti-tumor immune responses. The characterization of inhibitory molecules and<br />
pathways that are involved in these escape mechanisms may help in designing novel<br />
approaches to improve anticancer immunotherapy. One immunotherapy approach is<br />
adoptive transfer of tumor-reactive T cells. Tumor control requires expansion and<br />
survival of tumor-reactive T cells without exhaustion in vivo. This may best be<br />
achieved by transferring peripheral T cells into lymphopenic hosts, since transfer is<br />
followed by acute homeostatic proliferation (HP). We study this HP process with the<br />
aim to optimize adoptive T cell immunotherapy.<br />
Role of co-inhibitory molecules during tumor immune escape Sufficient T cell<br />
activation depends on TCR ligation and positive secondary signals to prevent anergy<br />
induction of the activated T cell. Recent work revealed that this secondary signal is<br />
not an on-off phenomenon but a gradually modulated signal intensity orchestrated by<br />
several co-stimulatory and co-inhibitory molecules inducibly expressed on the T cell<br />
surface. We and others have shown that one of the ligands (PD-L1) of such a coinhibitory<br />
molecule (PD-1) is highly expressed on tumor cells and impairs immune<br />
responses against tumor cells in animal models as well as when testing human<br />
T cells in vitro. We demonstrated further that susceptibility of T cells for PD-1<br />
mediated signals inversely depends on the tumor antigen-density on tumor cells.<br />
This indicates that a decreased tumor antigen expression combined with higher PD-<br />
L1 expression might be the initial change towards tumor immune escape.<br />
To show prognostic relevance of PD-L1 expression on melanomas, we currently<br />
examine tumor samples from melanoma patients treated at the <strong>NKI</strong>-AVL. The in<br />
vitro results and first screening of the melanomas have convinced us that interfering<br />
with PD-L1/PD-1 interaction should be tested in early phase clinical trials in humans.<br />
Possible technical approaches for a clinical implementation are either the blockade of<br />
PD-L1 or PD-1 by means of monoclonal antibodies or the suppression of PD-1 T cell<br />
surface expression via siRNA. Currently we are comparing all three approaches<br />
concerning efficacy and availability for transfer into the clinic.<br />
Homeostatically proliferating T cells for the treatment of cancer Transfer of<br />
naïve peripheral T cells into lymphopenic recipients results in a slow cytokine-driven<br />
proliferation of these T cells. During this HP, T cells acquire effector functions<br />
(IFN-production, lytic activity) while keeping characteristics of naïve T cells resulting<br />
in superior lymph node homing capabilities, decreased anergy induction and<br />
superior tumor growth control compared to naïve or antigen-activated counterparts.<br />
While the use of lymphopenia to induce HP by preconditioning the patient has been<br />
implemented recently in pilot studies elsewhere, we focus on dissecting characteristic<br />
of these HP T cells with the aim to induce HP in vitro, thus not requiring the<br />
intensive pretreatment of the patient. We have identified CD62L, an adhesion<br />
molecule, to be a pivotal molecule for homing of the transferred T cells to the<br />
peripheral lymph node, the only compartment acute HP relies on. Characterization<br />
of HP T cells using microarray analysis revealed several other molecules that are<br />
exclusively expressed in/on HP T cells. The identified molecules of the HP<br />
phenotype shall be tested for their functional relevance by means of transgenic<br />
overexpression or siRNA-mediated silencing.<br />
Development of an inducible spontaneous murine melanoma model Adoptive<br />
T cell therapies have been tested so far predominantly in transplantable tumor<br />
animals models. Such models often do not mimic the complex interaction between<br />
the tumor cell and the tumor microenvironment and may therefore be of little<br />
predictive value for cancer therapy. Mouse models for spontaneous melanoma better<br />
approximate the patient situation, but due to the late onset of tumor formation are<br />
less practical for long term immunotherapeutic experiments. To bypass this dilemma<br />
we aim to develop an inducible mouse melanoma model with a faster tumor onset.<br />
Group leader Christian Blank<br />
61<br />
IMMUNOLOGY<br />
Christian Blank MD PhD Group leader<br />
Kerstin Schuster PhD Post-doc<br />
Lisa Borkner MSc PhD student<br />
Lukas Kremmler PhD student<br />
Jules Gadiot Technical staff
62<br />
MOLECULAR BIOLOGY<br />
Division head, group leader Hein Te Riele<br />
Hein Te Riele PhD Group leader<br />
Rob Dekker PhD Post-doc<br />
Camiel Wielders PhD Post-doc<br />
Marieke Aarts MSc PhD student<br />
Sietske Bakker MSc PhD student<br />
Tinke Vormer MSc PhD student<br />
Eva Wielders Msc PhD student<br />
Marleen Dekker Technical staff<br />
Elly Delzenne-Goette Technical staff<br />
Sandra De Vries MSc Technical staff<br />
Anja Van der Wal Technical staff<br />
DIVISION OF MOLECULAR BIOLOGY<br />
Genetic instability and deregulated cell cycle control are hallmarks of human cancer.<br />
Our research involves both aspects focusing on (1) the role of DNA mismatch repair<br />
and the Fanconi anemia pathway in mutation avoidance and (2) the role of cell cycle<br />
checkpoints in tumor suppression.<br />
DNA MISMATCH REPAIR<br />
Inherited defects in DNA mismatch repair (MMR) underlie the cancer syndrome<br />
HNPCC (hereditary non-polyposis colorectal cancer). The primary function of MMR<br />
is correction of DNA replication errors. Mismatch repair is initiated by MSH2/<br />
MSH6 or MSH2/MSH3 protein complexes, which recognize base.base mismatches<br />
and small loops of unpaired bases. Subsequent steps involve recruitment of another<br />
heterodimeric protein complex, MLH1/PMS2, activation of exonucleolytic activity to<br />
remove the error-containing DNA strand and resynthesis of a new strand.<br />
Mismatches can also occur in heteroduplex DNA formed by exchange of homologous<br />
but not perfectly identical DNA strands. Recognition of such mismatches by MMR<br />
leads to dissociation of the heteroduplex and abortion of the recombination reaction.<br />
Finally, O 6 -methylguanine lesions in DNA are processed by the MMR system into toxic<br />
lesions causing cell death. Thus, DNA MMR has three main functions: it acts in an<br />
anti-mutagenic and anti-recombinogenic function, and it mediates the toxicity of<br />
methylating agents.<br />
Oligonucleotide-directed gene modification We have recently found that MMR<br />
imposes a strong impediment to subtle gene modification by short single-stranded<br />
DNA oligonucleotides in mouse embryonic stem (ES) cells. As readout for<br />
oligonucleotide-mediated gene targeting (‘oligo targeting’) we measure re-activation<br />
of a disabled neomycin (neo) reporter gene carrying a mutation in the start codon<br />
(ATG ➝ AAG). Single-stranded deoxyribo-oligonucleotides of ±38 residues<br />
containing 1-4 altered nucleotides could generate a novel ATG start codon, resulting<br />
in G418 resistance, albeit at extremely low frequency. We found that MMR deficiency<br />
increased the efficacy of oligo targeting 200-500-fold, reaching frequencies of up to<br />
10 -5 -10 -4 (Dekker et al., NAR 2003;31:e27).<br />
To alleviate the mutagenic effects of constitutive MMR deficiency, we render ES cells<br />
permissive for oligo targeting by transiently suppressing MSH2 protein levels by<br />
RNA interference. In an attempt to further increase the efficacy of oligo targeting, we<br />
have generated a library of short-hairpin-RNA-expressing vectors suppressing the<br />
activity of the major DNA repair pathways. Somewhat unexpectedly, none of the<br />
knock-down vectors affected the oligo targeting efficiency. These results seem to<br />
discard homologous recombination as a critical mediator of oligonucleotide-mediated<br />
gene modification. We also found that the level of transcription did not affect the<br />
efficacy of oligo targeting. Together, these results suggest that oligo targeting occurs<br />
in the context of DNA replication.<br />
Unclassified variants of MMR genes Besides mutations that fully disrupt gene<br />
function, subtle mutations affecting only a single codon are frequently found in<br />
(suspected) HNPCC. The consequences of single amino acid substitutions are often<br />
difficult to predict. Such allelic variants are therefore termed ‘Unclassified Variants’<br />
(UV). We have developed a three-step procedure to study the pathogenicity of<br />
missense variants of MSH2 and MSH6 found in the human population. First, we<br />
recreate the allelic variant in mouse ES cells by oligonucleotide-mediated codon<br />
substitution. Secondly, we have developed a method to make ES cells homozygous<br />
for the mutated allele. Thirdly, we study mutant ES cells for their capacity to sustain<br />
the three main MMR functions. By this method we could thus far identify one UV as<br />
deleterious, one as an innocent polymorphism and a third as a separation of function<br />
mutant with reduced MMR capacity. As we use mouse ES cells, we were able to<br />
introduce the latter mutant into the germ line of mice to study whether this variant<br />
predisposes to cancer.
THE FANCONI ANEMIA PATHWAY<br />
Genetic instability and cancer predisposition may also result from inherited defects<br />
in chromosome maintenance mechanisms. An example is Fanconi anemia (FA), a<br />
genetic disorder characterized by developmental abnormalities and a high incidence<br />
of AML and epithelial tumors. FA is caused by bi-allelic inherited defects in either<br />
one of 13 genes, FANCA, B, C, D1, D2, E, F, G, I, J, L, M, N. At the cellular level,<br />
defects in FA genes cause high sensitivity to crosslinking agents as manifested by G 2<br />
arrest, chromosomal aberrations and cell death. It has therefore been hypothesized<br />
that chromosomal instability caused by acquired defects in FA genes may underlie<br />
sporadic tumor development. Furthermore, such tumors may be exquisitely<br />
susceptible to crosslinking chemotherapeutics like cisplatin.<br />
To assess the significance of the FA genome maintenance pathway in suppression of<br />
cancer, we have generated mouse models carrying defects in the FA genes Fancf and<br />
Fancm. These mice are crossed into cancer prone Apc +/- mice to study whether FA<br />
defects affect the incidence, latency and spectrum of tumorigenesis.<br />
CELL CYCLE CHECKPOINTS<br />
Loss of G 1/S control is a frequent, if not mandatory event in development of cancer.<br />
However, we found that mouse embryonic fibroblasts (MEFs) devoid of proper G1/S<br />
control still rely on mitogens and attachment to proliferate indefinitely. We made<br />
such cells by genetically ablating the pocket proteins pRB, p107 and p130. These<br />
proteins impede the G 1/S transition of the cell cycle by binding to and inhibiting E2F<br />
transcription factors. Their activity is relieved upon phosphorylation by Cyclin D- and<br />
E-dependent kinases following mitogenic signaling. Complete phosphorylation of<br />
pocket proteins defines the so-called G 1 restriction point after which cells no longer<br />
require mitogens to enter S-phase. Ablation of pocket proteins in MEFs (TKO MEFs)<br />
alleviates the G 1 restriction point and allows cells to pass G1 and enter S phase in the<br />
absence of mitogens. We are using pocket-protein-defective cells to study<br />
mechanisms that restrict proliferation beyond the G1 restriction point.<br />
Growth-factor independence TKO MEFs can pass through S-phase in the absence<br />
of mitogenic signalling, but are then restricted by: (1) massive apoptosis and (2) G 2<br />
arrest that became apparent upon expression of the apoptosis inhibitor BCL2. G2<br />
arrest was effectuated through inhibitory interactions of the cyclin-dependent-kinase<br />
inhibitors p21 CIP1 and p27 KIP1 with Cyclins A and B1. The arrest was reversible: readdition<br />
of serum for 3-6 h led to mitotic entry about 15 h later, thus defining a<br />
restriction point operating in G 2. Caffeine accelerated mitogen-induced cell cycle reentry,<br />
which is indicative for activation of a DNA damage checkpoint. Indeed, we<br />
found mitogen-starved G 2-arrested TKO cells to accumulate g-H2AX/RAD51 foci that<br />
gradually disappeared upon mitogen stimulation (figure 1). Furthermore, cells<br />
entering mitosis showed excessive chromosomal aberrations. None of these effects<br />
were seen in TKO cells that were cultured in the presence of mitogens. Together,<br />
these results suggest that transient mitogen starvation of TKO cells induces DNA<br />
damage leading to chromosomal aberrations.<br />
Anchorage independence We also showed that partial or full pocket protein<br />
ablation was not sufficient to sustain anchorage-independent growth, even upon<br />
expression of RAS V12 , although DKO (Rb -/- p107 -/- or Rb -/- p130 -/- ) and TKO MEFs were<br />
immortal. Apparently, a cell cycle mechanism still operates to restrict proliferation of<br />
these cells in soft agar. We found anchorage-deprived RAS V12 -expressing MEFs<br />
arrested in G 1 or G 2, dependent on which pocket proteins were still active. Thus,<br />
TKO cells arrested in G 2 while DKO cells also arrested in G 1. By performing a screen<br />
for genetic events that permit anchorage-independent growth of RAS V12 -expressing<br />
pocket-protein-defective MEFs, we identified the immortalizing oncogene TBX2 that<br />
was shown to interfere with the p53 pathway through downregulation of p19 ARF .<br />
TBX2-mediated downregulation of p53 and p21 CIP1 rescued cyclin-dependent kinase<br />
acivities more effectively in DKO MEFs than Rb -/- MEFs, consistent with the higher<br />
transformability of DKO MEFs.<br />
Publications<br />
63<br />
MOLECULAR BIOLOGY<br />
Foijer F, Simonis M, Van Vliet M,<br />
Kerkhoven R, Sorger PK, Te Riele H.<br />
Oncogenic pathways impinging on the G2<br />
restriction point. Oncogene <strong>2008</strong>;27:1142-54<br />
Vormer TL, Foijer F, Wielders CLC,<br />
Te Riele H. Anchorage-independent growth<br />
of pocket-protein-deficient murine<br />
fibroblasts requires bypass of G2 arrest and<br />
can be accomplished by expression of TBX2.<br />
Mol Cell Biol <strong>2008</strong>;28:7263-73<br />
Figure 1: Mitogen-starved TKO MEFs (lacking<br />
Rb, p107 and p130) accumulate DNA damage.<br />
(A) g-H2AX foci in TKO MEFs mitogenstarved<br />
for 7 days. (B) Graphic representation<br />
of g-H2AX foci accumulation in mitogenstarved<br />
TKO MEFs. (C) Disappearance of<br />
g-H2AX foci upon mitogen addition.
64<br />
MOLECULAR BIOLOGY<br />
Group leader Piet Borst<br />
Piet Borst MD PhD Group leader<br />
Sven Rottenberg DVM PhD Dipl. ECVP<br />
Senior post-doc<br />
Henri Van Luenen PhD Academic staff<br />
Jayasree Iyer PhD Post-doc<br />
Marina Pajic PhD Post-doc<br />
Saara Vainio MD PhD Post-doc<br />
Koen Van de Wetering DVM PhD Post-doc<br />
Maaike Gonggrijp DVM PhD student<br />
Serge Zander DVM MSc PhD student<br />
Marcel De Haas Technical staff<br />
Liesbeth Van Deemter Technical staff<br />
Wouter Feddema Technical staff<br />
Ariena Kersbergen Technical staff<br />
Bas Ter Riet Technical staff<br />
Publications<br />
Borst P. Mega dose vitamin C as therapy<br />
for human cancer? Proc Natl Acad Sci<br />
USA <strong>2008</strong> (in press)<br />
Borst P, Rottenberg S, Jonkers J. How do<br />
real tumors become resistant to cisplatin?<br />
Cell Cycle <strong>2008</strong>;7:1353-9<br />
Borst P, Sabatini R. Base J: discovery,<br />
biosynthesis and possible functions. Ann<br />
Rev Microbiol <strong>2008</strong>;62:235-51<br />
Borst P, van de Wetering K,<br />
Schlingemann R. Does the absence of<br />
ABCC6 (multidrug resistance protein 6) in<br />
patients with Pseudoxanthoma elasticum<br />
prevent the liver from providing sufficient<br />
vitamin K to the periphery? Cell Cycle<br />
<strong>2008</strong>;7:1575-9<br />
De Wolf C, Jansen R, Yamaguchi H,<br />
de Haas M, van de Wetering K,<br />
Wijnholds J, Beijnen J, Borst P.<br />
Contribution of the drug transporter<br />
ABCG2 (breast cancer resistance protein)<br />
to resistance against anticancer nucleosides.<br />
Mol Cancer Ther <strong>2008</strong>;7:3092-102<br />
DNA BASE J<br />
Base J b-glucosyl-hydroxymethyluracil (base J), which we discovered in African<br />
trypanosomes in 1993 (Gommers-Ampt et al. Cell 1993;75:1129-36), is a base present<br />
in kinetoplastid flagellates and in Euglena. It replaces 1% of thymine in DNA and is<br />
predominantly located in repetitive sequences, such as telomeric repeats. We have<br />
characterized a J-binding protein (JBP1) that binds with high specificity to<br />
J-containing duplex DNA (Cross et al. EMBO J 1999;18:6573-81). Our recent work<br />
indicates that JBP1 is a thymidine hydroxylase that catalyses the first step of J<br />
biosynthesis, the conversion of T in DNA into hydroxymethyluracil. JBP1 appears to<br />
belong to the family of Fe 2+ and 2-oxoglutarate-requiring dioxygenases, as does a<br />
second putative hydroxylase, JBP2. In the kinetoplastid Leishmania, a JBP1 KO is<br />
lethal. In contrast, JBP2 is dispensable in Leishmania under normal growth<br />
conditions, but JBP2 KO strains are hypersensitive to bromodeoxyuridine (BrdU).<br />
During growth in BrdU, Leishmania loses its J, which is located for > 98% in<br />
telomeric repeats in this organism. How J loss leads to cell death is unclear. We do<br />
not find alterations in DNA integrity or cell cycle blocks. Solving the cause of death<br />
remains a top priority. We are also continuing attempts to set up assays for<br />
hydroxylase activity and for the putative glucosyl transferase catalyzing the second<br />
step in J biosynthesis. With Anastassis Perrakis (<strong>NKI</strong>-AVL) we are trying to determine<br />
the structure of JBP1-J-DNA complexes by crystallography; with Paul Wentworth<br />
(Scripps, California), we are looking for inhibitors of the binding of JBP1 to DNA and<br />
of its hydroxylase function.<br />
MULTIDRUG RESISTANCE OF CANCER CELLS<br />
We are interested in mechanisms of drug resistance in cancer cells and focus on<br />
resistance caused by increased ATP-dependent transport of drug out of the cell,<br />
mediated by ATP-binding cassette (ABC) transporters. We have isolated genes for<br />
these transporters and are characterizing their substrate specificity and sensitivity to<br />
inhibitors in transfected cells. We use the baculovirus system to produce insect cell<br />
membrane vesicles containing high amounts of transporter protein and suitable for<br />
vesicular transport studies. We also use transfected polarized kidney cell monolayers<br />
in which the transporters either route to the apical or the basolateral membrane,<br />
allowing the study of vectorial transport through the cell layer. To study the<br />
physiological function in metabolism and defense of the body against drugs and<br />
xenotoxins of these transporters, we have inactivated genes for several drug<br />
transporters by targeted gene disruption in mice. Initially we looked at<br />
P-glycoproteins (ABCB1 family); most recently we have studied the Multidrug<br />
Resistance Protein (ABCC) family members MRP2, 3, 4, 5 and 6.<br />
MRP3 (ABCC3) is an organic anion transporter contributing to the cellular export of<br />
endogenous or exogenous (toxic) compounds, conjugated to glutathione, sulphate or<br />
glucuronate. Using KO mice, we have shown that murine MRP3 is important for<br />
basolateral export of glucuronated compounds from the gut epithelium into blood.<br />
An interesting example is provided by resveratrol-glucuronide (R-gluc), which mainly<br />
enters the body via MRP3. We have shown that R-gluc is a high-affinity substrate for<br />
MRP3 and that R-mono-sulphate and R-di-sulphate are high-affinity substrates for<br />
another ABC-transporter: BCRP (ABCG2) (figure 2). We have initiated a systematic<br />
search for other glucuronides transported by MRPs by comparing the glucuronide<br />
derivatives in plasma/urine of WT and KO mice using Mass Spectrometry. We have<br />
identified several glucuronidated phyto-estrogens, derived from food, as novel MRP3<br />
substrates by this approach and anticipate that it may also be helpful to find<br />
substrates of other MRPs.<br />
MRP6 and PXE Pseudoxanthoma elasticum (PXE) is an autosomal recessive disease<br />
characterized by a progressive mineralization of connective tissue, resulting in skin,<br />
arterial and eye disease. Classical PXE is caused by mutations in the MRP6 (ABCC6)<br />
gene. Recent studies by Uitto et al. on Abcc6 -/- mice show that the absence of ABCC6<br />
in the liver is crucial for PXE and confirm the ‘metabolic disease hypothesis’ for PXE,<br />
which states that tissue calcification is due to the absence of a plasma factor secreted
from the basolateral hepatocyte membrane. We have proposed that this plasma factor<br />
is vitamin K (precursor). We think that vitamin K (precursor) is secreted by ABCC6<br />
from the liver as a glutathione-, glucuronide-, or sulphate conjugate and that this<br />
supplements the vitamin K need of peripheral tissues that receive insufficient<br />
vitamin from the diet, because dietary vitamin K is effectively extracted from blood by<br />
the liver. Peripheral tissue vitamin K is needed for the gamma-carboxylation of<br />
glutamate residues in proteins known to be required for counteracting calcification of<br />
connective tissue throughout the body.<br />
We have started an ambitious program to test this hypothesis in collaboration with<br />
Arthur Bergen (AMC), who generated an Abcc6 -/- mouse. We have made conjugates<br />
of menadione (Vit. K3) and are testing these in vesicular transport assays with MRP6vesicles.<br />
DRUG RESISTANCE IN ‘SPONTANEOUS’ MOUSE TUMORS<br />
We have started a project in collaboration with Jos Jonkers (<strong>NKI</strong>-AVL) to study<br />
resistance mechanisms in ‘spontaneous’ tumors arising in mice, conditionally<br />
defective in p53 and Brca1. These mice contain floxed alleles of these two genes and a<br />
Cre recombinase gene driven by a Keratin14 promotor, active only in epithelial cells,<br />
resulting in breast (and skin) cancer. When treated with the maximum tolerable dose<br />
of doxorubicin, docetaxel or topotecan, the breast tumors initially respond but<br />
eventually always develop resistance. Resistance is often associated with upregulation<br />
of the Mdr1a and Mdr1b genes (Abcb1), which encode drug-transporting P-glycoproteins<br />
and we have shown with specific inhibitors that remarkably low levels of<br />
Abcb1 upregulation (3-fold the levels in sensitive tumors) suffice to make the tumor<br />
multidrug resistant. We are also using this mouse model to test new anticancer drugs<br />
and drug combinations. Impressive tumor regression has been obtained with a new<br />
inhibitor of Poly-ADP-ribose polymerase I (PARPI) in collaboration with<br />
AstraZeneca. We are crossing disrupted alleles for the Abcb1 and Abcg2 genes into our<br />
mouse model to further test the importance of these transporters in drug resistance<br />
and to uncover other forms of resistance not mediated by transporters.<br />
In contrast to the results obtained with MDR drugs, we have been unable to obtain<br />
cisplatin resistance in this tumor model. The tumors respond to each new treatment<br />
with cisplatin, but are never fully eradicated.<br />
Although we have identified a tumor-initiating cell (‘stem cell’) in this tumor model<br />
characterized by high surface expression of CD24 and CD49f, this fraction does not<br />
appear to be enriched in the ‘remnants’ from which the tumors regrow after<br />
chemotherapy (figure 3). We are currently testing the hypothesis that the resistance<br />
of ‘remnants’ is not due to specific biochemical defense mechanisms of the putative<br />
tumor stem cells, but to the ability of a sub-fraction of the cells to go into<br />
‘hibernation’, i.e. stop cell cycle progression until the drug is gone.<br />
Figure 3: Fractionation of Brca1-/-;p53-/- tumors using FACS. A. Sorting of live (propidium iodidenegative),<br />
Lin- (non-endothelial, non-fibroblastic and non-hematopoietic) cells with CD24- and CD49fspecific<br />
antibodies. B. Tumorigenicity of limiting dilutions of sorted fractions after orthotopic<br />
transplantation into syngeneic animals. C. Tumor incidence summary of the graph shown in B. D.<br />
Percentage of cell fractions in tumors before treatment and in remnants 10 days after the injection of 6mg<br />
cisplatin (CDDP) per kg i.v.<br />
65<br />
MOLECULAR BIOLOGY<br />
Publications (continued)<br />
Rottenberg S, Jonkers J. Modeling therapy<br />
resistance in genetically engineered mouse<br />
cancer models. Drug Resist Updates<br />
<strong>2008</strong>;11:51-60<br />
Rottenberg S, Kersbergen A,<br />
van der Burg E, Nygren AOH, Zander S,<br />
Derksen PW, de Bruin M, Zevenhoven J,<br />
Lau A, Boulter R, Cranston A, O’Connor<br />
MJ, Martin NMB, Borst P, Jonkers J. High<br />
sensitivity of BRCA1-deficient mammary<br />
tumors to the PARP inhibitor AZD2281<br />
alone and in combination with platinum<br />
drugs. Proc Natl Acad Sci U S A<br />
<strong>2008</strong>;105:17079-84<br />
Figure 2: Transport of Resveratrol-3-sulphate<br />
(Res-3-S) and Resveratrol-di-sulphate (Resdi-S)<br />
by BCRP in vesicular transport<br />
experiments. (A) Schematic overview of the<br />
synthesis of [ 3 H]resveratrol-sulphates used in<br />
the vesicular transport experiments<br />
presented. [ 3 H]resveratrol was converted into<br />
[ 3 H]Res-3-S and [ 3 H]Res-di-S using mouse<br />
liver cystosol and the sulphate donor<br />
3’-phosphoadenosine-5’phosphosulphate<br />
(PAPS). Insets represent HPLC<br />
chromatograms of the purified [ 3 H]<br />
resveratrol-sulphates.<br />
Time course experiments of membrane<br />
vesicles containing human BCRP using 140<br />
nM [ 3 H]Res-3-S (B) or [ 3 H]Res-di-S (C).<br />
Transport was determined in the presence or<br />
absence of 4 mM ATP as indicated.<br />
Concentration-dependent transport of Res-<br />
3-S and Res-di-S by BCRP is shown in (D)<br />
and (E), respectively. Values are corrected<br />
for transport determined in absence of ATP.<br />
Each data point and error are the means ±<br />
SD of an experiment performed in<br />
triplicate.
66<br />
MOLECULAR BIOLOGY<br />
Group leader Jos Jonkers<br />
Jos Jonkers PhD Group leader<br />
Karin De Visser PhD Research associate<br />
Peter Bouwman PhD Post-doc<br />
Michiel De Bruin PhD Post-doc<br />
Gilles Doumont PhD Post-doc<br />
Marco Koudijs PhD Post-doc<br />
Xiaoling Liu PhD Post-doc<br />
Ewa Michalak PhD Post-doc<br />
Petra Ter Brugge PhD Post-doc<br />
Rinske Drost Msc PhD student<br />
Bastiaan Evers Msc PhD student<br />
Henne Holstege Msc PhD student<br />
Janneke Jaspers Msc PhD student<br />
Sjoerd Klarenbeek Msc PhD student<br />
Christiaan Klijn Msc PhD student<br />
Hanneke Van der Gulden Technical staff<br />
Ingrid Van der Heijden Technical staff<br />
Ellen Wientjens Technical staff<br />
Tanya Braumuller Research assistant<br />
Eva Kregel Research assistant<br />
Mark Pieterse Research assistant<br />
Ewoud Speksnijder Research assistant<br />
Eline Van der Burg Research assistant<br />
Publications<br />
Rottenberg S, Jonkers J. Modeling therapy<br />
resistance in genetically engineered mouse<br />
cancer models. Drug Resist Updat.<br />
<strong>2008</strong>;11:51-60<br />
Klijn C, Holstege H, de Ridder J, Liu X,<br />
Reinders M, Jonkers J*, Wessels L*.<br />
Identification of cancer genes using a<br />
statistical framework for multi-experiment<br />
analysis of non-discretized array CGH<br />
data. Nucleic Acids Res. <strong>2008</strong>;36:e13<br />
(* joint corresponding authors).<br />
Doumont G, de Visser KE, Derksen PW,<br />
Jonkers J. Models for angiogenesis:from<br />
fundamental mechanisms to anticancer<br />
treatment research. Drug Discov<br />
Today:Disease Models <strong>2008</strong>;4:75-82<br />
Borst P, Rottenberg S, Jonkers J. How do<br />
real tumors become resistant to cisplatin?<br />
Cell Cycle <strong>2008</strong>;7:1353-1359<br />
MOUSE MODELS OF BREAST CANCER<br />
The focus of our research group is on the genetic dissection of human breast cancer<br />
through the use of genetically engineered mouse models. For this, we have developed<br />
models for p53-induced breast cancer, BRCA1- and BRCA2- associated hereditary<br />
breast cancer, and E-cadherin-associated metastatic breast cancer. We are using these<br />
models to (1) investigate genotype-phenotype relations in mammary tumorigenesis;<br />
(2) perform therapeutic intervention studies; (3) identify genetic changes underlying<br />
breast tumorigenesis; (4) study the role of innate and adaptive immunity in breast<br />
cancer development.<br />
Conditional mouse models for BRCA-associated breast cancer We have<br />
previously generated conditional mouse mutants with tissue-specific loss of Brca1/2<br />
and p53 to establish models for BRCA1- and BRCA2-associated breast cancer. The<br />
Brca1 -/- ;p53 -/- mammary tumors share histopathological and molecular features with<br />
BRCA1-deficient breast cancers in women: they are highly proliferative, poorly<br />
differentiated, hormone receptor and HER2 negative mammary adenocarcinomas<br />
with pushing borders and a high degree of genomic instability. Interestingly, we have<br />
found that mammary tumor formation in our BRCA1 model is still estrogendependent.<br />
We are currently investigating whether this estrogen dependence is due<br />
to autocrine or paracrine mechanisms.<br />
Tumor intervention studies in the BRCA mammary tumor models The central<br />
role of BRCA1 and BRCA2 in DNA double-strand break (DSB) repair via homologous<br />
recombination (HR) implies that BRCA-deficient tumors are especially sensitive to<br />
DSB inducing chemotherapeutics. Indeed, in vitro cytotoxicity studies with BRCA2deficient<br />
mammary tumor cell lines and in vivo tumor intervention studies in our<br />
BRCA1 mammary tumor model showed high sensitivity of BRCA-deficient tumors to<br />
DNA-damaging agents such as doxorubicin or platinum drugs.<br />
In collaboration with Sven Rottenberg, Piet Borst and KuDOS Pharmaceuticals, we<br />
have used our BRCA1/2 models to test the anti-tumor effects of PARP inhibition,<br />
which may be selectively toxic to HR-deficient cells because it suppresses DNA<br />
single-strand break repair. Indeed, in vitro studies showed selective toxicity of the<br />
PARP inhibitor AZD2281 in Brca2 -/- ;p53 -/- mammary tumor cells, compared to p53 -/-<br />
cells. Administration of AZD2281 to mice with Brca1 -/- ;p53 -/- mammary tumors<br />
induced tumor regression without signs of toxicity, resulting in strongly increased<br />
survival. Long-term treatment with AZD2281, however, resulted in the development<br />
of drug resistance caused by upregulation of P-glycoprotein drug efflux pumps.<br />
Importantly, resistance could be reversed by co-administration of AZD2281 and the<br />
P-glycoprotein inhibitor tariquidar. Moreover, combination of AZD2281 with<br />
platinum drugs significantly prolonged recurrence-free survival, suggesting that<br />
AZD2281 potentiates the effect of these DNA-damaging agents. Together, these data<br />
demonstrate in vivo efficacy of AZD2281 against BRCA-deficient breast cancer, and<br />
illustrate how conditional mouse models of human cancer can be used for preclinical<br />
evaluation of novel therapeutics, for investigating drug resistance mechanisms and<br />
for testing ways to overcome therapy resistance.<br />
Conditional mouse models for E-cadherin-deficient metastatic breast cancer<br />
While metastatic disease is the main cause of death in breast cancer patients, the<br />
underlying mechanisms are poorly understood. Loss of E-cadherin is strongly<br />
associated with tumor invasion and metastasis, as well as with invasive lobular<br />
carcinoma (ILC), which accounts for 10-15% of all breast cancers. To study the role of<br />
E-cadherin in breast oncogenesis, we have generated a mouse model for invasive<br />
lobular breast carcinoma (ILC) based on epithelium-specific inactivation of<br />
E-cadherin and p53. Compared to p53 -/- mammary carcinomas, Ecad -/- ;p53 -/- mammary<br />
tumors show a significantly reduced latency, a morphological switch from ductal to<br />
lobular carcinoma, and a phenotypic change from non-invasive to highly invasive and<br />
metastatic tumors. Moreover, Ecad -/- ;p53 -/- mammary tumor cell lines – but not p53 -/-<br />
cell lines – are resistant to detachment induced apoptosis, aka anoikis.<br />
We have performed kinome-wide siRNA screens to identify factors that modulate<br />
anoikis resistance of Ecad -/- ;p53 -/- tumor cells, which may serve as a surrogate assay
for survival of circulating tumor cells. We have identified several kinases that are<br />
essential for survival of Ecad -/- ;p53 -/- cells under non-adherent culture conditions. In<br />
vivo validation studies will be performed using pharmacological inhibitors or<br />
inducible expression of shRNAs against these kinases in Ecad -/- ;p53 -/- mammary<br />
tumors in mice.<br />
Tumor intervention studies in the E-cadherin mammary tumor model We are<br />
also using the E-cadherin mammary tumor model for preclinical testing of novel<br />
therapeutics. For this purpose we have derived panels of clonal, luciferase-marked<br />
mammary tumor cell lines from our mouse models. Together with the spontaneous<br />
models, these reagents permit validation of candidate drug targets and testing of<br />
(combinations of) anticancer drugs on four different levels: (1) in vitro, on the panels<br />
of tumor cell lines; (2) in vivo, on tumor outgrowths from orthotopically grafted cell<br />
lines; (3) in vivo, on tumor outgrowths from orthotopically grafted tumor fragments<br />
(allowing parallel treatments on genetically identical tumors); (4) in vivo, on the<br />
sporadic tumors that develop in the mouse mammary tumor models (allowing<br />
intervention at early stages of tumor development). We are currently using this multilevel<br />
platform to test a number of conventional and targeted anti-cancer drugs.<br />
Array-CGH analysis of mouse mammary tumors We have performed array-based<br />
comparative genomic hybridization (aCGH) analysis of panels of mammary tumors<br />
derived from our conditional mouse models. To identify regions with significantly<br />
recurrent DNA copy number aberrations (CNAs), we have developed KC-SMART<br />
(Kernel Convolution – a Statistical Method for Aberrant Region deTection) for multiexperiment<br />
analysis of aCGH data. KC-SMART generates a Kernel Smoothened<br />
Estimate (KSE) of recurrent CNAs across the genome, aggregated over all tumors.<br />
The peaks in the KSE curves are tested against randomly permutated data to identify<br />
significantly recurrent CNAs. We have used this approach to significantly recurrent<br />
CNAs in mouse and human breast cancer panels and in human cancer cell line<br />
panels. We are using these data sets for (1) cross-species comparisons of CNAs in<br />
mouse and human tumors; (2) co-occurrence analysis of CNAs in human tumor cell<br />
lines; (3) integration of aCGH and gene expression data to identify candidate cancer<br />
genes in recurrent CNAs.<br />
The role of innate and adaptive immunity in breast cancer development<br />
Studies in a transgenic mouse model for skin carcinogenesis revealed an unexpected<br />
role for B lymphocytes and their soluble mediators in initiating chronic inflammation<br />
during premalignancy and subsequent progression to invasive skin cancer (De Visser<br />
et al. Cancer Cell 2005;7:411-23). We are currently investigating whether a similar link<br />
exists between innate/adaptive immunity and breast cancer development,<br />
progression and therapy resistance in our mouse model for E-cadherin deficient<br />
metastatic breast cancer. Similar to human breast cancer lesions, neoplastic<br />
mammary glands of these mice are characterized by leukocyte infiltration. To assess<br />
the roles of innate and adaptive immune systems in breast cancer development and<br />
progression, these mice have been crossed with various immune-deficient (Rag1 -/- ,<br />
Il2rg -/- ) mice. In the resulting compound mutant mice, chronic inflammation,<br />
angiogenesis, premalignant progression, cancer development and metastasis<br />
formation are being analyzed. We will also perform adoptive transfer experiments<br />
with bone marrow cells from mice with macrophage-specific expression of the<br />
diphtheria toxin receptor to study the effects of diphtheria toxin-mediated<br />
macrophage ablation on mammary tumor development and progression.<br />
67<br />
MOLECULAR BIOLOGY<br />
Publications (continued)<br />
Uren AG, Kool J, Matentzoglu K,<br />
de Ridder J, Mattison J, van Uitert M,<br />
Lagcher W, Sie D, Tanger E, Cox T,<br />
Reijnders M, Hubbard TJ, Rogers J,<br />
Jonkers J, Wessels L, Adams DJ,<br />
van Lohuizen M, Berns A. Large scale<br />
mutagenesis in p19ARF and p53 deficient<br />
mice identifies cancer genes and their<br />
collaborative networks. Cell <strong>2008</strong>;133:727-741<br />
Evers B, Drost R, Schut E, de Bruin M,<br />
van der Burg E, Derksen PW, Holstege H,<br />
Liu X, van Drunen E, Beverloo HB,<br />
Smith GC, Martin NM, Lau A,<br />
O’Connor MJ, Jonkers J. Selective inhibition<br />
of BRCA2-deficient mammary tumor cell<br />
growth by AZD2281 and cisplatin. Clin<br />
Cancer Res. <strong>2008</strong>;14:3916-3925<br />
Pietersen A, Evers B, Prasad AA,<br />
Tanger E, Cornelissen-Steijger P,<br />
Jonkers J, van Lohuizen M. Bmi1 regulates<br />
stem cells but also proliferation and<br />
differentiation of committed cells in<br />
mammary gland epithelium. Curr Biol.<br />
<strong>2008</strong>;18:1094-1099<br />
Bouwman P, Jonkers J. Mouse models for<br />
BRCA1 associated tumorigenesis:from<br />
fundamental insights to preclinical utility.<br />
Cell Cycle <strong>2008</strong>;7:2647-2653<br />
Rottenberg S, Jaspers JE, Kersbergen A,<br />
van der Burg E, Nygren AO, Zander SA,<br />
Derksen PW, de Bruin M, Zevenhoven J,<br />
Lau A, Boulter R, Cranston A,<br />
O’Connor MJ, Martin NM, Borst P,<br />
Jonkers J. High sensitivity of BRCA1deficient<br />
mammary tumors to the PARP1<br />
inhibitor AZD2281 alone and in<br />
combination with platinum drugs. Proc<br />
Natl Acad Sci USA. <strong>2008</strong>;105:17079-17084<br />
Lehembre F, Yilmaz M, Wicki A,<br />
Schomber T, Strittmatter K, Ziegler D,<br />
Kren A, Went P, Derksen PW, Berns A,<br />
Jonkers J, Gerhard Christofori G. NCAMinduced<br />
focal adhesion assembly:a functional<br />
switch upon loss of E-cadherin. EMBO J.<br />
<strong>2008</strong>;27:2603-2615<br />
Pietersen AM, Horlings HM,<br />
Hauptmann M, Langerod A, Ajouaou A,<br />
Cornelissen-Steijger P, Wessels LF,<br />
Jonkers J, van de Vijver M, van Lohuizen M.<br />
Ezh2 and Bmi1 inversely correlate with<br />
prognosis and p53 mutation in breast<br />
cancer. Breast Cancer Res. <strong>2008</strong> (in press)
68<br />
MOLECULAR BIOLOGY<br />
Group leader Sabine Linn<br />
Sabine Linn MD PhD Group leader<br />
Chiel De Bruin PhD Post-doc<br />
Michael Knauer MD PhD Post-doc<br />
Jolien Bueno de Mesquita MD PhD student<br />
Marleen Kok MD PhD student<br />
Marieke Vollebergh MD PhD student<br />
Rutger Koornstra MD PhD student<br />
Ingrid Van der Heijden Technical Staff<br />
Publications<br />
Bueno-de-Mesquita JM, Linn SC, Keijzer R,<br />
Wesseling J, Nuyten DS, van KC, Meijers C,<br />
de Graaf PW, Bos MM, Hart AA, Rutgers EJ,<br />
Peterse JL, Halfwerk H, de GR, Pronk A,<br />
Floore AN, Glas AM, Van’t Veer LJ,<br />
van de Vijver MJ. Validation of 70-gene<br />
prognosis signature in node-negative breast<br />
cancer. Breast Cancer Res Treat <strong>2008</strong>; DOI<br />
10.1007/s10549-008-0191-2 (e-pub ahead of<br />
print)<br />
Heerema-McKenney A, Wijnaendts LC,<br />
Pulliam JF, Lopez-Terrada D,<br />
McKenney JK, Zhu S, Montgomery K,<br />
Mitchell J, Marinelli RJ, Hart AA,<br />
van de Rijn M, Linn SC. Diffuse myogenin<br />
expression by immunohistochemistry is an<br />
independent marker of poor survival in<br />
pediatric rhabdomyosarcoma: a tissue<br />
microarray study of 71 primary tumors<br />
including correlation with molecular<br />
phenotype. Am J Surg Pathol <strong>2008</strong>;32:1513-22<br />
Holm C, Kok M, Michalides R, Fles R,<br />
Koornstra R, Wesseling J, Hauptmann M,<br />
Neefjes J, Peterse J, Stal O, Landberg G,<br />
Linn S. Phosphorylation of the oestrogen<br />
receptor alpha at serine 305 and prediction<br />
of tamoxifen resistance in breast cancer. J<br />
Pathol <strong>2008</strong>; DOI 10.1002/path.2455<br />
(e-pub ahead of print)<br />
Schilder CM, Linn SC, Van Dam FS,<br />
Schagen SB. [The effect of hormone therapy<br />
on cognitive function in patients with breast<br />
cancer]. Ned Tijdschr Geneeskd<br />
<strong>2008</strong>;152:494-8<br />
MOLECULAR DISSECTION OF BREAST AND LUNG CANCER BY<br />
DIFFERENTIAL DRUG SENSITIVITY<br />
In the clinic, we generally use anticancer drugs selected by clinical trials to perform<br />
best for the whole group of breast and non-small cell lung cancer (NSCLC) patients,<br />
whereas little is known about the molecular mechanisms underlying differential drug<br />
sensitivity. The focus of our research line is to unravel these mechanisms in order to<br />
develop tests that may guide treatment decisions in the clinic and ultimately improve<br />
survival. For this purpose we use several genome-wide approaches and molecular<br />
techniques, in order to dissect the mechanisms that divide clinically well-defined<br />
cohorts of breast and NSCLC patients into resistant and sensitive to a particular drug.<br />
In addition, we use conditional mouse models for breast cancer, and derived clonal<br />
cell lines, to study differential chemosensitivity in a controlled fashion.<br />
A second research line focuses on the impact of prognostic molecular classifiers on<br />
adjuvant systemic treatment advice in breast cancer.<br />
Compliance with national guidelines, revision of guidelines and 70-gene<br />
prognosis signature We have demonstrated that the introduction of national<br />
guidelines for adjuvant systemic treatment of breast cancer in 2002 led to a more<br />
uniform treatment advice in The Netherlands. The 70-gene prognosis signature did<br />
not lead to a decrease in adjuvant systemic treatment prescription before <strong>2008</strong>.<br />
However, we calculated that the revision of the national guidelines in <strong>2008</strong> would<br />
lead to an increased use of adjuvant systemic therapy in lymph-node negative<br />
patients from 50% to 75%. Based on this, we calculated that use of the<br />
70-gene prognosis signature would lead to a 10-25% decrease in the use of adjuvant<br />
systemic treatment without compromising survival (see also Division of<br />
Experimental Therapy).<br />
Value of 70-gene prognosis signature in defined breast cancer subgroups In<br />
collaboration with Division of Experimental Therapy and Agendia BV, using a pooled<br />
database of ~1800 breast cancer patients with data on the 70-gene prognosis<br />
signature, we have demonstrated that the 70-gene prognosis signature retained its<br />
prognostic value in patients with tumors smaller than 20 mm (N=224).<br />
Impact of inter-observer variation in tumor grade on adjuvant systemic<br />
treatment advice We studied inter-observer variation in ~700 lymph-node negative<br />
breast cancer patients. The local pathological examination was discordant with the<br />
central review for histologic grade in 28% (kappa 0.56; grade 2 tumours 35%<br />
discordant). If Adjuvant Online version 8.0 was applied, 8% of patients (kappa 0.83)<br />
would have been assigned to a different clinical risk group (see also Division of<br />
Experimental Therapy).<br />
Development of a predictive test for tamoxifen resistance in breast cancer<br />
Experimental studies have shown that phosphorylation of ERa at serine 118<br />
(ERaS118-P) is required for downregulation of ERa-induced gene expression by<br />
tamoxifen. In collaboration with the group of Rob Michalides (Division of Cell<br />
Biology II) and the group of Göran Landberg (Lund University, Lund, Sweden and<br />
Breakthrough Breast Cancer Research Unit, Paterson Institute for Cancer Research,<br />
Manchester, UK) we have studied the expression pattern of ERaS118-P in 239 breast<br />
cancer patients, who participated in a randomized trial of adjuvant tamoxifen versus<br />
no endocrine therapy. Patients with ERaS118-P high tumors benefited from adjuvant<br />
tamoxifen (HR=0.36, 95%CI 0.20-0.65, p=0.001) while patients with ERaS118-P low<br />
tumors did not (HR=0.87, 95%CI 0.51-1.48, p=0.60). In multivariate analysis, the<br />
interaction between ERaS118-P and treatment was significant (p=0.037).<br />
We are currently collecting tumor material of approximately 1000 patients who<br />
participated in another randomized trial of adjuvant tamoxifen versus no endocrine<br />
therapy, in order to confirm our findings in a second, independent series.<br />
CYP450 enzyme system, tamoxifen and breast cancer outcome Tamoxifen is<br />
metabolized by several cytochrome P450 (CYP450) enzymes into compounds with<br />
altered affinity for the estrogen receptor, among which the very active metabolite
Figure 4: Relation of ERaS118-P with outcome after tamoxifen treatment. Kaplan-Meier survival analysis<br />
according to ERaS118-P levels. Recurrence-free survival (RFS) of premenopausal patients who had been<br />
randomly assigned to tamoxifen or no adjuvant systemic treatment. ERaS118-P Low (Allred score 0-6)<br />
(A) and ERaS118-P High tumors (Allred score 7-8) (B) were analyzed separately.<br />
Hazard ratios and p-values are based on univariate Cox regression analysis.<br />
endoxifen. CYP450 analysis of estrogen receptor positive tumors has suggested that<br />
polymorphisms of 2D6 and 2C19 are predictive of outcome after adjuvant tamoxifen<br />
therapy in patients with early breast cancer. In collaboration with Rob van Schaik and<br />
Els Berns (Erasmus Medical Center, Rotterdam) we are investigating the predictive<br />
and prognostic role of these polymorphisms in relation to tamoxifen treatment and<br />
breast cancer outcome. In addition, in collaboration with Jos Beijnen (Division of<br />
Medical Oncology) and Hans Bonfrer (Division of Diagnostic Oncology) we are<br />
investigating correlations of these polymorphisms with steady-state serum<br />
concentrations of tamoxifen and its metabolites in 143 patients who have been treated<br />
with adjuvant tamoxifen in the nineties (side study of EORTC 10901; accrual from<br />
1995-1999). Breast cancer outcome of these patients will also be correlated with<br />
CYP450 polymorphisms and serum concentrations of tamoxifen and metabolites.<br />
Molecular mechanisms underlying sensitivity for high dose alkylating agents<br />
Using an array comparative genomic hybridization (aCGH) classifier initially<br />
constructed to identify BRCA1-mutated tumours we were able to identify a group of<br />
metastatic breast cancer patients treated with platinum-based chemotherapy with a<br />
high complete remission-rate and long progression free survival.<br />
We found a high correlation between our BRCA1-like classifier, an expansive growth<br />
pattern on histology and an estrogen receptor (ER) negative status. To test the<br />
performance of this classifier in an independent patient series, we used a<br />
randomized clinical trial comparing 5 x FEC with 4 x FEC followed by 1x carboplatinthiotepa-cyclophosphamide<br />
(CTC) for high risk, primary operable breast cancer<br />
[Rodenhuis et al., New Engl J Med, 2003]. We first evaluated the performance of an<br />
expansive growth pattern versus an invasive growth pattern in de subgroup of ER<br />
negative, HER2 negative breast cancer patients. Indeed, patients with an expansive<br />
growth pattern had an extremely good outcome after high dose CTC. Further<br />
analyses are ongoing. (Collaboration with Divisions of Experimental Therapy,<br />
Molecular Biology and Medical Oncology).<br />
Screening for drugs active against lobular breast cancer In collaboration with<br />
the Jonkers group, we have generated and characterized a panel of 27 murine breast<br />
cancer cell lines derived from tumors with a Trp53 -/- or Cdh1 -/- ;Trp53 -/- genotype,<br />
modeling ductal and lobular breast carcinoma, respectively. When comparing drug<br />
sensitivity under cluster versus low cluster plate growth conditions, differential<br />
sensitivity was observed for src inhibitors in cell lines with a Cdh1 -/- ;Trp53 -/- genotype,<br />
displaying greater sensitivity under low cluster growth conditions. Further analyses<br />
are ongoing, in order to unravel the underlying mechanisms that can explain this<br />
differential sensitivity.<br />
Serum concentrations of EGFR ligands and sensitivity for erlotinib in NSCLC<br />
In 2003 approximately 7200 patients were diagnosed with NSCLC in the Netherlands<br />
of which 44% was older than 70 years. Besides relatively toxic palliative chemotherapy,<br />
targeted therapies (i.e. EGFR-inhibitors) may be used in these patients. However,<br />
EGFR-inhibitors have a low response rate and have side-affects. We have shown that<br />
the EGFR ligands amphiregulin and transforming growth factor alpha, measured in<br />
serum, can be used as predictive markers of resistance to EGFR-inhibitors.<br />
69<br />
MOLECULAR BIOLOGY<br />
Publications (continued)<br />
Kok M, Linn SC, Van Laar RK, Jansen MP,<br />
van den Berg TM, Delahaye LJ, Glas AM,<br />
Peterse JL, Hauptmann M, Foekens JA,<br />
Klijn JG, Wessels LF, Van’t Veer LJ,<br />
Berns EM. Comparison of gene expression<br />
profiles predicting progression in breast<br />
cancer patients treated with tamoxifen.<br />
Breast Cancer Res Treat <strong>2008</strong>; DOI<br />
10.1007/s10549-008-9939-y (e-pub ahead<br />
of print)<br />
Kok M, Linn SC, van de Vijver MJ.<br />
Estrogen receptor phenotypes defined by gene<br />
expression profiling. In: Fuqua SAW (ed).<br />
Hormone receptors in breast cancer. New<br />
York, NY: Springer, <strong>2008</strong> (in press)<br />
Linn SC, Bueno-de-Mesquita JM, van de<br />
Vijver MJ. Are gene signatures better than<br />
traditional clinical factors? – Authors’ reply.<br />
Lancet Oncol <strong>2008</strong>;9:198-9<br />
Schilder CM, Eggens PC, Seynaeve C,<br />
Linn SC, Boogerd W, Gundy CM, Beex LV,<br />
Van Dam FS, Schagen SB.<br />
Neuropsychological functioning in<br />
postmenopausal breast cancer patients<br />
treated with tamoxifen or exemestane after<br />
AC-chemotherapy: Cross-sectional findings<br />
from the neuropsychological TEAM-side<br />
study. Acta Oncol <strong>2008</strong>;1-10
70<br />
MOLECULAR BIOLOGY<br />
Group leader Alfred Schinkel<br />
Alfred Schinkel PhD Group leader<br />
Dilek Iusuf MSc PhD student<br />
Jurjen Lagas MSc PhD student<br />
Evita Van de Steeg MSc PhD student<br />
Robert Van Waterschoot MSc PhD student<br />
Marijn Vlaming MSc PhD student<br />
Corina Van der Kruijssen MSc Technical staff<br />
Anita Van Esch Technical staff<br />
Els Wagenaar Technical staff<br />
Publications<br />
Marchetti S, Oostendorp RL, Pluim D,<br />
van Eijndhoven M, van Tellingen O,<br />
Schinkel AH, Versace R, Beijnen JH,<br />
Mazzanti R, Schellens JH. In vitro<br />
transport of gimatecan<br />
(7-t-butoxyiminomethylcamptothecin) by<br />
breast cancer resistance protein,<br />
P-glycoprotein, and multidrug resistance<br />
protein 2. Mol Cancer Ther 2007;6:3307-13<br />
Van Waterschoot RA, van Herwaarden AE,<br />
Lagas JS, Sparidans RW, Wagenaar E,<br />
van der Kruijssen CMM, Goldstein JA,<br />
Zeldin DC, Beijnen JH, Schinkel AH.<br />
Midazolam metabolism in Cytochrome<br />
P450 3A knockout mice can be attributed to<br />
upregulated CYP2C enzymes. Mol<br />
Pharmacol <strong>2008</strong>;73,1029-36<br />
Zimmermann C, van de Wetering K,<br />
van de Steeg E, Wagenaar E, Vens C,<br />
Schinkel AH. Species-dependent transport<br />
and modulation properties of human and<br />
mouse multidrug resistance protein 2<br />
(MRP2/Mrp2, ABCC2/Abcc2). Drug<br />
Metab Dispos <strong>2008</strong>;36:631-40<br />
Lagas JS, Sparidans RW,<br />
van Waterschoot RAB, Wagenaar E,<br />
Beijnen JH, Schinkel AH. P-glycoprotein<br />
limits oral availability, brain penetration<br />
and toxicity of an anionic drug, the<br />
antibiotic salinomycin. Antimicrob Agents<br />
Chemother. <strong>2008</strong>;52:1034-9<br />
GENES AND PROTEINS INVOLVED IN ANTICANCER DRUG<br />
RESISTANCE AND PHARMACOKINETICS<br />
Our research focuses on genes and proteins that cause drug resistance or drug<br />
susceptibility in tumors, or influence the pharmacological and toxicological behavior<br />
of anticancer and many other drugs and toxins, including carcinogens. Insight into<br />
these systems may: i) improve chemotherapy and more generally pharmacotherapy<br />
approaches for cancer and other diseases; ii) increase insights into factors<br />
determining susceptibility to carcinogens, and; iii) allow elucidation of physiological<br />
functions. To study the physiological, pharmacological and toxicological roles of the<br />
proteins involved, and their interactions, we generate and analyze knockout or<br />
transgenic mice lacking or overexpressing the relevant genes.<br />
Impact of active drug transporters We have a long-standing interest in plasma<br />
membrane proteins of the ATP binding cassette (ABC) multidrug transporter family,<br />
including P-glycoprotein (P-gp, ABCB1), MRP2 (ABCC2) and BCRP (ABCG2) (figure 6).<br />
These proteins actively export a wide range of anticancer, anti-HIV/AIDS, and many<br />
other drugs from cells. This ATP-dependent drug extrusion can cause multidrug<br />
resistance (MDR) in tumor cells. P-gp, MRP2 and BCRP all localize to the apical<br />
membrane of polarized epithelial cells, resulting in apically directed export of drug<br />
substrates, and there is considerable overlap in substrate specificity between these<br />
transporters. Previous experiments in P-gp and Bcrp1 knockout mice indicated that<br />
these transporters can protect an organism against exogenous toxins and drugs by<br />
limiting penetration of substrates into brain, testis, and fetus, by restricting uptake of<br />
orally administered substrates, and by mediating excretion of substrates via liver and<br />
intestine. To extend these analyses we have generated Mrp2 knockout mice, and<br />
crossed these with existing P-gp, Bcrp1 and Mrp3 knockout mice in order to elucidate<br />
the separate and combined contributions of these transporters to pharmacological,<br />
toxicological and physiological functions.<br />
Figure 5: Putative structure of a prototypic ABC drug<br />
transporter<br />
P-gp, Bcrp1, Mrp2 and Mrp3 (compound) knockout mice We found that<br />
salinomycin, an anionic antibiotic, was transported by P-gp in vitro, and we could<br />
subsequently demonstrate that its oral availability, brain penetration and toxicity was<br />
markedly increased in P-gp knockout mice. This was surprising, as it was previously<br />
thought that P-gp affected only cationic or neutral amphiphilic compounds. This<br />
finding thus further expands the already wide spectrum of drugs that are potentially<br />
affected by P-gp activity in vivo. We further found that the important analgesic<br />
diclofenac was transported by BCRP and Bcrp1. Moreover, diclofenac and the<br />
uricosuric benzbromarone could efficiently stimulate human MRP2-mediated drug<br />
transport already at very low concentrations. Efforts to show these effects also in vivo<br />
in mice failed, and retrospective analysis indicated that this is likely due to the fact<br />
that mouse Mrp2 shows sometimes marked differences with human MRP2 in<br />
stimulation by various compounds.<br />
We generated Mrp2/Mrp3 combination knockout mice, which were viable and fertile.<br />
The in vivo pharmacokinetics of the anticancer drug methotrexate (MTX) and its<br />
main toxic metabolite 7OH-MTX were analyzed in these mice. This indicated an
important role of Mrp3, present in the basolateral membrane of the hepatocytes, in<br />
transporting compounds back from the liver into the circulation, thus affecting the<br />
urinary elimination of the drugs. This was especially evident when Mrp2, present in<br />
the bile canalicular membrane, was absent, potentially leading to marked<br />
accumulation of compounds in the liver. Mrp3 thus seems to function as an<br />
important alternative clearance pathway for compounds that are potentially<br />
accumulating in the liver. Together, these results suggest that variation in MRP2 and<br />
MRP3 activity in patients might have profound effects on elimination and toxicity of<br />
MTX and 7OH-MTX, and thus on the therapeutic use of MTX.<br />
CYP3A transgenic and knockout mice Cytochrome P450 3A (CYP3A) enzymes<br />
metabolize >50% of prescribed drugs, and represent one of the most important<br />
detoxifying systems. As CYP3A activity shows high inter- and intra-patient variability,<br />
it can have a profound influence on variable drug behavior (pharmacodynamics) and<br />
drug toxicity. Moreover, its substrates overlap extensively with those of the drug<br />
transporters P-gp, BCRP and MRP2. To investigate the physiological and<br />
pharmacological roles of CYP3A, we previously generated Cyp3a knockout mice, and<br />
showed a pronounced effect of Cyp3a deficiency on the oral bioavailability, i.v.<br />
clearance and toxicity of the anticancer drug and CYP3A substrate docetaxel. Using<br />
transgenic mice that specifically overexpressed human CYP3A4 in either liver or<br />
intestine of Cyp3a knockout mice we could further show a dominant role of intestinal<br />
CYP3A4 in oral bioavailability of docetaxel, whereas hepatic CYP3A4 dominates<br />
clearance of i.v. docetaxel.<br />
We subsequently characterized the Cyp3a knockout mice by studying the metabolism<br />
of midazolam, one of the most widely used probes to assess CYP3A activity. We<br />
expected that midazolam metabolism would be severely reduced in the absence of<br />
Cyp3a. We used hepatic and intestinal microsomal preparations from Cyp3a knockout<br />
and wild-type mice to assess the midazolam metabolism in vitro. In addition, in vivo<br />
metabolite formation was determined after intravenous administration of<br />
midazolam. Surprisingly, there was still marked midazolam metabolism in hepatic<br />
(but not intestinal) microsomes from Cyp3a knockout mice. Accordingly, we found<br />
comparable amounts of midazolam as well as its major metabolites in plasma after<br />
intravenous administration in Cyp3a knockout mice when compared to wild-type<br />
mice. These data suggest that other hepatic cytochrome P450 enzymes could take<br />
over the midazolam metabolism in Cyp3a knockout mice. We next provided evidence<br />
that Cyp2c enzymes, which are upregulated in the Cyp3a knockout mice, are<br />
primarily responsible for this metabolism and that several but not all murine Cyp2c<br />
enzymes are capable of metabolizing midazolam to its 1’-OH and/or 4-OH<br />
derivatives. These data illustrate compensatory changes in detoxifying systems that<br />
may occur in Cyp3a knockout mice. Studies in these mice with drugs that are partly<br />
metabolized by Cyp2c should therefore be interpreted with caution.<br />
We then investigated the mechanism of regulation of CYP2C55 (the most highly<br />
upregulated Cyp2c gene in the Cyp3a knockout mice) and other detoxifying systems<br />
in Cyp3a knockout mice further. Induction studies with prototypical inducers<br />
demonstrated an important role for the nuclear receptors PXR and CAR in the upregulation<br />
of CYP2C55. Subsequent diet-switch experiments revealed that foodderived<br />
xenobiotics are primarily responsible for the increased induction of CYP2C55<br />
as well as of several other primary detoxifying systems in Cyp3a knockout mice. Our<br />
data suggest that CYP3A normally metabolizes food-derived activators of PXR and/or<br />
CAR, explaining the increased levels of such activators in Cyp3a knockout mice and<br />
subsequent up-regulation of a range of detoxifying systems. Interestingly, our studies<br />
with tissue-specific CYP3A4 transgenic Cyp3a knockout mice revealed that not only<br />
hepatic but also intestinal expression of CYP3A4 could reduce the hepatic expression<br />
of detoxifying systems to near wild-type levels. Apparently, intestinal CYP3A4 can<br />
limit the hepatic exposure to food-derived activators of nuclear receptors, thereby<br />
regulating the expression of a range of detoxifying systems in the liver. This broad<br />
biological effect further emphasizes the importance of intestinal CYP3A activity and<br />
could have profound implications for the prediction of drug exposure.<br />
71<br />
MOLECULAR BIOLOGY<br />
Publications (continued)<br />
Matsushima S, Maeda K, Hayashi H,<br />
Debori Y, Schinkel AH, Schuetz JD,<br />
Kusuhara H, Sugiyama Y. Involvement of<br />
multiple efflux transporters in hepatic<br />
disposition of fexofenadine. Mol Pharmacol<br />
<strong>2008</strong>;73:1474-83<br />
Damen CWN, Rosing H, Tibben MM,<br />
Van Maanen MJ, Lagas JS, Schinkel AH,<br />
Schellens JHM, Beijnen JH. A sensitive<br />
assay for the quantitative analysis of<br />
vinorelbine in mouse and human EDTA<br />
plasma by high-performance liquid<br />
chromatography coupled with electrospray<br />
tandem mass spectrometry. J Chromatogr B<br />
Analyt Technol Biomed Life Sci<br />
<strong>2008</strong>;868:102-9<br />
Enokizono J, Kusuhara H, Ose A,<br />
Schinkel AH, Sugiyama Y. Quantitative<br />
investigation of the role of breast cancer<br />
resistance protein (Bcrp/Abcg2) in limiting<br />
brain and testis penetration of xenobiotic<br />
compounds. Drug Metab Disp<br />
<strong>2008</strong>;36:995-1002<br />
Sparidans RW, Lagas JS, Schinkel AH,<br />
Schellens JHM, Beijnen JH. Liquid<br />
chromatography-tandem mass spectrometric<br />
assay for diclofenac and three primary<br />
metabolites in mouse plasma. J Chromatogr<br />
B Analyt Technol Biomed Life Sci<br />
<strong>2008</strong>;872:77-82<br />
Vlaming MLH, Pala Z, van Esch AM,<br />
Wagenaar E, van Tellingen O,<br />
de Waart DR, Oude Elferink RPJ,<br />
van de Wetering K, Schinkel AH. Impact<br />
of Mrp2 (Abcc2) and Mrp3 (Abcc3) on the<br />
in vivo elimination of methotrexate and its<br />
main metabolite 7-hydroxymethotrexate.<br />
Clin Cancer Res <strong>2008</strong>;14:8152-60<br />
Lagas JS, van der Kruijssen CMM,<br />
Beijnen JH, Schinkel AH. Transport of<br />
diclofenac by BCRP (ABCG2) and<br />
stimulation of MRP2- (ABCC2-) mediated<br />
drug transport by diclofenac and<br />
benzbromarone. Drug Metab Disp<br />
2009;37:129-36<br />
Van Waterschoot AB, Rooswinkel RW,<br />
Wagenaar E, van der Kruijssen CMM,<br />
van Herwaarden AE, Schinkel AH.<br />
Intestinal Cytochrome P450 3A plays an<br />
important role in the regulation of<br />
detoxifying systems in the liver. FASEB J.<br />
2009;23:224-31
72<br />
MOLECULAR BIOLOGY<br />
Group leader Lodewyk Wessels<br />
Lodewyk Wessels PhD Group leader<br />
Nicola Armstrong PhD Academic staff<br />
Michael Hauptmann PhD Academic staff<br />
Christiaan Klijn MSc PhD student<br />
Wouter Meuleman MSc PhD student<br />
Jeroen De Ridder MSc PhD student<br />
Martin Van Vliet MSc PhD student<br />
Jorma De Ronde PhD student<br />
Bram Gerritsen Bioinformatician<br />
Publications<br />
Van Uitert M, Meuleman W, Wessels LFA.<br />
Biclustering Sparse Binary Genomic Data.<br />
Journal of Computational Biology<br />
<strong>2008</strong>;15:1329-45<br />
Reyal F, Van Vliet MH, Armstrong NJ,<br />
Horlings HM, de Visser KE, Kok MJT,<br />
Teschendorff AE, Mook S, Van’t Veer L,<br />
Caldas C, Salmon RJ, Van de Vijver MJ,<br />
Wessels LFA. A comprehensive analysis of<br />
prognostic signatures reveals the high<br />
predictive capacity of Proliferation,<br />
Immune response and RNA splicing<br />
modules in breast cancer. Breast Cancer Res<br />
<strong>2008</strong>;10:R93<br />
Van Vliet MH, Reyal F, Horlings HM,<br />
van de Vijver MJ, Reinders MJT,<br />
Wessels LFA. Pooling breast cancer<br />
datasets has a synergetic effect on<br />
classification performance and improves<br />
signature stability. BMC Genomics<br />
<strong>2008</strong>;9:375<br />
Knijnenburg TA, Wessels LFA and<br />
Reinders MJT Combinatorial influence of<br />
environmental parameters on transcription<br />
factor activity. Bioinformatics.<br />
<strong>2008</strong>;24:i172-81<br />
Klijn C, Holstege H, de Ridder J, Liu X,<br />
Reinders MJT, Jonkers J., Wessels LFA.<br />
Identification of cancer genes using a<br />
statistical framework for multi-experiment<br />
analysis of non-discretized array CGH<br />
data. Nucleic Acids Res <strong>2008</strong>;36:e13<br />
BIOINFORMATICS AND STATISTICS<br />
Our group focuses on developing novel computational approaches which exploit a<br />
wide variety of (cancer) biological and epidemiological questions. These include, but<br />
are not limited to, stratifying tumors into groups with distinct and homogeneous<br />
outcome and therapy response, elucidation the function of genes – and especially<br />
pathways – involved in tumorigenesis and understanding molecular regulatory<br />
mechanisms, in general. Below a number of exemplary projects are presented in<br />
more detail.<br />
Extracting oncogenes and oncogenic pathways from insertional mutagenesis<br />
screens To find oncogenic lesions which are collaborating events in tumorigenesis,<br />
we developed an approach to detect the significant occurrences of multiple<br />
independent insertions within one tumor. This approach employs a predefined<br />
significance level while controlling the family-wise error. It operates at multiple<br />
biologically relevant scales, providing insight in the behavior of co-occurrences across<br />
these scales. This framework was augmented by an approach which reprioritizes the<br />
co-occurrences to compensate for co-occurrences with frequently hit targets. An<br />
approach was also developed to detect co-occurrences between single gene inserts<br />
and inserts associated with a family of genes. This provides a powerful technique to<br />
detect gene families where the family members can substitute for each other during<br />
oncogenesis. The results were validated by comparing these to co-occurrences of<br />
genes obtained through literature mining of published abstracts and expert<br />
evaluation. We have extended this approach to capture more complex interactions<br />
between insertion loci. More specifically, we detect simple logic circuits modeling<br />
combinations of co-occurring and mutually exclusive insertions that accurately<br />
predict the expression pattern of downstream targets.<br />
Detecting cancer genes in aCGH data derived from tumor panels We have<br />
developed a new approach which inputs non-discretized array comparative genomic<br />
hybridization (aCGH) data to identify regions that are significantly aberrant across an<br />
entire tumor set. In an analysis of 89 human breast tumors, our method showed<br />
enrichment for known cancer genes in the detected regions and identified<br />
aberrations that are strongly associated with breast cancer subtypes and clinical<br />
parameters. Furthermore, we identified 18 recurrent aberrant regions in a new<br />
dataset of 19 p53-deficient mouse mammary tumors. These regions, combined with<br />
gene expression microarray data, point to known cancer genes and novel candidate<br />
cancer genes. This framework for aberrant region detection has been extended to also<br />
capture significantly co-occurring and mutually exclusive aberrations. Application of<br />
this approach to a large cell line panel revealed a number of known as well as<br />
interesting new oncogenic interactions.<br />
Functional analysis of expression signatures on a large breast cancer<br />
compendium In order to better understand the relative lack of overlap between<br />
breast cancer prognostic signatures that are seemingly similar in terms of predictive<br />
capacity, we performed a comprehensive analysis of the performance of nine gene<br />
expression signatures on seven different breast cancer datasets. To minimize interdataset<br />
variability, we only employed a collection of 947 breast cancer samples<br />
hybridized on the Affymetrix platform, collected the raw data, and pre-processed and<br />
normalized these samples jointly. For 44% of these patients all nine classifiers are<br />
concordant. In the group where all signatures predict a good outcome, only seven<br />
percent of the tumors recur. In the group where all signatures predict a poor<br />
outcome, 35% of the tumors recur, indicating a large degree of heterogeneity in this<br />
group that is not captured by gene expression. The percentage of ‘poor outcome’<br />
events relative to the number of ‘poor outcome’ calls is correlated, indicating a benefit<br />
when combining the separate classifiers. To better characterize the functional<br />
processes associated with the largely non-overlapping signatures, we created an<br />
enlarged signature for each of them by including genes that are highly correlated<br />
with the genes in the signature. Identification of functional gene sets that are overrepresented<br />
in the intersection of the enlarged signatures revealed a common core of<br />
ten different functional modules. The identified modules include: Immune response,
KRAS, proliferation, RNA splicing, Rb pathway, sterol biosynthesis, extra-cellular<br />
matrix constituent, focal adhesion, negative regulation of proliferation and apoptosis.<br />
All modules are associated with survival in an independent test set. The combination<br />
of the RNA splicing and immune response modules resulted in a high prognostic<br />
performance on an independent validation set.<br />
Classifier signature stability Pooling the abovementioned collection of 947 breast<br />
cancer samples from seven studies would limit the effect of any sampling biases<br />
from the individual datasets, and alleviate small sample size problems. We showed<br />
that pooling datasets and deriving a classifier from the pooled data leads to more<br />
accurate classifiers. More specifically, when starting with six breast cancer datasets,<br />
and pooling pairs of datasets, we observed a synergetic effect on the classification<br />
performance in 73% of the cases. By increasing the number of datasets pooled (from<br />
three to six), we observed a significant association between the number of datasets<br />
that are pooled, the validation performance and the number of genes selected.<br />
Functional enrichment of the signatures derived from pooled datasets indicates high<br />
significance for the proliferation categories. Microtubule-associated pathways only<br />
emerge when pooling more than five datasets, while a plethora of gene sets that are<br />
only significant in signatures derived from single datasets, were no longer significant<br />
in pooled variants, thus identifying those as likely false positives.<br />
Computational Analysis of Lamina Associated Domains (LADs) LADs are very<br />
large (0.1-10Mb), sharply defined segments of the human genome that are associated<br />
with the lamina. These domains provide insight in how the genome is organized<br />
within the cell nucleus. On a more general level it is possible that LADs encode<br />
mechanisms involved in, e.g., higher level gene regulation and/or higher order<br />
packaging of the DNA. LADs are demarcated by specific sequence elements,<br />
indicating that the folding of DNA is in part hard-wired in the genome. Some of the<br />
more prevalent elements identified at the borders of LADs include an abundance of<br />
CpG-islands, particular transcription factor binding site motifs and promoters of<br />
specifically oriented genes. The sequence elements found so far are, unfortunately,<br />
insufficient to fully characterize LADs. Initial experiments show that LAD structures<br />
change across cell types. We aim to employ machine learning approaches to integrate<br />
various data sources (lamin binding profiles, expression data, genomic features and<br />
chromatin features) to formulate testable hypotheses regarding mechanistic and<br />
functional properties of LADs.<br />
Flexible statistical modeling of exposure-time-response relationships An<br />
adequate depiction of exposure-time-response relationships is important in assessing<br />
public health implications of an occupational or environmental exposure. Recent<br />
advances have focused on flexible modeling of the overall shape of latency. However,<br />
methods are needed to allow for varying shapes of latency under different exposure<br />
profiles. We developed a tensor product spline model for describing exposureresponse<br />
relationships for protracted time-dependent exposure histories in<br />
epidemiologic studies. The methods use flexible multi-dimensional techniques to<br />
jointly model age, latency and exposure-response effects. In analyzing lung cancer<br />
mortality and exposure to radioactive radon gas based on data from the Colorado<br />
Plateau Uranium Miners cohort, a model that allows for varying exposure-dependent<br />
latency shapes is found to be superior to models that only allowed for an overall<br />
latency curve. Specifically, the model suggests that, at low exposure levels risk<br />
increased at short latencies followed by a slow decline for longer latency periods.<br />
On the other hand, risk was higher but did not change much by latency for higher<br />
exposure levels. The proposed methodology has the advantage of allowing for latency<br />
functions that vary by exposure levels and, conversely, exposure-response<br />
relationships that are influenced by the latency structure.<br />
73<br />
MOLECULAR BIOLOGY<br />
Publications (continued)<br />
Meuleman W, Engwegen JYMN,<br />
Gast M-CW, Beijnen JH, Reinders MJT<br />
and Wessels LFA. Comparison of<br />
normalisation methods for Surface-<br />
Enhanced Laser Desorption and Ionisation<br />
Time-Of-Flight Mass Spectrometry data<br />
BMC Bioinformatics <strong>2008</strong>;9:88<br />
Weigelt B, Horlings HM, Kreike B,<br />
Hayes MM, Hauptmann M, Wessels LFA,<br />
de Jong D, van de Vijver MJ,<br />
van ‘t Veer LJ, and Peterse JL. Refinement<br />
of breast cancer classification by molecular<br />
characterisation of histological special types.<br />
J Pathol <strong>2008</strong>;216:141-50<br />
Berhane K, Hauptmann M, Langholz B.<br />
Using tensor product splines in modeling<br />
exposure-time-response relationships:<br />
application to the Colorado Plateau<br />
uranium miners cohort. Stat Med<br />
<strong>2008</strong>;27:5484-96<br />
Sigurdson AJ, Ha M, Hauptmann M,<br />
Bhatti P, Sram R, Beskid O, Tawn EJ,<br />
Whitehouse CA, Lindholm C, Nakano M,<br />
Kodama Y, Nakamura N, Vorobstova I,<br />
Oestreicher U, Stephan G, Yong LC,<br />
Bauchinger M, Schmid E, Chung HW,<br />
Darroudi F, Roy L, Voisin P, Barquinero JF,<br />
Livingston G, Blakey D, Hayata I, Zhang W,<br />
Wang C, Bennett LM, Littlefield LG,<br />
Edwards AA, Kleinerman RA, Tucker JD.<br />
International study of factors affecting<br />
human chromosome translocations. Mutat<br />
Res <strong>2008</strong>;652:112-21<br />
Hooning MJ, Aleman BMP, Hauptmann M,<br />
Baaijens MH, Klijn JG, Noyon R, Stovall M,<br />
Van Leeuwen FE. Roles of radiotherapy<br />
and chemotherapy in the development of<br />
contralateral breast cancer. J Clin Oncol<br />
(in press)<br />
Holm C, Kok M, Michalides R, Fles R,<br />
Koornstra RH, Van ‘t Veer LJ, Neefjes J,<br />
Peterse JL, Hauptmann M, Stal O,<br />
Landberg G, Linn SC. Phosphorylation of<br />
the estrogen receptor alpha at serine 305<br />
and prediction of tamoxifen response in<br />
breast cancer. J Pathol (in press)
74<br />
MOLECULAR BIOLOGY<br />
Group leader Rob Wolthuis<br />
Rob Wolthuis Group Leader<br />
Linda Clijsters PhD student<br />
Wouter Van Zon PhD student<br />
Erik Voets PhD student<br />
Janneke Ogink Technical staff<br />
Bas Ter Riet Technical staff<br />
Publications<br />
Wolthuis R, Clay-Farrace L, van Zon W,<br />
Yekezare M, Koop L, Ogink J, Medema R<br />
and Pines J. (<strong>2008</strong>) Cdc20 and Cks Direct<br />
the Spindle Checkpoint-Independent<br />
Destruction of Cyclin A. Molecular Cell<br />
<strong>2008</strong>;30:290-302<br />
van Leuken RJ, Clijsters L, Wolthuis RMF<br />
(<strong>2008</strong>) To cell cycle, swing the APC/C.<br />
BBA Reviews in Cancer <strong>2008</strong>;1786:49-59<br />
van Leuken RJ, Luna-Vargas MP,<br />
Sixma TK, Wolthuis RMF and Medema RH<br />
Usp39 is essential for mitotic spindle<br />
checkpoint integrity and controls<br />
mRNA-levels of Aurora B. Cell Cycle<br />
<strong>2008</strong>;17:2710-19<br />
Lindqvist A, van Zon W,<br />
Karlsson Rosenthal C, Wolthuis RMF.<br />
(2007) Cyclin B1-Cdk1 activation continues<br />
after centrosome separation to control<br />
mitotic progression. PLoS Biology<br />
2007;5:e123<br />
REGULATION OF MITOSIS IN NORMAL AND CANCER CELLS<br />
In our group we aim to understand how mitosis works in both normal and cancer<br />
cells, by using a combinatorial approach of biochemistry, molecular genetics in<br />
human cells, and dedicated live-cell fluorescence imaging. We are focussing on two<br />
problems that cells face at the end of the cell cycle: a) how to enter mitosis from G2<br />
phase and b) how to successfully exit mitosis and divide. Equally important is how<br />
cells respond to insults or mistakes in G2 or mitosis.<br />
Mitosis: decisive finish of the cell cycle Cells duplicate their chromosomes and<br />
increase their size during the S and G2 phases. Mitosis forms the spectacular end of<br />
the cell cycle when sister chromatids are irreversibly separated and two new cells are<br />
formed. Cells should delay mitotic entry under conditions that are unfavourable for<br />
safe and successful division, such as after DNA damage, or when cells are too small.<br />
The decision to enter mitosis is controlled by signalling pathways called the G2<br />
checkpoints that direct the activation of the principal mitotic kinase, Cyclin B1-Cdk1.<br />
Once cells are in mitosis, they need to ensure that all the duplicated chromosomes<br />
are properly attached to the mitotic spindle. This is safe-guarded by the Mitotic<br />
Spindle Checkpoint, which works by inhibiting another key enzyme of our interest, the<br />
Anaphase-Promoting Complex or Cyclososme (APC/C). Once active, the APC/C triggers<br />
destruction of regulatory proteins, to initiate sister chromatid separation and mitotic<br />
exit. Progression through mitosis needs to be swift to prevent DNA damage as a<br />
result of the hyper-condensed state of the chromosomes, as well to avoid cellular<br />
damage caused by transcriptional and translational silencing during mitosis. Rapid<br />
dynamics are thus critical to mitosis. This also makes mitosis the most exciting stage<br />
of the cell cycle, yet the most difficult to study by biochemical means.<br />
Getting into Mitosis: look before you leap The transition from G2 to mitosis is<br />
catalyzed by activation of Cyclin B1-Cdk1. Because Cdk1 levels remain fairly constant<br />
throughout the cell cycle, and are in excess over Cyclin, all Cyclin B1 that is newly<br />
synthesized in G2 phase binds Cdk1. At this stage Cyclin B1-bound Cdk1 is kept<br />
inactive by phosphorylation of Thr14 and Tyr15, resulting in slow accumulation of<br />
inactive Cyclin B1-Cdk1 complexes. In Cyclin B1 immunoprecipitates of synchronized<br />
cells, Cdk1 activity rises very gradually in time, reaching no more than 25% of its<br />
maximum activity at the end of G2. The Cdc25 phosphatases are activated by the<br />
slowly rising Cyclin B1-Cdk1 activity, which enhances Cyclin B1-Cdk1 activation.<br />
In contrast, the inactivating kinases Wee1 and Myt1 are inactivated by rising Cyclin<br />
B1-Cdk1 activity. The result is a double positive-feedback loop leading to steep<br />
activation of Cyclin B1-Cdk1. Only at the very end of G2 this auto-catalyzing feed-back<br />
loop is initiated and cells become committed to mitosis. The protein levels,<br />
intracellular localisations and intrinsic activities of the Wee1/Myt1 inhibitory kinases<br />
and Cdc25 activating phosphatases which control Cyclin B1-Cdk1 are intensely<br />
regulated. We aim to create further insights into the mechanisms that trigger abrupt<br />
activation in late G2 and the parameters of an ‘activation threshold’ for mitosis<br />
(figure 7). Recently, in collaboration with Arne Lindqvist and Christina Karlsson-<br />
Rosenthal, we used a novel assay to monitor the activation of Cyclin B1-Cdk1 in single<br />
cells. We discovered that human Cyclin B1-Cdk1 activity most likely has a ‘bi-stable’<br />
response curve in vivo. Apparently, Cyclin B1-Cdk1 activation develops in a non-linear<br />
fashion after the activation threshold. Similar models are required to understand the<br />
threshold for mitotic entry and progression, either under normal conditions or after<br />
cellular insults in G2. By determining these conditions in both normal and cancer<br />
cells, we aim to create useful models for Cyclin B1-Cdk1 control and understand the<br />
effects of interfering with Cyclin B1-Cdk1 activity as an approach for cancer therapy.
Figure 6: Requirement of Cdk1 for Mitotic Entry and Mitotic Progression in Human Cells. (A) Cdk1<br />
knock-down cells were synchronized in G2/M and mitotic cells were isolated. Separated G2 and mitotic<br />
pools were analyzed for Cdk1 expression by Western blotting. The percentage of remaining Cdk1 protein is<br />
indicated. (B) Mitotic cells were lysed (lanes 1 and 2) or released from nocodazole and incubated in fresh<br />
medium for 3 h, recollected, and lysed (lanes 3 and 4). Differences in mitotic phosphorylation shift of APC3<br />
and Cdc25C, depending on the Cdk1 levels, are shown (lanes 1 and 2). (C) The impaired phosphorylation<br />
of APC3 in Cdk1-attenuated mitotic cells (lane 1) was rescued by co-expressed Cdk1-YFP with a silent<br />
mutation in the RNAi targeting region (lane 2). (D) Metaphase duration in Cdk1 RNAi cells (right) or<br />
Cdk1 RNAi cells rescued by co-expression of non–RNAi-sensitive Cdk1-YFP (left). (E) Time-lapse<br />
microscopy analysis of mitotic progression after entry with normal or impaired Cdk1 levels. Bottom panels<br />
are consecutive images of tubulin-YFP in a pS-control cell in mitosis; top panels show delayed chromosome<br />
alignment (frames 2 and 3) and stalled metaphase (frames 4–6) after Cdk1 shRNA. From: Lindqvist A,<br />
van Zon W, Karlsson Rosenthal C, Wolthuis RMF Cyclin B1-Cdk1 activation continues after centrosome<br />
separation to control mitotic progression. PLoS Biology 2007;5:e123.<br />
Mitotic Exit: coordination of events by protein destruction Mitotic entry<br />
requires accumulation of regulatory proteins, such as the mitotic Cyclins. In contrast,<br />
these proteins must be destroyed at distinct, sequential time points in mitosis. A<br />
multi-subunit ubiquitin ligase –the APC/C- ubiquitinates these mitotic proteins at<br />
highly defined mitotic time points, which initiates their proteolysis by the 26S<br />
proteasome. Sequential degradation of for instance Cyclin A, Cyclin B1 and Securin<br />
coordinates cell division with segregation of sister chromatids. Therefore, precise<br />
regulation of protein destruction by the APC/C is essential to guarantee cell viability<br />
and genomic integrity.<br />
The question remains how the APC/C recognizes a critical substrate at the right<br />
time. To address this important issue in human cells, we are using a combination of<br />
gene silencing, biochemistry, in vitro ubiquitination assays, and live cell imaging to<br />
identify regulation of the APC/C activation (figure 7). Our recent work revealed<br />
unexpected roles for the potentially oncogenic Cks protein family in directing Cdc20dependent<br />
destruction of Cyclin A, and in coordinating the concurrent destruction of<br />
Cyclin B1 and Securin. We are using these new findings to search for roles of<br />
individual APC/C subunits, phosphorylation events as well as the intracellular<br />
localization of APC/C-complexes, in controlling mitosis. Furthermore, we think that<br />
depending on the way the APC/C is inhibited, distinct molecular programs may be<br />
activated which affect the cellular responses to a prolonged arrest in mitosis.<br />
The next research goal is to understand how oncogenic mutations may impinge on<br />
critical steps in mitotic entry, mitotic progression and mitotic exit, but also how they<br />
determine the way cells respond to errors in mitosis, or to anti-mitotic drugs. It is<br />
anticipated that answers to these questions could create exciting opportunities to<br />
improve anti-cancer therapies.<br />
75<br />
MOLECULAR BIOLOGY<br />
Figure 7: Measuring APC/C activity by<br />
time-lapse fluorescence microscopy of Cyclin<br />
B1 destruction. A plasmid encoding for<br />
fluorescent Cyclin B1 was injected into G2phase<br />
cells, a few hours before they entered<br />
mitosis. The top panel shows a cell<br />
undergoing mitotic division, followed over<br />
time by differential interference contrast<br />
(DIC). The bottom panel shows the<br />
localisation and quantitative levels of Cyclin<br />
B1 in the fluorescence channel of the same<br />
cells. The cell undergoing division rapidly<br />
degrades Cyclin B1, as measured by a<br />
decrease in fluorescence signal. This assay<br />
allows the quantitation of ubiquitindependent<br />
protein destruction in live cells<br />
undergoing division and was first<br />
established in the laboratory of Jon Pines,<br />
Gurdon Institute, Cambridge, UK.
76<br />
MOLECULAR CARCINOGENESIS<br />
Division head, group leader René Bernards<br />
René Bernards PhD Group leader<br />
Katrien Berns PhD Post-doc<br />
Annette Dirac PhD Post-doc<br />
Michael Hölzel MD PhD Post-doc<br />
Sidong Huang PhD Post-doc<br />
Rianne Oosterkamp MD Clinical fellow<br />
Miguel Rubio PhD Post-doc<br />
Linda Smit PhD Post-doc<br />
Mirjam Epping MSc PhD student<br />
Ernst-Jan Geutjes MSc PhD student<br />
Guus Heynen MSc PhD student<br />
Roderik Kortlever MSc PhD student<br />
Jasper Mullenders Msc PhD student<br />
Miranda van Dongen Technical staff<br />
Marielle Hijmans MSc Technical staff<br />
Mandy Madiredjo Technical staff<br />
Publications<br />
Bernards R. Cancer: Entangled pathways.<br />
Nature <strong>2008</strong>;455:479-80<br />
Van ‘t Veer LJ, Bernards R. Enabling<br />
personalized cancer medicine through<br />
analysis of gene-expression patterns. Nature<br />
<strong>2008</strong>;452:564-70<br />
Eichhorn PJ, Gili M, Scaltriti M, Serra V,<br />
Guzman M, Nijkamp W, Beijersbergen RL,<br />
Valero V, Seoane J, Bernards R, Baselga J.<br />
Phosphatidylinositol 3-kinase<br />
hyperactivation results in lapatinib<br />
resistance that is reversed by the mTOR/<br />
phosphatidylinositol 3-kinase inhibitor<br />
NVP-BEZ235. Cancer research<br />
<strong>2008</strong>;68:9221-30<br />
Epping MT, Bernards R. Molecular basis of<br />
the anti-cancer effects of histone deacetylase<br />
inhibitors. The international journal of<br />
biochemistry & cell biology 2009;41:16-20<br />
Epping MT, Hart AA, Glas AM,<br />
Krijgsman O, Bernards R. PRAME<br />
expression and clinical outcome of breast<br />
cancer. British journal of cancer<br />
<strong>2008</strong>;99:398-403<br />
DIVISION OF MOLECULAR<br />
CARCINOGENESIS<br />
FUNCTIONAL GENOMICS<br />
My group uses functional genomics technologies to find novel cancer-relevant genes<br />
and to identify mechanisms of resistance to cancer drugs. We use both highthroughput<br />
gain-of-function genetic screens and RNA interference-based loss-offunction<br />
genetic screens to achieve these goals.<br />
Identification of mechanisms of drug resistance Unresponsiveness to therapy is<br />
a recurring problem in the treatment of cancer. It is therefore important to identify<br />
the molecular pathways that contribute to unresponsiveness to cancer therapeutics.<br />
We use both gain-of-function and loss-of-function genetic screens to identify genes<br />
that contribute to drug resistance, in particular to new classes of targeted<br />
therapeutics. For the loss-of-function genetic screens, we use our vast collection of<br />
RNA interference vectors, which at present consists of 55,000 shRNA vectors that<br />
together target over 23,000 mouse and human genes for suppression. We use this<br />
vector set in conjunction with an efficient way to screen shRNA libraries: siRNA bar<br />
code screening (in collaboration with Roderick Beijersbergen, division of Molecular<br />
Carcinogenesis, see below). In the past year, we have focused on the identification of<br />
genes whose suppression contributes to resistance to retinoic acid (RA)-based<br />
therapies. Retinoic acid is a key morphogen in early vertebrate development and<br />
tissue regeneration, which induces growth arrest, differentiation and apoptosis. In<br />
the clinic, retinoids are increasingly being used for the treatment of cancer, including<br />
leukemias and neuroblastoma. However, the molecular mechanisms of resistance to<br />
retinoid-based therapies remain obscure.<br />
To address this, we performed a large-scale RNA interference screen with a<br />
30,000-vector mouse shRNA library to identify genes whose suppression confers<br />
resistance to RA-mediated differentiation in mouse F9 teratocarcinoma cells. To date,<br />
we have validated two genes whose suppression confers resistance to RA-induced<br />
differentiation and growth arrest in these F9 cells: ZNF423 and CtBP2 (figure 1). For<br />
ZNF423, a large zinc finger protein, we have shown that it binds directly to the RAR/<br />
RXR receptor heterodimer in chromatin and that this binding is required for receptor<br />
activation by RA. ZNF423 knockdown also prevents differentiation of neuroblastoma<br />
cell lines in vitro. Conversely, ectopic expression of ZNF423 enhances RAdifferentiation<br />
in human neuroblastoma cells. Significantly, we found that ZNF423 is<br />
a marker of poor outcome in two independent cohorts of human neuroblastoma<br />
tumor samples. Our data are consistent with a model in which high levels of ZNF423<br />
expression allow neuroblastoma cells to respond to differentiation-inducing signals,<br />
rending such cells less malignant than cells having low levels of ZNF423, which are<br />
more resistant to such signals. We have recently performed an additional loss of<br />
function genetic screen in human neuroblastoma to find additional genes whose<br />
suppression confers resistance to RA. Unexpectedly, we found here that suppression<br />
of the tumor suppressor gene NF1 causes resistance to RA-induced differentiation.<br />
This suggests that RAS signaling can modify responses of neuroblastoma cells to<br />
retinoids. We are currently asking if NF1 is mutated in human neuroblastoma and<br />
how loss of NF1 contributes to RA resistance.<br />
We are also using a similar approach to identify mechanisms of resistance to drugs<br />
that act downstream of HER2 in breast cancer: inhibitors of PIK3CA and mTOR.<br />
Drugs that inhibit these kinases are currently in early stage clinical trials. Uncovering<br />
mechanisms of resistance against these drugs may help identify those patients that<br />
benefit most from these drugs. We have identified and validated one gene whose<br />
suppression can cause resistance to the mTOR inhibitor rapamycin in breast cancer<br />
cell lines. The mechanism leading to resistance to rapamycin is under investigation.
Figure 1: Znf423 is required for RA-induced differentiation in mouse ES cells.<br />
Down-regulation of Znf423 by RNAi in mouse Embryo Stem (ES) cells confers resistance to RA-induced<br />
differentiation. ES cells expressing two different shRNAs against Znf423 or GFP (control) were grown in<br />
the absence or presence of exogenous RA (1 µM) for 1 week, after which cells were fixed, stained for<br />
alkaline phosphatase (AP) and photographed. The alkaline phosphatase stain represents a marker of the<br />
undifferentiated state of ES cells, demonstrating that knockdown of Znf423 blocks differentiation.<br />
Functional studies on de-ubiquitinating enzymes My laboratory has established a<br />
collection of shRNA vectors that target deubiquitinating enzymes (DUBs) for<br />
suppression. Currently, we have a set to some 100 DUB shRNA vectors and a similar<br />
set of siRNA reagents. We have used these RNA interference reagents to identify<br />
novel DUBs in cancer relevant pathways. In a screen aimed at identifying DUBs that<br />
can remove ubiquitin moieties from the androgen receptor, we identified USP26.<br />
Remarkably, mutations in USP26 have been reported in patients suffering from male<br />
infertility syndromes, underscoring the potential role of USP26 in androgen receptor<br />
signaling.<br />
Using a single-well siRNA screening approach, we have identified DUBs whose<br />
suppression either inhibits or enhances TNF-alpha mediated apoptosis. We are<br />
currently studying how these DUBs affect signaling through their respective<br />
pathways.<br />
77<br />
MOLECULAR CARCINOGENESIS<br />
Publications (continued)<br />
Herold S, Hock A, Herkert B, Berns K,<br />
Mullenders J, Beijersbergen R, Bernards R,<br />
Eilers M. Miz1 and HectH9 regulate the<br />
stability of the checkpoint protein, TopBP1.<br />
Embo J <strong>2008</strong>;27:2851-61<br />
Kortlever RM, Nijwening JH, Bernards R.<br />
Transforming growth factor-beta requires its<br />
target plasminogen activator inhibitor-1 for<br />
cytostatic activity. J Biol Chem<br />
<strong>2008</strong>;283:24308-13<br />
Kortlever RM, Brummelkamp TR,<br />
van Meeteren LA, Moolenaar WH,<br />
Bernards R. Suppression of the p53dependent<br />
replicative senescence response by<br />
lysophosphatidic acid signaling. Mol Cancer<br />
Res <strong>2008</strong>;6:1452-60<br />
Stemke-Hale K, Gonzalez-Angulo AM,<br />
Lluch A, Neve RM, Kuo WL, Davies M,<br />
Carey M, Hu Z, Guan Y, Sahin A,<br />
Symmans WF, Pusztai L, Nolden LK,<br />
Horlings H, Berns K, Hung MC,<br />
van de Vijver MJ, Valero V, Gray JW,<br />
Bernards R, Mills GB, Hennessy BT. An<br />
integrative genomic and proteomic analysis<br />
of PIK3CA, PTEN, and AKT mutations in<br />
breast cancer. Cancer research<br />
<strong>2008</strong>;68:6084-91<br />
Wittner BS, Sgroi DC, Ryan PD,<br />
Bruinsma TJ, Glas AM, Male A, Dahiya S,<br />
Habin K, Bernards R, Haber DA,<br />
Van ‘t Veer LJ, Ramaswamy S. Analysis of<br />
the MammaPrint breast cancer assay in a<br />
predominantly postmenopausal cohort. Clin<br />
Cancer Res <strong>2008</strong>;14:2988-93<br />
Bernards R. Reaction to American Society<br />
of Clinical Oncology 2007 update of<br />
recommendations for the use of tumor<br />
markers in breast cancer. J Clin Oncol<br />
<strong>2008</strong>;26:2057-8<br />
Rutgers EJ, Pusztai L, Bernards R. Are<br />
short-term or long-term recurrence rates<br />
more important in breast cancer screening?<br />
Annals of internal medicine <strong>2008</strong>;149:357<br />
Eichhorn PJ, Creyghton MP, Bernards R.<br />
Protein phosphatase 2A regulatory subunits<br />
and cancer. Biochim Biophys Acta <strong>2008</strong> (in<br />
press)<br />
Bernards R. RNAi delivers insight into liver<br />
cancer. Cell <strong>2008</strong>;135:793-5
78<br />
MOLECULAR CARCINOGENESIS<br />
Group leader Roderick Beijersbergen<br />
Roderick Beijersbergen PhD Group leader<br />
David Egan PhD Post-doc<br />
Armida Fabius MSc PhD student<br />
Johan Kuiken MSc PhD student<br />
Jeroen Nijwening MSc PhD student<br />
Cor Lieftink MSc Bioinformatician<br />
Bram Gerritsen MSc Bioinformatician<br />
Wouter Nijkamp Technical staff<br />
THE RNAI STRATEGY IN TARGET DISCOVERY<br />
The research in our lab continues to evolve around the identification of novel drug<br />
targets in cancer using large scale cell-based screening technologies. The goal of our<br />
work is to identify essential components in disease-related pathways that can be<br />
explored as drug targets in cancer therapy and to identify potential mechanisms of<br />
resistance and to achieve a more complete understanding of drug action, thereby<br />
enabling more rational decisions on drug application.<br />
Synthetic lethal interactions For the effective treatment of cancer, there is a great<br />
need for drugs that specifically target tumor cells without affecting normal cells. With<br />
the use of RNA interference, we have begun to explore synthetic lethal phenotypes in<br />
mammalian cells. Synthetic lethal phenotypes are defined as a combination of two<br />
mutations, which by themselves are non-lethal, but together result in a lethal<br />
phenotype. These interactions can lead to the identification of novel cancer drug<br />
targets that are only cytotoxic in the background of a tumor specific alteration and<br />
represent ‘genotype specific’ drugs. We have generated a panel of isogenic cell lines<br />
derived from primary human BJ fibroblasts that contain single or multiple defined<br />
genetic alterations that together are required for tumorigenic transformation. These<br />
genetic alterations include among others, loss-of-p53, activation of RAS or activation<br />
of PI3K. These cell lines have been used in high throughput single well assays in<br />
combination with large siRNA collections targeting more than 8000 genes, to<br />
identify siRNAs that result in enhanced lethality only in the background of these<br />
tumor specific genetic alterations. We have completed these screens and have<br />
identified several siRNAs whose lethality is dependent on a specific genotype<br />
(figure 2). We are in the process of extensive validation and elucidation of the<br />
mechanisms underlying these phenotypes.<br />
Figure 2: Synthetic lethal interaction with loss of p53<br />
Scatter plot representing the results of inactivation of 8000 genes by siRNA in BJ cell in the presence<br />
(X-axis) or absence (Y-axis) of p53 expression and a low dose of cis-platin. Each dot represents 1 gene<br />
targeted by 4 individual siRNAs and indicates the relative effect on cell viability in each of the 2 different<br />
genotypes. Depicted in dark grey is a panel of control siRNAs, depicted in light grey are potential synthetic<br />
lethal interactions with loss-of-p53. In addition to siRNAs that specifically decrease viability in p53 null<br />
cells, we have also identified siRNAs causing enhanced proliferation only in the presence of p53 expression.<br />
Bar code screens The bar code screening technology has been developed to monitor<br />
the effect of the inactivation of gene expression in large complex populations of cells<br />
(figure 3). In this way the effect of gene inactivation on the phenotype of individual<br />
cells can be followed. The design of the <strong>NKI</strong> shRNA libraries allows for the<br />
identification of individual shRNA vectors in a large pool of shRNA vector containing<br />
cells. Each hairpin vector contains a unique 19-mer sequence (“barcode”) designed to<br />
target one specific cellular gene. This 19-mer sequence can be used to determine the<br />
relative abundance of a shRNA vector by hybridization to DNA micro-arrays
containing the 19-mer barcode sequences. By using this technique, large complex<br />
populations of shRNA expressing cells can be monitored for the abundance of each<br />
individual shRNA expressing cell (see figure 3) reflecting the effect of specific gene<br />
inactivation on cellular behavior. The bar code screening technology facilitates the<br />
identification of genes that contribute to drug resistance in cancer cells, in particular<br />
to new classes of targeted therapeutics. An example of this strength is the recent<br />
finding that PI3 kinase activation contributes to resistance to lapatinib treatment. At<br />
this moment several novel cancer drugs, in early stages of clinical development, are<br />
used with our bar code screening technology to identify genes that either enhance the<br />
drug’s effect or result in resistance to drug treatment.<br />
Figure 3: Bar code screen technology<br />
Large populations of shRNA expressing cells are analyzed in parallel using molecular barcode screens.<br />
Each shRNA vector is tagged with a unique ‘barcode’ sequence (the 19-mer targeting sequence) that is used<br />
to monitor the representation of each vector in a complex population by hybridizing to an oligonucleotide<br />
microarray containing the 19-mer barcode sequences. When a population of cells is subjected to a selective<br />
pressure, there will be selection for those cells that express a shRNA that modulates the cellular response. As<br />
a result the representation of individual shRNA constructs is expected to change. This change in the<br />
relative abundance of individual shRNA vectors can be determined by comparison of a control population<br />
versus the population exposed to selection. The signal intensity of shRNAs that confer resistance to the<br />
selection will increase compared to the control population.<br />
siRNA screens and High Content Imaging Over the last year we have expanded<br />
our capabilities to perform large scale single well siRNA screens. An example of<br />
these efforts is a screen for the identification of novel components of the TGFb<br />
signaling pathway using a TGFb responsive reporter controlling the expression of<br />
luciferase. We have identified several siRNAs that either increase or decrease TGFb<br />
signaling. These hits are used for follow-up experiments to elucidate their<br />
mechanism of action.<br />
79<br />
MOLECULAR CARCINOGENESIS<br />
Publications<br />
Eichhorn PJ, Gili M, Scaltriti M, Serra V,<br />
Guzman M, Nijkamp W, Beijersbergen RL,<br />
Valero V, Seoane J, Bernards R, Baselga J.<br />
Phosphatidylinositol 3-kinase<br />
hyperactivation results in lapatinib<br />
resistance that is reversed by the mTOR/<br />
phosphatidylinositol 3-kinase inhibitor<br />
NVP-BEZ235. Cancer research<br />
<strong>2008</strong>;68:9221-30<br />
Herold S, Hock A, Herkert B, Berns K,<br />
Mullenders J, Beijersbergen R, Bernards R,<br />
Eilers M. Miz1 and HectH9 regulate the<br />
stability of the checkpoint protein, TopBP1.<br />
Embo J <strong>2008</strong>;27:2851-61<br />
OttoT, Horn S, Brockmann S, Eilers U,<br />
Schüttrumpf L, Popov N, Kenney A,<br />
Schulte JH, Beijersbergen RL,<br />
Christiansen H, Berwanger B, Eilers M.<br />
Stabilization of N-Myc is a critical function<br />
of Aurora-A in human neuroblastoma.<br />
Cancer Cell (in press)
80<br />
MOLECULAR GENETICS<br />
Division head, group leader Maarten Van Lohuizen<br />
Maarten Van Lohuizen PhD Group leader<br />
Elisabetta Citterio PhD Senior Post-doc<br />
Jean Bernard Beaudry PhD Post-doc<br />
Jaap Kool PhD Post-doc<br />
Karim Nacerdine PhD Post-doc<br />
Inka Pawlitzky PhD Post-doc<br />
Alexandra Pietersen PhD Post-doc<br />
Anke Sparmann PhD Post-doc<br />
Bas Tolhuis PhD Post-doc<br />
Bart Westerman PhD Post-doc<br />
Sophia Bruggeman MSc PhD student<br />
Panthea Taghavi MSc PhD student<br />
Joep Vissers MSc PhD student<br />
Marleen Blom Technical staff<br />
Paulien Cornelissen Technical staff<br />
Danielle Hulsman Technical staff<br />
Ellen Tanger Technical staff<br />
Hans Teunissen Technical staff<br />
Els Verhoeven Technical staff<br />
Huub van Vugt Technical staff<br />
Julian Puppe MD Visiting scientist<br />
Publications<br />
Terranova R, Yokobayashi S, Stadler MB,<br />
Otte AP, Van Lohuizen M, Orkin SH,<br />
Peters AH. Polycomb group proteins Ezh2<br />
and Rnf2 direct genomic contraction and<br />
imprinted repression in early mouse<br />
embryos. Dev Cell. <strong>2008</strong>:15:668-79<br />
Taghavi P, Verhoeven E, Jacobs JJ,<br />
Lambooij JP, Stortelers C, Tanger E,<br />
Moolenaar WH, Van Lohuizen M. In vitro<br />
genetic screen identifies a cooperative role<br />
for LPA signaling and c-Myc in cell<br />
transformation. Oncogene <strong>2008</strong>:27:6806-16<br />
Pietersen AM, Evers B, Prasad AA,<br />
Tanger E, Cornelissen-Steijger P, Jonkers J,<br />
Van Lohuizen M. Bmi1 regulates stem cells<br />
and proliferation and differentiation of<br />
committed cells in mammary epithelium.<br />
Curr Biol. <strong>2008</strong>:18:1094-9<br />
Boutsma E, Noback S, Van Lohuizen M.<br />
The Polycomb protein and E3 ubiquitin<br />
ligase Ring1B harbors an IRES in its highly<br />
conserved 5’ UTR. PLoS ONE <strong>2008</strong>:3:e2322<br />
DIVISION OF MOLECULAR GENETICS<br />
ROLE OF POLYCOMB-GROUP GENES IN TRANSCRIPTIONAL<br />
REPRESSION, STEM CELL FATE AND TUMORIGENESIS<br />
Our lab has a long-standing interest in epigenetic gene regulation dictated by<br />
chromatin modifications. We study the mechanism of transcriptional repression by<br />
Polycomb-group (Pc-G) protein complexes, and the effects of deregulation of Pc-G<br />
genes on development, Cell cycle control, cancer formation and stem cell<br />
maintenance. In addition, we are performing large-scale genetic screens in primary<br />
cells and in cancer-predisposed mice to identify cancer-relevant networks of<br />
oncogenes and tumor-suppressor genes. Model organisms comprise Mouse and<br />
Drosophila.<br />
Functional characterization of Pc-G protein complexes Repressive Pc-G proteins<br />
and the counteracting Trithorax-group (Trx-G) of nucleosome remodeling factors are<br />
involved in the maintenance of proper gene expression patterns during development<br />
at the level of chromatin structure. Pc-G protein complexes control large sets of genes<br />
including Hox gene clusters and the INK4a/ARF tumor suppressor locus. At least<br />
two biochemical distinct evolutionary highly conserved Pc-G protein complexes can<br />
be distinguished. The first (PRC2) contains EzH1/EzH2 (SET domain proteins acting<br />
as Histone H3 methylases), Su(z)12, Eed and histone deacetylases. The second large<br />
complex (PRC1) encompasses Bmi1/Mel18, M33/MPc2, Mph1/Mph2 and Ring1b/<br />
Ring1a together with other more loosely associated proteins is required throughout<br />
development. To study Pc-G, function we focus on representative members of PRC1<br />
and PRC2 in gain- and loss-of-function studies in mice and Drosophila. In genetic<br />
and biochemical experiments we identified the Bmi1/Ring1b heterodimer as an E3<br />
ubiquitin ligase for monoubiquitination of histone H2A (collaboration with Gretel<br />
Buchwald and Titia Sixma, Division of Biochemistry). This enzymatic activity is part<br />
of the core PRC1 complex and conditional Ring1b loss-of-function experiments<br />
indicate an essential role for maintenance of Pc-G repression in development and<br />
stem cell maintenance. In addition we are studying the function of the<br />
deubequitinating enzyme Usp3 that binds to mono-ubiquinated H2A and can<br />
remove this mark. A major unresolved question is where and how Pc-G complexes<br />
bind to chromatin. We have performed a genome-wide survey of where PRC1 and<br />
PRC2 complexes bind to the Drosophila genome using DamID profiling on cDNA<br />
and tilling arrays (collaboration with Bas van Steensel, Division of Gene Regulation).<br />
This highlighted binding of both PRC1 and PRC2 to distinct domains of 10-140 Kb<br />
containing ± 400 target genes. These comprise developmental regulators,<br />
transcriptional repressors as well as key stem cell signaling pathways such as Wnt,<br />
Hh, Notch and several new distinct functional categories. Currently we are<br />
investigating the possibility that these domains may interact in 3D nuclear space<br />
using optimized in vivo 4C (chromatin conformation capture on Chip).<br />
Connections between Pc-G gene repression, control of stem cell fate and<br />
cancer formation We originally identified Bmi1 as an oncogene that cooperates with<br />
cMyc in the induction of B and T-cell lymphomas in mice, underscoring the<br />
connection between deregulation of Pc-G repression and cancer. In contrast, Bmi1<br />
knockout mice show severe progressive proliferation defects and increased apoptosis<br />
of lymphoid and myeloid cells, resulting in severe lymphopenia. In addition, also<br />
primary embryo fibroblasts (MEFs) and neural precursors in the cerebellum of Bmi1<br />
knockouts show proliferation defects. We demonstrated that these defects are in part<br />
due to increased levels of the tumor suppressors p16INK4a and p19ARF, that are<br />
critical regulators of the RB and the p53 tumor suppressor pathways. As such, the<br />
INK4a/ARF locus acts as an important tumor-prevention mechanism in normal cells<br />
and stem/precursor cells. Using the well-established mammary fat pad<br />
transplantation model, we recently revealed the essential role of Bmi1 in mammary<br />
epithelial stem cells and precursors and ductal tree development (figure 1). Genetic<br />
studies showed that the proliferative defects but not the observed premature
Figure 1: Mammary fat pad transplantation assay.<br />
Ductal outgrowth is severely impaired upon<br />
transplantation of Bmi1-/- (KO) when compared to<br />
Bmi1+/- (Het). This suggests a severe mammary<br />
epithelial precursor (stem) cell defect in Bmi1<br />
deficient mice.<br />
differentiation upon loss of Bmi1 in mammary epithelial precursors is in part<br />
mediated via INK4a/ARF. Importantly, these studies revealed a dual role for Bmi1/<br />
Pc-G: controlling both proliferation and differentiation. A key characteristic of cancer<br />
cells is their unlimited self-renewal. In this respect, cancer cells resemble stem cells,<br />
and accumulating evidence suggests that many forms of cancer may indeed contain<br />
cells carrying stem cell markers. In studying the proliferation defects and progressive<br />
loss of cells in Bmi1 deficient mice we discovered that Bmi1 is required for<br />
proliferation and self-renewal of neural stem cells. Importantly, loss of the INK4a/<br />
ARF locus rescues the proliferation & renewal defects, indicating it also is a critical<br />
Pc-G target in neural stem cells. Using a transplantable Glioma model we recently<br />
demonstrated a critical role for Bmi1 in maintenance of brain tumor formation<br />
(figure 2). Interestingly, Bmi1 acts in this tumor setting in an Ink4a/ARFindependent<br />
manner on cell adhesion and migration. These results, together with<br />
the recently established role of Bmi1 in hemapoietic stem cells and leukaemic stem<br />
cells, suggest a common conserved role for Bmi1-containing Polycomb complexes in<br />
maintenance and expansion of stem cells or committed progenitors and in the<br />
pathogenesis of tumors originating from the neoplastic transformation of these cells.<br />
The possible broader relevance of these findings for human cancer is further<br />
underscored by the amplification of BMI1 in Mantle cell lymphomas and a subset of<br />
brain cancers and the overexpression of BMI1 in various tumor types including nonsmall<br />
cell lung cancer, breast cancer and liver cancer. Conditional transgenic- and<br />
knockout models are currently used to investigate the role of Pc-G genes in various<br />
tissue stem/progenitors and in solid cancers that develop in these tissues.<br />
In vivo genetic screens to identify new groups of collaborating oncogenes or<br />
tumor suppressors In close collaboration with Anton Berns (this division), Jos<br />
Jonkers (Division of Molecular Biology) and David Adams and Allen Bradley from<br />
the Sanger Centre (Hinxton, UK) we have developed high-throughput insertional<br />
mutagenesis techniques and are now extending and optimizing these types of<br />
screens to other cancer relevant models such as breast cancer. The power of this<br />
approach as a cancer gene discovery platform is highlighted by the first completed<br />
screens in hemapoietic tumors induced in wild type, p53 or p19Arf deficient mice.<br />
These screens yielded over 10.000 insertion sites implicating over 300 loci in<br />
tumorigenesis and uncovered new pathway-specific oncogenes and candidate tumorsuppressors.<br />
Cross species comparative analysis with a large array-CGH dataset of<br />
human cancer cell lines revealed both new and novel candidate oncogenes and<br />
tumor-suppressor genes. The role and mechanism of action of several of these new<br />
putative oncogenes or tumor suppressors, is under investigation using bone marrow<br />
or fetal liver hemapoietic cell-transplatation systems.<br />
Figure 2: Severely reduced Glioma formation of Bmi1-/- transformed astrocytes. Survival curves indicate<br />
that astrocytes oncogenically transformed by loss of INK4a/ARF and activation of EGF-receptor signaling<br />
rapidly form agressive gliomas whereas tumor formation is delayed upon transplantation of Bmi1-deficient<br />
transformed astrocytes orthotopically transplanted in the forebrain of recipient mice.<br />
81<br />
MOLECULAR GENETICS<br />
Publications (continued)<br />
Van der Stoop P, Boutsma EA, Hulsman D,<br />
Noback S, Heimerikx M, Kerkhoven RM,<br />
Voncken JW, Wessels LF, Van Lohuizen M.<br />
Ubiquitin E3 ligase Ring1b/Rnf2 of<br />
polycomb repressive complex 1 contributes to<br />
stable maintenance of mouse embryonic<br />
stem cells. PLoS ONE. <strong>2008</strong>:3:e2235<br />
Uren AG, Kool J, Matentzoglu K,<br />
De Ridder J, Mattison J, Van Uitert M,<br />
Lagcher W, Sie D, Tanger E, Cox T,<br />
Reinders M, Hubbard TJ, Rogers J,<br />
Jonkers J, Wessels L, Adams DJ,<br />
Van Lohuizen M, Berns A.<br />
Large-scale mutagenesis in p19(ARF)- and<br />
p53-deficient mice identifies cancer genes<br />
and their collaborative networks. Cell<br />
<strong>2008</strong>:133:727-41<br />
Vissers JH, Nicassio F, van Lohuizen M,<br />
Di Fiore PP, Citterio E. The many faces of<br />
ubiquitinated histone H2A: insights from<br />
the DUBs. Cell Div. <strong>2008</strong>:3:8<br />
Vékony H, Raaphorst FM, Otte AP,<br />
Van Lohuizen M, Leemans CR,<br />
Van der Waal I, Bloemena E. High<br />
expression of Polycomb group protein EZH2<br />
predicts poor survival in salivary gland<br />
adenoid cystic carcinoma. J Clin Pathol.<br />
<strong>2008</strong>:61:744-9<br />
Puschendorf M, Terranova R, Boutsma E,<br />
Mao X, Isono K, Brykczynska U, Kolb C,<br />
Otte AP, Koseki H, Orkin SH,<br />
Van Lohuizen M, Peters AH. PRC1 and<br />
Suv39h specify parental asymmetry at<br />
constitutive heterochromatin in early mouse<br />
embryos. Nat Genet. <strong>2008</strong>:40:411-20<br />
Pietersen AM, Van Lohuizen M. Stem cell<br />
regulation by polycomb repressors:<br />
postponing commitment. Curr Opin Cell<br />
Biol. <strong>2008</strong>:20:201-7<br />
Miyazaki M, Miyazaki K, Itoi M, Katoh Y,<br />
Guo Y, Kanno R, Katoh-Fukui Y, Honda H,<br />
Amagai T, Van Lohuizen M, Kawamoto H,<br />
Kanno M. Thymocyte proliferation<br />
induced by pre-T cell receptor signaling is<br />
maintained through polycomb gene product<br />
Bmi-1-mediated Cdkn2a repression.<br />
Immunity <strong>2008</strong>:28:231-45
82<br />
MOLECULAR GENETICS<br />
Group leader Anton Berns<br />
Anton Berns PhD Group leader<br />
Paul Krimpenfort PhD Academic staff<br />
Margriet Snoek PhD Academic staff<br />
Joaquim Calbo PhD Post-doc<br />
Hilda De Vries PhD Post-doc<br />
Johan Jongsma PhD Post-doc<br />
Jaap Kool PhD Post-doc<br />
Andor Kranenburg PhD Post-doc<br />
Kate Sutherland PhD Post-doc<br />
Anthony Uren PhD Post-doc<br />
Andrej Alendar MSc PhD student<br />
Erwin Van Montfort MSc PhD student<br />
Colin Pritchard MSc Research assistant<br />
Rahmen Bin Ali Technical staff<br />
Jan Paul Lambooij Technical staff<br />
Natalie Proost Technical staff<br />
Johanna Blitz Technical staff<br />
Fina Van de Ahé Technical staff<br />
John Zevenhoven Technical staff<br />
Figure 3: Mapping interaction networks<br />
between Common Insertion Sites.<br />
Co-occurrence between the top 25 CISs. CIS<br />
names and CIS rank are indicated on<br />
vertical axis, numbers on horizontal axis<br />
are CIS rank. The horizontal axis<br />
represents CISs that are assumed to be the<br />
predisposing, more clonal event and the<br />
vertical axis represents CISs that are<br />
presumed to be subsequent, subclonal events.<br />
MOUSE MODELS FOR CANCER<br />
The mouse is used as a model organism for establishing the role of oncogenes and<br />
tumor suppressor genes in tumor development. Using Cre/Lox mediated switching,<br />
taking advantage of somatic gene transfer methods, expression of multiple<br />
oncogenes and tumor suppressor genes can be regulated in a tissue-specific and<br />
spatial-temporal fashion. This permits a more accurate modeling of tumorigenesis as<br />
it occurs in man, and therefore provides the opportunity for establishing relevant<br />
genotype-phenotype correlations. These models also provide an excellent<br />
experimental tool to test prevention and intervention strategies especially when<br />
combined with sensitive in vivo imaging techniques. Finally, these models permit us<br />
to identify new oncogenes and tumor suppressor genes involved in tumor<br />
progression using a variety of techniques, such as array CGH, expression profiling<br />
and proviral insertional mutagenesis. Some of the gene families identified in our<br />
models are being studied in more depth.<br />
Functional analysis of oncogenes and tumor suppressor genes We utilize mice<br />
carrying combinations of different oncogene and conditional tumor suppressor gene<br />
alleles to model a range of tumors. Our current focus is on several lung cancers<br />
subtypes and mesotheliomas. To achieve (sporadic) activation of oncogenes and<br />
inactivation of tumor suppressor genes we use Adeno-Cre or Lentivirus-mediated<br />
gene transfer to switch the conditional oncogenes and tumor suppressor gene alleles.<br />
Subsequently, tumor initiation and progression is monitored in longitudinal studies<br />
in which noninvasive imaging techniques are used.<br />
Lung tumors We focus on small cell lung cancer (SCLC), non-small cell lung cancer<br />
(NSCLC) and squamous cell carcinoma (SCC). When Rb and p53 are inactivated<br />
specifically in lung, SCLC ensues. The marker profile of these tumors is<br />
indistinguishable from that of human SCLC. The tumors also metastasize to the<br />
same sites. Array CGH showed frequent amplification of L-Myc further supporting<br />
their resemblance with the human counterpart. These tumors are heterogeneous and<br />
consist of different cell types. Cloning of the different cells from a single tumor<br />
showed very different marker profiles. Both cells with neuroendocrine and progenitor<br />
markers were found. Interestingly, these phenotypically highly diverse cell lines<br />
shared some highly distinct genetic aberrations indicating that they were derived<br />
from a common progenitor. Some known recurrent lesions were found that<br />
specifically marked the tumor cells with neuroendocrine markers, such as L-myc<br />
amplification. Forced overexpression of L-myc in the non-neuroendocrine cells did<br />
not substantially change their phenotype. However, K-ras introduction into<br />
neuroendocrine cells profoundly altered these cells. They now more closely<br />
resembled NSCLC. This is of interest since this has also been reported for relapses of<br />
SCLC. This raises the question about the cell type in which tumorigenesis is initiated<br />
and that can differentiate to seemingly quite different lineages. Therefore, we have<br />
designed a series of cell-type specific Adeno-Cre viruses that enable us to switch<br />
oncogenes and tumor suppressor genes in distinct lung cell types in vivo. Using<br />
promoters specific for Clara cells, Alveolar type I and II cells, and neuroendocrine<br />
cells to drive Cre expression upon Adeno-Cre infection we will establish the marker<br />
profile of cells in lung that give rise to SCLC and NSCLC. Preliminary results indicate<br />
that distinct subsets of cells serve as a compartment from which these tumors derive.<br />
This presents a proof of concept for the approach and therefore, we are now<br />
generating a larger set of Adeno-Cre viruses to more precisely define the target cells<br />
that give rise to the different types of lung cancer.<br />
Mesotheliomas Inactivation of Nf2 and Ink4a has been reported in a subset of<br />
human mesotheliomas. This has served as a basis to design a mesothelioma mouse<br />
model. We have inactivated Nf2 in combination with either Ink4a/p19Arf, p53, or p53/<br />
Ink4a loss by Adeno-Cre infection of mesothelia of the thoracic and intraperitoneal<br />
cavity. Concomitant loss of Ink4a/p19Arf or p53 strongly accelerated mesothelioma<br />
development. We found that p53 deficiency or proficiency does not result in a distinct<br />
resistance profile towards cytotoxic drugs. Initially we have performed the studies<br />
with cell lines cloned from these tumors. However, these cell lines appear irreversibly
altered, as measured by expression profiling, likely as a result of in vitro propagation.<br />
Therefore, we have set up a primary tumor grafting model system that allows us to<br />
test various intervention strategies following subcutaneous and orthotopic grafting of<br />
primary tumor cells. These tumors appear highly refractory to most intervention<br />
protocols. We are making an inventory of the activated signaling pathways in the<br />
individual tumors to gain better insight in combination therapies that might<br />
specifically target the signaling pathways activated these tumors.<br />
Ink4 proteins We have shown that disrupting Ink4a, Ink4b and p19Arf together, a<br />
situation often seen in human tumors, results in very aggressive tumor development,<br />
not seen in Ink4a/p19Arf knockout mice. It appears that Ink4b is a critical backup<br />
for Ink4a. The backup function of Ink4b for Ink4a raises the question whether Ink4c<br />
and Ink4d might fulfill similar backup functions. Therefore we have begun to cross<br />
the Ink4c and Ink4d knockout alleles into the conditional Inka-/-;Ink4b-/-;p19Arflox/<br />
lox mice. Although in mouse embryo fibroblasts a constitutive-active Cdk4 mutant<br />
shows a similar phenotype as the Ink4a-/-;Ink4b-/- alleles in a Ras transformation<br />
assay, it is evident from studies of others that in vivo Ink4c and Ink4d have tumor<br />
suppressor functions in specific tissues. The knockout of different combinations of<br />
all four genes should teach us to what extent this tumor suppressor family plays a<br />
role in tumor suppression in additional tissues. In addition, this might reveal<br />
unexpected physiological functions of these proteins.<br />
Identification of new oncogenic pathways Proviral insertional mutagenesis<br />
screens marks genes instrumental in tumorigenesis. In collaboration with the groups<br />
of Maarten van Lohuizen, Lodewyk Wessels, Jos Jonkers, and John Hilkens we have<br />
performed a large-scale proviral insertional mutagenesis study in tumor suppressor<br />
knockout mice. In collaboration with the group of David Adams of the Sanger Centre<br />
we have sequenced the insertion sites of approximately 1000 lymphoid tumors<br />
induced in wild-type and genetically-predisposed genetic backgrounds. The largest<br />
subgroup study was performed in p53 and p19Arf mutant mice. p53 and p19Arf are<br />
tumor suppressors frequently mutated in human tumors. In a high throughput<br />
screen in mice for mutations collaborating with either p53 or p19Arf deficiency,<br />
we identified 10.806 retroviral insertion sites, implicating over 300 loci in<br />
tumorigenesis. This dataset reveals 20 genes that are specifically mutated in either<br />
p19Arf -deficient, p53-deficient or wild-type mice (including Flt3, mmu-mir-106a-363,<br />
Smg6 and Ccnd3), as well as networks of significant collaborative and mutually<br />
exclusive interactions between cancer genes (see figure 4). Furthermore, we found<br />
candidate tumor suppressor genes, as well as distinct clusters of insertions within<br />
genes like Flt3 and Notch1 that induce mutants with different spectra of genetic<br />
interactions. Cross species comparative analysis with aCGH data of human cancer<br />
cell lines revealed known and candidate oncogenes (Mmp13, Slamf6, Rreb1) and<br />
tumor suppressors (Wwox, Arfrp2). This dataset should prove to be a rich resource<br />
for the study of genetic interactions that underlie tumorigenesis.<br />
Figure 4: Common Insertion site interaction network representing the co-occurrence or mutual exclusivity<br />
of the 20 most significant CISs. Co-occurring CISs are connected by uninterupted lines (thin line, 0.001<<br />
p-value < 0.05; heavy line, p-value < 0.001), mutual exclusive CISs are connected with broken lines (thin<br />
line, 0.001< p-value < 0.05; heavy line, p-value < 0.001).<br />
Publications<br />
83<br />
MOLECULAR GENETICS<br />
Berns A. Kras and Hras – What is the<br />
difference. Nature Genetics <strong>2008</strong>;40:1149-50<br />
Boyle P, Anderson BO, Andersson LC,<br />
Ariyaratne Y, Auleleyb GR, Barbacid M,<br />
Bartelink H, Baselga J, Behbehani K,<br />
Belardelli F, et al. Need for global action for<br />
cancer control. Ann Oncol <strong>2008</strong>;19:1519-<br />
1521<br />
Lehembre F, Yilmaz M, Wicki A,<br />
Schomber T, Strittmatter K, Ziegler D,<br />
Kren A, Went P, Derksen PW, Berns A, et<br />
al. NCAM-induced focal adhesion assembly:<br />
a functional switch upon loss of E-cadherin.<br />
EMBO J. <strong>2008</strong>;27:2603-15<br />
Uren AG, Kool J, ,Matentzogly K,<br />
de Ridder J, Mattison J, van Uitert M,<br />
Lagcher W, Sie D, Tanger E, Cox T,<br />
Reinders M, Hubbard TJ, Rogers J,<br />
Jonkers J, Wessels L, Adams DJ,<br />
van Lohuizen M, Berns A. Large-scale<br />
mutagenesis in p19 ARF and p53 deficient<br />
mice identifies cancer genes and their<br />
collaborative networks. Cell <strong>2008</strong>;133:727-41<br />
Berns A. A tRNA with oncogenic capacity.<br />
Cell <strong>2008</strong>; 133:29-30<br />
Jongsma J, van Montfort E, Vooijs M,<br />
Zevenhoven J, Krimpenfort P,<br />
Van der Valk M, Van de Vijver M, Berns A.<br />
A conditionele mouse model for malignant<br />
mesothelioma. Cancer Cell <strong>2008</strong>;13:261-271<br />
Van Amerongen R, Berns A. Targeted<br />
anticancer therapies: mouse models help<br />
uncover the mechanisms of tumor escape.<br />
Cancer Cell <strong>2008</strong>;13:5-7
84<br />
MOLECULAR GENETICS<br />
Group leader Daniel Peeper<br />
Daniel Peeper PhD Group leader<br />
Gireesh Anirudhan PhD Post-doc<br />
Christophe Desmet PhD Post-doc<br />
Tristan Gallenne PhD Post-doc<br />
Alexandre Prieur PhD Post-doc<br />
Cornelia Hömig PhD Post-doc<br />
Christelle Lenain MD PhD Post-doc<br />
Katrin Meissl PhD Post-doc<br />
Celia Vogel MSc Post-doc<br />
Thomas Geiger MSc PhD student<br />
Joanna Kaplon MSc PhD student<br />
Thomas Kuilman Msc PhD student<br />
Chrysiis Michaloglou BSc PhD student<br />
Marjon Smit MSc PhD student<br />
Liesbeth Vredeveld MSc PhD student<br />
Sirith Douma MSc Technical staff<br />
Aranzazu Rosado Technical staff<br />
Publications<br />
Kuilman T, Peeper DS. Senescence-<br />
Messaging Secretome: SMS-ing cellular<br />
stress. Nature Reviews Cancer (in press).<br />
Smit MA, Peeper DS. Deregulating EMT<br />
and senescence: double impact by a single<br />
twist. Cancer Cell <strong>2008</strong>;14:5-7<br />
Kuilman T, Michaloglou C, Vredeveld LC,<br />
Douma S, van Doorn R, Desmet CJ,<br />
Aarden LA, Mooi WJ, Peeper DS.<br />
Oncogene-induced senescence relayed by an<br />
interleukin-dependent inflammatory<br />
network. Cell <strong>2008</strong>;133:1019-31<br />
Prieur A, Peeper DS. Cellular senescence in<br />
vivo: a barrier to tumorigenesis. Curr Opin<br />
Cell Biol. <strong>2008</strong>;20:150-5<br />
Schlisio S, Kenchappa RS, Vredeveld LC,<br />
George RE, Stewart R, Greulich H, Shahriari K,<br />
Nguyen NV, Pigny P, Dahia PL, Pomeroy SL,<br />
Maris JM, Look AT, Meyerson M, Peeper DS,<br />
Carter BD, Kaelin WG Jr. The kinesin KIF1Bbeta<br />
acts downstream from EglN3 to induce apoptosis<br />
and is a potential 1p36 tumor suppressor. Genes<br />
Dev. <strong>2008</strong>;22:884-93<br />
Michaloglou C, Vredeveld LC, Mooi WJ,<br />
Peeper DS. BRAF(E600) in benign and malignant<br />
human tumours. Oncogene. <strong>2008</strong>;27:877-95<br />
RESOLVING CANCER CELL NETWORKS AND IDENTIFYING<br />
CANCER DRUG TARGETS<br />
The Peeper laboratory is dedicated to resolving mechanisms that protect mammalian<br />
cells against oncogenic transformation, aiming to identify novel cancer drug targets.<br />
Non-malignant cells require multiple genetic mutations to transform into tumor<br />
cells. With both classical biochemical and genetic approaches, as well as with<br />
functional screens (based on both cDNA expression and RNA interference genomewide<br />
libraries), we have been focusing on two of these events: bypass of oncogeneinduced<br />
growth arrest (‘premature senescence’, which limits the proliferative capacity<br />
of cells) and suppression of ‘anoikis’ (apoptosis resulting from lack of adhesion,<br />
suppression of which may contribute to metastasis). In addition, we are studying the<br />
role of senescence as an in-vivo mechanism protecting against tumorigenesis. We<br />
anticipate that these approaches, together, will lead to the identification of a network<br />
of gene products, deregulation of which allows for tumorigenesis and metastasis. In<br />
turn, these proteins may represent novel therapeutic drug targets.<br />
Function-based genomic screens for metastasis genes Metastasis commonly<br />
underlies the malignancy of cancers, representing the principal cause of cancertreatment<br />
failure. Tumor colonization is prevented by several physiologic barriers,<br />
including ‘anoikis’: apoptosis resulting from lack of adhesion. Indeed, acquiring<br />
resistance to anoikis has been proposed to represent a general prerequisite for<br />
survival of metastases upon tumor and dissemination. In an attempt to identify<br />
metastasis-associated oncogenes we designed an unbiased, functional genome-wide<br />
screen solely based on anoikis suppression. With this approach, we previously<br />
identified TrkB, a neurotrophic tyrosine kinase receptor, as a potent suppressor of<br />
anoikis. Whereas non-malignant intestinal epithelial cells underwent caspaseassociated<br />
anoikis in vitro, this was completely prevented by TrkB, allowing formation<br />
of spheroid aggregates growing in suspension. Mice readily cleared parental cells, but<br />
TrkB-expressing cells formed many lung and heart metastases. Co-expression of<br />
BDNF, the prototypic TrkB ligand, caused highly invasive metastases in virtually<br />
every tissue, with an extremely short latency.<br />
We have made significant progression in identifying downstream signaling targets of<br />
this receptor, as they, in addition to TrkB, may serve as targets for therapeutic<br />
intervention. RNAi knockdown of some of these newly identified targets impairs the<br />
tumorigenic potential of TrkB. We have recently identified several transcription<br />
factors, including Twist and Snail, whose RNAi-mediated silencing significantly<br />
suppressed the metastatic potential of the tumor cells as well. Furthermore, we found<br />
that the transcriptome that was associated with the activity of one of these<br />
transcription factors had strong predictive value on the clinical outcome of human<br />
breast cancer. These results reveal a key role for this particular gene in breast cancer<br />
metastasis and indicate that its transcriptome can be exploited prognostically.<br />
Moreover, these data merit efforts to inactivate the components of this signaling<br />
pathway for clinical intervention of human breast cancer.<br />
We are also in the process of identifying cancer-associated mutations of TrkB,<br />
which we will analyze for biological significance. Finally, we have engineered<br />
TrkB transgenic mouse models, to further explore the role of TrkB in cancer and<br />
metastasis, and to serve as a model system for TrkB in cancer, enabling the<br />
development and testing of TrkB-inhibitory therapeutic drugs.<br />
Oncogene-Induced cellular Senescence (OIS) as a tumor-suppressing<br />
mechanism Most normal mammalian cells have a finite lifespan, thought to<br />
constitute a protective mechanism against unlimited proliferation. This phenomenon<br />
(‘senescence’) is driven by telomere attrition, which triggers the induction of tumor<br />
suppressors, including p16INK4a. In cultured cells, senescence can be elicited also<br />
prematurely, by oncogenes. However, whether such Oncogene-Induced Senescence<br />
(OIS) represents a physiological process has long been debated. Human nevi<br />
(‘moles’), benign tumors of melanocytes, frequently harbor oncogenic mutations in<br />
BRAF (predominantly V600E). Nonetheless, nevi typically remain growth-arrested
for decades and only rarely progress into malignancy (melanoma). We previously<br />
found that nevi have many hallmarks of senescent cells, and proposed that they are<br />
restricted from progression to malignant melanoma by virtue of their ability to<br />
undergo OIS.<br />
Currently, we are using various functional genomic approaches to identify novel OIS<br />
pathways whose deregulation contributes to melanoma. In addition to taking a<br />
candidate gene approach, we are combining gene expression analysis and RNAi in<br />
combination with function-based genomic screens. For example, we postulated that<br />
putative tumor suppressors, similar to p16 INK4A , are transcriptionally upregulated<br />
during the senescence response. Indeed, microarray expression analysis revealed the<br />
induction of various signaling networks that are induced specifically in BRAF E600 -<br />
expressing, senescent human cells. Among the top outliers was interleukin 6 (IL-6).<br />
RNAi-mediated knockdown of IL-6 abrogated the senescence response and led to<br />
continuous proliferation of BRAF E600 -expressing cells. Similar findings were made<br />
for other interleukins, which prompted us to search for a common denominator of<br />
BRAF E600 -regulated interleukins. We found that C/EBPb acts as a major player in<br />
this pathway. Consistently, knockdown of C/EBPb effectively bypassed BRAF E600 -<br />
induced senescence. In collaboration with prof. L. Aarden (Sanquin) we found that in<br />
OIS IL-6 had a cell-autonomous role, being required in an autocrine fashion both for<br />
the induction and maintenance of cell cycle arrest of cells exposed to oncogenic<br />
stress. Upon IL-6 depletion, the inflammatory network collapsed and cells bypassed<br />
senescence. C/EBPb was identified as an OIS integrator, acting with IL-6 to amplify<br />
the activation of the inflammatory network, including IL-8. In collaboration with<br />
prof. Wolter Mooi (VUmc), we observed that in human colon adenomas, IL-8<br />
specifically co-localized with arrested, p16 INK4A -positive epithelium. Our results<br />
suggest a model in which the antagonistic functions of IL-6 contribute to connect<br />
senescence with an inflammatory phenotype and cancer. We anticipate that this<br />
integrative genomics approach will identify novel tumor suppressors involved in<br />
melanoma and other (BRAF E600 -associated) cancers.<br />
We noted that the activation of the inflammatory transcriptome in cellular senescence<br />
resulted in the secretion of several factors, including cytokines and chemokines. This<br />
Senescence-Messaging Secretome, or SMS, as we propose to call this, plays an<br />
important role in the communication between senescent cells and their<br />
microenvironment. Although the selective advantage of OIS to the organism seems<br />
obvious, it is less straightforward to explain why the SMS contributes to this. We<br />
believe that the evolutionary advantage to utilize the SMS messaging system is that it<br />
does just that: messaging, allowing for communication both within and between<br />
cells. One might hypothesize that, for example, through SMS signals, a senescent cell<br />
sends a ‘danger signal’ to its microenvironment. This could be advantageous early in<br />
life in several ways, but this system could also have adverse effects for the organism,<br />
at later age (figure 5).<br />
85<br />
MOLECULAR GENETICS<br />
Figure 5: Potential benefits and threats of<br />
association of secreted factors with<br />
senescence. The use of SMS factors for<br />
senescence has potential ‘bright’ and ‘dark’<br />
sides. a-c | Several potential advantages<br />
(‘bright’ side) could explain the evolutionary<br />
pressure for the SMS. a | SMS factors could<br />
constitute a ‘danger signal’, which sensitizes<br />
normal neighbouring cells (pink) to senesce.<br />
For example, this may reduce the chance<br />
that damaged cells at risk for neoplastic<br />
transformation would fail to enter<br />
senescence. b | Another possible benefit is<br />
that also (normal) stromal cells could<br />
receive the ‘danger signal’ and stop<br />
supporting growth of the incipient neoplastic<br />
cell, thereby helping to implement a full<br />
senescence response in this cell. Alternatively,<br />
they could start secreting proliferationinhibitory<br />
(SMS) factors. c | A third<br />
advantage is the possible attraction of the<br />
innate immune system, which can dispose of<br />
the senescent cell. d-e | In later stages of<br />
tumourigenesis, tumour cells can misuse<br />
SMS signalling (‘dark’ side). d | By secreting<br />
SMS factors, cancer cells can induce<br />
senescence in stromal cells, such as<br />
fibroblasts. These in turn secrete factors that<br />
contribute to tumour growth. This model of<br />
two-way communication between tumour<br />
cells and stromal cells may explain why<br />
tumour cells commonly express several SMS<br />
factors. e | Stress and/or aging can lead to<br />
an increase in the number of senescent cells,<br />
thereby potentially stimulating expansion of<br />
premalignant cells as a function of the SMS.
86<br />
MOLECULAR GENETICS<br />
Group leader John Hilkens<br />
John Hilkens PhD Group leader<br />
Annabel Zwaagstra MSc PhD student<br />
Mandy Boer Technical staff<br />
Figure 6: Irs4 overexpression in HC11<br />
mammary epithelial cells induces anchorage<br />
independent growth. HC11 mammary<br />
epithelial cells retrovirally transduced with<br />
Irs4 or with an empty control vector<br />
(pMSCV) were grown in soft agar. Colonies<br />
were counted after 3 weeks in culture.<br />
GENES INVOLVED IN BREAST CANCER PROGRESSION<br />
AND METASTASIS<br />
Cancer is the result of sequential accumulation of multiple genetic changes in<br />
normal somatic cells affecting oncogenes and tumor suppressor genes. However,<br />
only a limited number of these genes have been identified. Complete knowledge of<br />
the cancer causing genes and the pathways they control is essential for the<br />
development of more effective novel therapeutic strategies. The aim of our laboratory<br />
is to identify and study novel genes involved in breast cancer and unravel the<br />
collaborating genetic pathways leading to breast cancer and breast cancer progression<br />
using mouse mammary tumor virus (MMTV) induced insertional mutagenesis in<br />
mouse models.<br />
Identification of mammary cancer genes in various mammary tumor models<br />
by MMTV tagging MMTV proviral insertion in the genome of murine mammary<br />
epithelial cells can activate flanking proto-oncogenes leading to mammary tumor<br />
induction. We have searched for common MMTV insertion sites (CISs, which<br />
indicate the presence of a nearby oncogene) in primary tumors from MMTV infected<br />
BALB/c mice, BALB/c mice conditionally deficient for p53 in the mammary gland<br />
and transgenic FVB mice that overexpress Erbb2 in the mammary gland. We<br />
identified over 50 CISs in a screen of almost 400 mammary tumors from these<br />
strains. Approximately 50% of the CIS associated genes are novel candidate cancer<br />
genes not previously implicated in mammary or other cancers. The number of<br />
tumors with proviral insertions in specific CISs differed strongly in each strain.<br />
A detailed expression analysis of the most frequently tagged genes revealed that<br />
oncogenic (i.e. those genes that are tagged by MMTV) Wnt and Fgf genes were<br />
activated in almost all wild type BALB/c tumors, less frequently in P53 deficient<br />
tumors and rarely in Erbb2 overexpressing tumors. These results suggest that the<br />
initial oncogenic event determines the subsequent oncogenic changes. Two genes<br />
belonging to the R-spondin gene family, Rspo2 and Rspo3, were also among the most<br />
frequently MMTV tagged genes in the tumors from wild type and conditionally<br />
deficient P53 mice. Rspo3, but not Rspo2, was upregulated in all Erbb2 tumors but<br />
rarely MMTV tagged. Most likely, Rspo3 can also be epigenetically upregulated or is<br />
downstream Erbb2. Eras is frequently tagged in the Erbb2 transgenic mice.<br />
Expression analysis showed that the gene was significantly more frequently<br />
expressed in MMTV induced tumors from the Erbb2 transgenic mice than in MMTV<br />
induced tumors from the FVB background strain, suggesting that Eras maybe<br />
specifically cooperating with Erbb2 in FVB mice.<br />
Irs4 is a novel mammary tumor oncogene We identified the Irs4 locus as a novel<br />
CIS in all three tumor models. Subsequently, we have validated Irs4 as an oncogene<br />
by showing that overexpression of this gene in NMuMG normal mammary epithelial<br />
cells rendered these cells highly tumorigenic in vivo. In addition to Irs4 that is<br />
activated in approximately 20% of the mammary tumors, also Igf2 is targeted by<br />
MMTV in approximately 10% of the tumors, indicating that the insulin-like growth<br />
factor pathway is a major oncogenic pathway in mouse mammary tumorigenesis.<br />
Interestingly, most mammary tumors showing overexpression of Irs4 frequently also<br />
expressed high levels of Irs1 and Irs2 suggesting that the downstream oncogenic<br />
activity of Irs4 is different from that of its family members in mammary tumor cells.<br />
In vitro studies showed that Irs4 overexpression in HC11 mammary epithelial cells<br />
induces anchorage independent growth and protects NMuMG cells against apoptosis<br />
induced by serum deprivation. Both properties are hallmarks for tumorigenesis. Irs4<br />
overexpression did not stimulate cell proliferation but reduced insulin requirement<br />
for growth of NMuMG mammary epithelial cells. Unexpectedly, Irs4 overexpression<br />
did not have a major effect on the Akt and MAP kinase pathways using MAPK and<br />
AKT phosphorylation as read outs, suggesting that Irs4 affects different pathways<br />
than Irs1 and Irs2.
THE ROLE OF CLASS I HDACS IN TRANSCRIPTION,<br />
DEVELOPMENT AND TUMORIGENESIS<br />
Epigenetic control of gene expression is known to play a prominent role in the<br />
suppression and development of cancer. E.g., histone modifications, including<br />
acetylation, are central to the transcriptional control of cancer-relevant pathways<br />
governing processes of cell proliferation, apoptosis, differentiation and DNA-repair.<br />
A growing list of non-histone targets is also subject to regulation through reversible<br />
acetylation (e.g. p53) via the opposing enzymatic actions of the same set of acetylases<br />
(HATs) and deacetylases (HDACs) that act on histones. Histone deacetylases<br />
enzymes involved in removing acetyl groups from lysine residues, are promising<br />
drug targets in cancer therapy. Despite evidence of clinical efficacy, the molecular<br />
mechanisms underlying the selective anti-tumorigenic effect of HDAC-inhibitors<br />
(HDACi) are unclear. HDACi are relatively non-specific, targeting multiple HDACs,<br />
of which only a subset may be important in tumor biology, resulting in undesirable<br />
side-effects. Furthermore, HDAC-function in normal and tumor cells is not fully<br />
understood, hampering understanding of the inhibitor’s anti-tumor selectivity and<br />
identification of critical HDAC targets responsible for anti-tumor effects. Complete<br />
knowledge of HDAC function will aid in designing more efficacious inhibitors,<br />
understanding mechanisms of resistance and the identification of responders within<br />
a patient population.<br />
Our research group wishes to address these issues in a systematic, genetic approach<br />
using conditional knockout alleles of two class I HDACs, Hdac1 and Hdac2. By<br />
deleting these genes in normal cells as well as tumor cells we wish to discover antitumor<br />
relevant targets of these HDACs and study their specific and redundant<br />
functions in transcription regulation, normal and tumor development and tumor<br />
maintenance. Studies aimed at unraveling the transcriptome of these transcriptional<br />
co-repressors in combination with the identification of new components of the<br />
HDAC1/2 mediated pathways may reveal new drug targets for anti-cancer therapy.<br />
Class I HDACs in development and cell cycle regulation Whereas deletion of<br />
Hdac1 results in early embryonic lethality, we found that Hdac2 ablation results in<br />
viable mice. Hdac2 -/- mice are born with a two-fold lower Mendelian frequency<br />
indicating that some Hdac2 -/- mice die during embryonic development. Also, Hdac2deficient<br />
mice display a 25% reduction in total bodyweight. Although Hdac2 -/- MEFs<br />
displayed no obvious phenotype, we observed a significant increase in Hdac1<br />
expression, suggesting a compensatory function of Hdac1 in the absence of Hdac2.<br />
Indeed, inactivation of Hdac1 in Hdac2 -/- MEFs, resulted in a G1 cell cycle arrest<br />
accompanied by senescence-associated b-galactosidase activity (SA-b-gal). Whereas<br />
the expression of several cell cycle inhibitors was not altered in cells lacking Hdac1<br />
and Hdac2, p21 Cip was significantly upregulated in Hdac2-null MEFs and even more<br />
in cells lacking both HDACs. Using p21 Cip shRNA vectors in Hdac1/2 deficient MEFs<br />
we observed an almost complete rescue of the cell cycle arrest, indicating that Hdac1<br />
and Hdac2 collectively suppress p21 Cip in order to maintain cell cycle progression. In<br />
contrast to the results obtained in MEFs we did not observe a reversal of the Hdac2defiency<br />
linked growth-defect in mice that lacked both Hdac2 and p21 Cip , suggesting<br />
that the growth-defect observed in Hdac2 -/- mice is p21 Cip -independent.<br />
Identification of novel HDAC1- and HDAC2-associated transcriptional nodes<br />
and cis-regulatory elements. Inhibition of class I HDAC mediated transcription is<br />
believed to be crucial for the efficacy of HDACi in tumor treatment. The<br />
identification of HDAC1 and HDAC2 target genes will therefore be of importance to<br />
identify pathways targeted by HDACi. Since it is likely that HDAC1 and HDAC2<br />
regulate many genes and the effects of HDAC inhibition can be contributed to the<br />
collaborative action of various genes we established a system to identify HDAC1- and<br />
HDAC2-associated transcriptional nodes through analysis of HDAC1 and HDAC2<br />
transcriptomes using bioinformatics. By determining over-represented cis-regulatory<br />
elements in the promoters of validated HDAC1 and HDAC2 target genes we will<br />
identify DNA binding proteins that recruit HDAC1 and HDAC2 to accomplish their<br />
transcriptional function.<br />
87<br />
MOLECULAR GENETICS<br />
Junior group leader Jan-Hermen Dannenberg<br />
Jan-Hermen Dannenberg PhD Junior<br />
group leader<br />
Inès Bejaoui MSc PhD student<br />
Eva Yanover Technical Staff<br />
Figure 7: Simulatenous ablation of Hdac1<br />
and Hdac2 results in a cell cycle arrest (left<br />
panel, bottom left) accompanied by<br />
senescence-associated b-galactosidase<br />
activity (SA-b-gal) (panel below). p21 Cip<br />
knockdown results in the rescue of this cell<br />
cycle arrest (upper panel, bottom right)
88<br />
MOLECULAR GENETICS<br />
Junior group leader Jacqueline Jacobs<br />
Jacqueline Jacobs PhD Junior group leader<br />
Paul-André Genest PhD Post-doc<br />
Marieke Peuscher MSc PhD student<br />
Jaco Van der Torre MSc PhD student<br />
Janet Van Noord Technical staff<br />
Publication<br />
Taghavi P, Verhoeven E, Jacobs JJL,<br />
Lambooij JP, Stortelers C, Tanger E,<br />
Moolenaar WH and van Lohuizen M.<br />
In Vitro Genetic Screen Identifies a<br />
Cooperative Role for LPA Signaling and<br />
c-Myc in Cell Transformation. Oncogene<br />
<strong>2008</strong>;27:6806-6816<br />
Figure 8: Severe fusion of mouse<br />
chromosomes upon loss of telomere capping<br />
due to inactivation of TRF2 (bright white<br />
signals mark telomeric sequences present at<br />
the ends of each chromosome)<br />
CHROMOSOME END PROTECTION BY TELOMERES<br />
Telomeres are protein-DNA complexes that protect natural chromosome ends from<br />
being treated as damaged DNA. Telomeres progressively shorten with every cell<br />
division until they become too short to function properly. The subsequent recognition<br />
of chromosome ends as broken DNA has important consequences for cellular and<br />
organismal life span but also for tumor development, and telomere maintenance is<br />
therefore a target of several recently developed anti-cancer strategies. Our main aim<br />
is to increase our understanding of how mammalian cells precisely perceive and<br />
respond to loss of telomere function and how telomere maintenance is controlled.<br />
Telomere-induced cellular senescence Loss of telomere function triggers a DNA<br />
damage response that causes cells to undergo apoptosis or to enter an irreversible<br />
growth arrest called senescence. This response limits the replicative life span of cells<br />
and thereby contributes to organismal aging. In addition, it represents an important<br />
tumor suppressor mechanism as it eliminates potentially cancerous cells. We have<br />
designed different RNAi loss-of-function genetic screens in order to identify novel<br />
factors involved in (the activation of) telomere-induced senescence. As a frequent tool<br />
in these ongoing screens or in subsequent validation experiments we mimic<br />
replicative telomere shortening by rendering telomeres dysfunctional through<br />
removal of the telomere capping protein TRF2 from telomeres.<br />
As an alternative genome-wide approach to investigate the consequences of loss of<br />
telomere protection we have performed micro-array analysis of the gene expression<br />
changes induced by loss of telomere function upon TRF2 inhibition. Although TRF2<br />
inhibition, oncogenic stress and DNA double-strand breaks (DSBs) all induce<br />
phenotypically similar senescence responses, we found that on gene expression level<br />
the telomere damage response is very different from oncogene-induced senescence<br />
and highly similar to the response to DSBs. Extensive analysis of the signaling pathways<br />
activated upon TRF2 inhibition is ongoing and has helped us identify a signaling<br />
pathway not known before to directly respond to telomere dysfunction. We are currently<br />
investigating this pathway for its contribution to telomere-induced senescence.<br />
Telomere-induced senescence involves activation of the p53 pathway and, as we and<br />
others have shown, also of the p16INK4a/RB pathway. By rendering telomeres<br />
dysfunctional by partially removing TRF2 from telomeres we were previously able to<br />
demonstrate that, despite its relatively late activation, p16INK4a significantly<br />
contributes to the senescence response triggered by such dysfunctional telomeres.<br />
P16INK4a most likely acts as a backup to p53 in maintaining human cells growth<br />
arrested upon loss of telomere function. We are now investigating the mechanism by<br />
which p16INK4a is induced upon telomere damage.<br />
Telomere maintenance and telomere-induced chromosome instability If cells<br />
with uncapped telomeres fail to undergo senescence or apoptosis and continue<br />
proliferating in the absence of a mechanism that replenishes telomeric repeats, DNA<br />
repair activities acting on chromosome ends cause chromosome fusions, anaphase<br />
bridges and nonreciprocal translocations. Such cells are at high risk of developing<br />
into cancer cells. Although telomere dysfunction is thought to be a major mechanism<br />
underlying chromosomal instability in human cancers, little is know about the<br />
precise structure of an uncapped telomere, how it is recognized, what precise ‘repair’<br />
events occur and how these events are controlled. To identify novel factors that are<br />
involved in telomere-induced genome instability we have developed an RNAi loss-offunction<br />
genetic screen in mouse cells in which we can instantly uncap telomeres by<br />
inactivating TRF2 to induce severe telomere fusion.<br />
An increasing number of micro-RNAs (miRNA) are being found to regulate an<br />
increasing number of pathways and changes in miRNA levels have been associated<br />
with several cancers. MiRNAs might also affect aging and cancer by affecting the<br />
expression levels of genes involved in telomere maintenance. To identify such<br />
miRNAs we have initiated two types of screens in which we use a collection of<br />
miRNAs constructed by the group of Reuven Agami (Division of Gene Regulation).
THE DEVELOPMENTAL ROLES OF ONCOGENES AND<br />
TUMOR SUPPRESSORS IN ZEBRAFISH<br />
Our research focuses on understanding the role of signaling pathways that regulate<br />
vertebrate development and disease using the zebrafish as a genetic model. Cancer is<br />
a multifactorial disease and exhibits many features of embryonic development.<br />
Deregulation of the genetic programs that guide growth and differentiation of cells<br />
and tissues during organogenesis may contribute to carcinogenesis.<br />
The short generation time (3 months), large progeny size (females typically lay 200<br />
eggs at a time), and availability of readily accessible transparent embryos combine to<br />
make the zebrafish an ideal system for dissecting developmental pathways and<br />
cellular mechanisms that when deregulated underlie malignant transformation.<br />
Analysis of cell polarity and energy metabolism during development and<br />
cancer Mutations in the serine-threonine kinase LKB1 lead to a gastrointestinal<br />
hamartomatous polyposis disorder with increased predisposition to malignancies of<br />
epithelial origin, in particular of the gastro-intestinal tract in humans. LKB1 activates<br />
AMPK under low energy conditions and that leads to growth suppression through<br />
several pathways including inhibition of the mTOR pathway. Additionally, activation<br />
of LKB1 induces complete polarization of human intestinal epithelial cells. To gain<br />
insight into how LKB1 mediates its effects during development and cancer, we have<br />
identified two mutations in the single zebrafish LKB1 ortholog by TILLING. Both<br />
mutations are stop codons in the kinase domain and fortuitously the Y246 mutation<br />
has also been identified in Peutz-Jeghers patients and has been shown to abolish the<br />
catalytic activity of LKB1.<br />
Lkb1 mutant zebrafish die at the larval stage owing to severe problems in maintaining<br />
intestinal architecture. Mutant larvae are indistinguishable from wt siblings the first<br />
5 days of development. Soon after yolk absorption, we observe an abrupt collapse of<br />
the intestinal villi and flattening of the epithelium. Perhaps surprisingly, cell<br />
polarization of intestinal epithelial cells is not affected as localization and levels of<br />
actin and aPKC on the apical membrane are normal. Interestingly, lkb1 mutant<br />
intestines resemble those of starved wild-type larvae at around day 12 pf. Consistent<br />
with this finding we observe premature depletion of liver glycogen. We are currently<br />
investigating whether also at the molecular level the lkb1 mutants exhibit accelerated<br />
metabolism. We are also comparing transcriptional profiles of wt and lkb1<br />
mutants with the aim to unravel the pathways regulated by LKB1 and a possible<br />
interconnection between maintenance of cell polarity, physiology and energy status<br />
as well as implications for cancer. Preliminary analysis of adult aged lkb1/+ fish,<br />
indicates that they are susceptible to bile duct adenocarcinomas, establishing that<br />
LKB1 acts as a tumor suppressor also in fish.<br />
The developmental roles of the Polycomb group proteins of transcriptional<br />
repressors (Collaboration with Maarten Van Lohuizen) Polycomb-mediated<br />
transcriptional repression has critical roles in embryonic development, maintenance<br />
of stem cell fate and cancer. Important targets of repression include developmental<br />
regulators, the Homeobox gene cluster and the Ink4a/ARF tumor suppressor locus.<br />
We aim to uncover the developmental pathways regulated by Polycomb repression.<br />
To this end, we perform morpholino (MO)-mediated knock-down of function and<br />
characterize the resulting phenotype. In particular, we are investigating the effects of<br />
transient inactivation of the E3 ubiquitin ligase, Ring1b. Targeted inactivation of<br />
Ring1b in mice leads to early embryonic lethality precluding analysis of the role of<br />
Ring1b in embryonic development. Fortuitously, maternal deposition of Ring1b<br />
mRNA in zebrafish allows them to complete gastrulation. Transient inactivation of<br />
Ring1b in zebrafish results in heart abnormalities, aberrant heart looping and<br />
diminished blood circulation. We have established that microinjection of three<br />
independent MOs leads to the same phenotype, results in dramatic reduction of<br />
protein levels and reduced ubiquitination of H2A. We are currently characterizing<br />
the phenotype further. In addition, we are generating a stable knock-out line by<br />
employing zinc finger nucleases methodology.<br />
Junior group leader Anna-Pavlina Haramis<br />
Anna-Pavlina Haramis PhD Junior group<br />
leader<br />
Yme van der Velden MSc PhD student<br />
Liqin (Bruce) Wang Research assistant<br />
Publications<br />
89<br />
MOLECULAR GENETICS<br />
Goessling W, North TE, Lord AM, Ceol C,<br />
Lee S, Weidinger G, Bourque C,<br />
Strijbosch R, Haramis AP, Puder M,<br />
Clevers H, Moon RT, Zon LI. APC mutant<br />
zebrafish uncover a changing temporal<br />
requirement for wnt signaling in liver<br />
development. Dev Biol. <strong>2008</strong>;320:161-74<br />
Hawkins TA, Haramis AP, Etard C,<br />
Prodromou C, Vaughan CK, Ashworth R,<br />
Ray S, Behra M, Holder N, Talbot WS,<br />
Pearl LH, Strähle U, Wilson SW. The<br />
ATPase-dependent chaperoning activity of<br />
Hsp90a regulates thick filament formation<br />
and integration during skeletal muscle<br />
myofibrillogenesis. Development<br />
<strong>2008</strong>;135:1147-56<br />
Figure 9: Vibratome section of a zebrafish<br />
intestine at day 7 post-fertilization stained<br />
with an antibody against aPKC (red).<br />
aPKC is localized at the apical surface of<br />
intestinal epithelial cells. The nuclei are<br />
stained for DAPI (blue).
90<br />
PSYCHOSOCIAL RESEARCH<br />
Division head, group leader Neil Aaronson<br />
Neil Aaronson PhD Group leader<br />
Hester Oldenburg MD PhD Academic staff<br />
Marc Van Beurden MD PhD Academic staff<br />
Emiel Rutgers MD PhD Academic staff<br />
Senno Verhoef MD Academic staff<br />
Saskia Duijts PhD Post-doc<br />
Eric Vermeulen PhD Post-doc<br />
Kirsten Douma MSc PhD student<br />
Karin Gehring MSc PhD student<br />
Doranne Hilarius MSc PhD student<br />
Rianne Hoopman MSc PhD student<br />
Ruud Knols MSc PhD student<br />
Chantal Lammens MSc PhD student<br />
Marijke Wevers MSc PhD student<br />
Chad Gundy MSc Senior statistical analyst<br />
Miranda Gerritsma Resarch assistant<br />
Tanja Nagtegaal Research assistant<br />
Marianne Kuenen Research assistant<br />
Publications<br />
Buijs C, Rodenhuis S, Bontenbal M, Beex LVAM,<br />
van der Wal E, Smit W, Nooij MA, Voest E,<br />
Hupperets P, ten Vergert EM, van Tinteren H,<br />
Willemse PHB, Mourits MJE, Aaronson NK,<br />
Post WJ, de Vries EGE. Prospective study of<br />
long-term impact of adjuvant high-dose and<br />
conventional chemotherapy on health-related<br />
quality of life. J Clin Oncol 2007;25:5403-9<br />
Gehring K, Sitskoorn MM, Aaronson NK,<br />
Taphoorn MJB. Interventions for cognitive<br />
deficits in adults with brain tumours.<br />
Lancet Neurol <strong>2008</strong>;7:548-60<br />
Gundy CM, Aaronson NK. The influence<br />
of proxy perspective on the evaluation of<br />
health-related quality of life: an empirical<br />
study. Med Care <strong>2008</strong>;46:209-16<br />
Hilarius DL, Kloeg PH, Gundy CM,<br />
Aaronson NK. The use of health-related quality<br />
of life assessments in daily clinical oncology<br />
practice: A community hospital-based<br />
intervention study. Cancer <strong>2008</strong>;113:628-637<br />
Hoopman R, Terwee CB, Aaronson NK.<br />
Translated COOP/WONCA charts found<br />
appropriate for use among Turkish and<br />
Moroccan ethnic minority cancer patients.<br />
J Clin Epidemiol <strong>2008</strong>;61:1036-48<br />
DIVISION OF PSYCHOSOCIAL<br />
RESEARCH AND EPIDEMIOLOGY<br />
PSYCHOSOCIAL ONCOLOGY<br />
The subdivision of psychosocial research is pursuing three major research lines:<br />
(1) health-related quality of life (HRQL) assessment in clinical research and clinical<br />
practice (group Neil Aaronson); (2) symptom perception and management (group<br />
Sanne Schagen and group Neil Aaronson); and (3) psychosocial issues in cancer<br />
clinical genetics (group Eveline Bleiker and group Neil Aaronson).<br />
Development and validation of a prostate cancer-specific health-related<br />
quality of life (HRQL) questionnaire for use in international clinical trials<br />
This multinational study evaluated the psychometrics of the EORTC QLQ-PR25, a<br />
questionnaire assessing the HRQL of prostate cancer patients. The QLQ-PR25 and<br />
the QLQ-C30 were administered to 642 prostate cancer patients from 13 countries<br />
treated with curative or palliative intent. The QLQ-PR25 assesses urinary, bowel, and<br />
sexual symptoms and functioning, and the side-effects of hormonal treatment. 509<br />
patients were available for the final analysis. Multi-trait scaling analyses confirmed<br />
the hypothesized scale structure of the QLQ-PR25. Internal consistency reliability<br />
was good (coefficient a = 0.70-0.86) for the urinary symptoms and sexual function<br />
scales, but lower for the bowel function and side-effects of hormonal treatment scales<br />
(a < 0.70). The module discriminated clearly between clinically distinct patient<br />
subgroups, and was responsive to changes in health status over time. In general, the<br />
QLQ-PR25 demonstrates acceptable psychometric properties and clinical validity.<br />
Some caution should be used in interpreting the bowel function and side-effects of<br />
hormonal therapy scales; results can be reported at the individual item and scale level.<br />
Cognitive rehabilitation of glioma patients: A prospective, randomized study<br />
This multicenter, randomized trial, carried out in collaboration with the University<br />
Medical Center Utrecht (M Sitskoorn, K Gehring) and the VU Medical Center (M.<br />
Taphoorn), evaluated the effectiveness of a cognitive rehabilitation program (CRP) on<br />
the neuropsychological (NP) test performance, cognitive complaints, and HRQL of<br />
patients with brain cancer. In total, 140 adult patients with low-grade and anaplastic<br />
gliomas, favorable prognostic factors, and both subjective cognitive symptoms and<br />
objective cognitive deficits were recruited from 11 hospitals. Patients were<br />
randomized to either an intervention group or a waiting-list control group. The<br />
intervention incorporated both computer-based attention retraining and<br />
compensatory skills training of attention, memory and executive functioning. All<br />
patients completed a battery of NP tests, and self-report questionnaires at baseline,<br />
immediately following completion of the CRP, and at 6 month follow-up.<br />
At immediate post-treatment, statistically significant intervention effects were<br />
observed for measures of subjective cognitive functioning and its perceived burden,<br />
but not for the objective NP outcomes or for any of the other self-report measures.<br />
At 6 month follow-up, the CRP group performed significantly better than the control<br />
group on NP tests of attention and verbal memory. Group differences in subjective<br />
outcomes were no longer statistically significant at 6 months.<br />
These results indicate that the CRP has a salutary effect on short-term cognitive<br />
complaints and on longer-term cognitive performance. Additional research is needed<br />
to identify which elements of the intervention are most effective, and to enhance its<br />
effect on other health outcomes.<br />
A randomized controlled trial of exercise training in hematological cancer<br />
patients following peripheral blood stem cell transplantation This RCT is being<br />
carried out by R Knols, a PhD student of ours in Zurich, Switzerland. Adult<br />
hematologic cancer patients who have undergone peripheral blood stem cell<br />
transplantation are randomly assigned to a 12-week, ambulatory, supervised<br />
endurance and repetitive strength exercise program or a usual care control group.<br />
Primary outcomes include musculoskeletal performance (knee extension and grip
strength), 6-minute walk test and 15-meter walking speed, and self-reported physical<br />
functioning. Secondary outcomes include self-reported HRQL and fatigue, selfreported<br />
and objectively assessed physical walking activity, whole body composition<br />
and hematological values. Patient recruitment has recently been completed (N = 128).<br />
To date, 107 patients have completed the study, including 6-month follow-up. The<br />
overall adherence to the exercise program is high (87% of the 24 physical exercise<br />
session attended; range = 21% to 100%). Loss to follow-up during the course of the<br />
study is approximately 15%, primarily due to disease recurrence. It is anticipated that<br />
the final follow-up will be completed by the end of 2009.<br />
Cognitive behavioral therapy and physical exercise for climacteric symptoms<br />
in breast cancer patients experiencing treatment-induced menopause This<br />
multicenter, randomized trial is evaluating the effectiveness of cognitive behavioral<br />
therapy (CBT), physical exercise (PE) or the combination of CBT/PE in alleviating<br />
climacteric symptoms in breast cancer patients experiencing treatment-induced<br />
menopause. In total, 325 consenting women, younger than 50 years of age, with<br />
histologically confirmed primary breast cancer, will be randomized to one of the three<br />
intervention groups or to a usual care, ‘waiting list’ control group. All participating<br />
women will be asked to complete a battery of questionnaires prior to randomization<br />
(T0), at 12 weeks (T1) and at 6 months (T2) post-entry study. Primary outcome<br />
measures are overall levels of vasomotor symptoms. Secondary outcomes are urinary<br />
symptoms, sexuality, body- and self-image and psychological distress. Patient accrual<br />
started in January <strong>2008</strong>. To date, 1835 women have been identified as being<br />
potentially eligible for study participation, of whom 1046 returned the initial brief<br />
screening questionnaire. Of these 1046 women, 431 met the eligiblity criteria. To<br />
date, 268 women have been randomly allocated to the one of the 4 study groups.<br />
Patient recruitment, intervention, and data collection (including follow-up) will<br />
continue until approximately mid-2010.<br />
Behavioral and psychosocial effects of rapid genetic counseling and testing in<br />
newly diagnosed breast cancer patients: A multicenter, randomized trial This<br />
multicenter, randomized trial, being carried out in collaboration with the University<br />
Medical Center Utrecht (Margreet Ausems), is investigating the uptake of rapid<br />
genetic counseling and testing (RGCT) when offered routinely to newly diagnosed<br />
breast cancer patients who, prior to receiving primary treatment, are identified as<br />
having at least a 10% risk of carrying a mutation in the BRCA1 or BRCA2 gene, and<br />
the impact of RGCT on a range of outcomes (see below).<br />
During an 18 month period, 255 eligible, consenting women from 13 hospitals in the<br />
Amsterdam and Utrecht regions of the Netherlands will be randomized either to a<br />
RGCT group or a “usual care” (UC) group at a ratio of 2:1. Women in the RGCT<br />
group will be referred for genetic counseling within days after diagnosis, and those<br />
who follow through on this referral will receive genetic counseling within 1 week of<br />
diagnosis. If a DNA-test is indicated, the results can be generated within 3 to 6 weeks.<br />
Women in the UC condition will receive standard advice and care. In some cases,<br />
patients may be referred by the treating surgeon or self-refer to genetic counseling.<br />
However, in current practice, this occurs rarely during the pre-surgery period.<br />
The study endpoints include: (1) uptake of RGCT; (2) choice of clinical management<br />
strategy, including the uptake of direct bilateral mastectomy (BLM) or of delayed<br />
preventive contralateral mastectomy (PCM); (3) cancer risk perception, and cancerrelated<br />
distress; (4) knowledge of genetic aspects of breast cancer; (5) decisional<br />
satisfaction; and (6) health-related quality of life (HRQL); and (6) women’s experience<br />
of and satisfaction with RGCT. Standardized questionnaires will be administered to<br />
all patients at study entry, and at 6 and 12 months to assess all psychosocial<br />
outcomes. A subset of women will be interviewed to obtain supplementary,<br />
qualitative data about the RGCT experience.<br />
In <strong>2008</strong>, the study protocol and all study materials were finalized, and ethical<br />
approval for the study was obtained from the <strong>NKI</strong>-AVL institutional review board.<br />
The protocol is currently under review by the UMCU and other participating<br />
hospitals. The first patient from the <strong>NKI</strong>-AVL was recently randomized.<br />
91<br />
PSYCHOSOCIAL RESEARCH<br />
Publications (continued)<br />
Knols RH, de Bruin ED, Aufdemkampe G,<br />
Uebelhart D, Aaronson NK. Reliability of<br />
ambulatory walking activity in patients<br />
with hematological malignancies. Arch<br />
Phys Med Rehab (in press)<br />
Langendijk JA, Doornaert P, Aaronson NK,<br />
Verdonck-de Leeuw IM, Leemans CR,<br />
Slotman BJ. The impact of late treatmentrelated<br />
toxicity on quality of life (EORTC<br />
QLQ-C30) among patients with head and<br />
neck cancer treated with radiotherapy.<br />
J Clin Oncol <strong>2008</strong>;26:3770-3776<br />
Mols F, Aquarius AE, Essink-Bot ML,<br />
Aaronson NK, Kil PJM, van de Poll-<br />
Franse LV. Does Diabetes Mellitus as<br />
comorbid condition affect health-related<br />
quality of life in prostate cancer survivors?<br />
Results of a population-based observational<br />
study. BJU International, <strong>2008</strong> June 3;<br />
(Epub ahead of print)<br />
Scott NW, Fayers PM, Aaronson NK,<br />
Bottomley A, de Graeff A, Groenvold M,<br />
Koller M, Petersen MA, Sprangers MAG.<br />
The relationship between overall quality of<br />
life and its subdimensions in cancer patients<br />
was influenced by culture: analysis, of an<br />
international database. J Clin Epidemiol<br />
<strong>2008</strong>;61;868-74<br />
Scott NW, Fayers PM, Aaronson NK,<br />
Bottomley A, de Graeff A, Groenvold M,<br />
Gundy C, Koller M, Petersen MA,<br />
Sprangers MAG. A simulation study<br />
provided sample size guidance for<br />
differential item functioning (DIF) studies<br />
using short scales. J Clin Epidemiol <strong>2008</strong><br />
Sept. 5: (Epub ahead of print)<br />
Van Andel G, Bottomley A, Fosså SD,<br />
Efficace F, Coens C, Guerif S, Kynaston H,<br />
Gontero P, Thalmann G, Akdas A,<br />
D’Haese S, Aaronson NK. An<br />
international field study of the EORTC<br />
QLQ-PR25: A questionnaire for assessing<br />
the health-related quality of life of patients<br />
with prostate cancer. Eur J Cancer<br />
<strong>2008</strong>;44:2418-2424<br />
Van Seters M, van Beurden M,<br />
ten Kate FJW, Ewing PC, Eijkemans MJC,<br />
Kagie MJ, Meijer CJM, Aaronson NK,<br />
Burger MPM, Helmerhorst THM.<br />
Effectiveness of imiquimod 5% cream in<br />
women with multifocal high-grade vulvar<br />
intrapithelial neoplasia (VIN): results of a<br />
randomized controlled trial. New Engl<br />
J Med <strong>2008</strong>;358:1465-73
92<br />
PSYCHOSOCIAL RESEARCH<br />
Group leader Sanne Schagen<br />
Sanne Schagen PhD Group leader<br />
Frits Van Dam PhD Academic staff<br />
Willem Boogerd MD PhD Academic staff<br />
Dieta Brandsma MD PhD Academic staff<br />
Sabine Linn MD PhD Academic staff<br />
Michiel De Ruiter PhD Post-doc<br />
Christien Schilder MSc PhD student<br />
Vincent Koppelmans MSc PhD student<br />
Riejanne Seigers MSc PhD student<br />
Chad Gundy MSc Senior statistical analyst<br />
Publications<br />
Schagen SB, Das E, van Dam FS. The<br />
influence of priming and pre-existing<br />
knowledge of chemotherapy-associated<br />
cognitive complaints on the reporting of<br />
such complaints in breast cancer patients.<br />
Psycho-oncology (in press)<br />
Schilder CMT, Eggens PC, Seynaeve C,<br />
Linn SC, Boogerd W, Gundy C, Beex LV ,<br />
van Dam FS, Schagen SB.<br />
Neuropsychological effects of tamoxifen and<br />
exemestane after AC-chemotherapy in<br />
postmenopausal women with early breast<br />
cancer: cross-sectional findings from the<br />
neuropsychological TEAM-side study. Acta<br />
Oncol <strong>2008</strong>;5:1-10<br />
Kreukels BP, Van Dam FS, Ridderinkhof RK,<br />
Boogerd W, Schagen SB. Persistent<br />
neurocognitive problems after adjuvant<br />
chemotherapy for breast cancer. Clin Breast<br />
Cancer <strong>2008</strong>;8:80-7<br />
Schilder CMT, Linn SC, Van Dam, FSAM,<br />
Schagen SB. De invloed van hormonale<br />
therapie op het cognitief functioneren van<br />
patiënten met het mammacarcinoom. Ned<br />
Tijdschr Geneeskd <strong>2008</strong>;152:494-8<br />
Kreukels BP, Hamburger HL, de Ruiter MB,<br />
van Dam FS, Ridderinkhof KR, Boogerd W,<br />
Schagen SB. ERP amplitude and latency<br />
in breast cancer survivors treated with<br />
adjuvant chemotherapy. Clin Neurophysiol.<br />
<strong>2008</strong>;119:533-41<br />
COGNITIVE FUNCTION IN CANCER PATIENTS<br />
Significant proportions of cancer patients report cognitive changes following therapy<br />
that interfere with their daily life activities and that can persist well into the<br />
survivorship period. The projects and collaborations constituting this research line<br />
center around the investigation of the prevalence, nature and cause of cognitive<br />
problems associated with systemic therapies. Understanding who is at risk and the<br />
emotional, cognitive and biological mechanisms associated with the development<br />
and maintenance of these effects is critical to treatment and prevention.<br />
Effects of endocrine treatment on cognitive function This prospective<br />
multicenter study is examining the neuropsychological sequelae of exemestane and<br />
tamoxifen in postmenopausal breast cancer patients compared to healthy controls.<br />
Patients were recruited from the TEAM trial, an open label, randomized multicenter<br />
study of exemestane treatment (25 mg once daily) or 2-3 years of adjuvant tamoxifen<br />
(20 mg once daily) followed by 3-2 years exemestane treatment (25 mg once daily).<br />
Patients were assessed at T1, before the start of endocrine treatment, and at T2 after<br />
12 months of treatment. At T2, after adjustment for T1 performance, exemestane<br />
users (n=99) did not perform significantly worse than healthy controls (n=120) on<br />
any of 8 cognitive domains. Tamoxifen users (n=80) performed worse than healthy<br />
controls on verbal memory (p=.003) and executive functioning (p=.005), and worse<br />
than exemestane users on information processing speed (p=.02).These findings<br />
underscore the need to include cognitive effects of endocrine treatment in long-term<br />
safety studies and stress the need for more knowledge about differential effects on<br />
cognitive function of these treatments.<br />
Nature and mechanism of cognitive deficits following chemotherapy:<br />
an (f )MRI study (<strong>NKI</strong>-AMC) In this ‘seed money’ project, 18 breast cancer patients<br />
treated with high-dose CTC chemotherapy (CT) and 15 breast cancer patients not<br />
treated with chemotherapy (non-CT) were scanned while performing neuropsychological<br />
tests at the 3 Tesla MRI facility of the Academic Medical Centre in<br />
Amsterdam, combining structural, functional and biochemical imaging. Results<br />
indicate that, 10 years after treatment, CT patients performed significantly worse<br />
than non-CT patients on several cognitive tests while being scanned. These<br />
impairments where accompanied by pronounced hypoactivation of bilateral posterial<br />
parietal cortex, indicating dysfunction of the dorsal attention network during memory<br />
encoding and executive functioning. 1H-MR spectroscopy of cerebral white matter, in<br />
addition, indicated significantly lower levels of N-acetyl aspartate, a marker of<br />
neuronal viability, suggesting decreased axonal integrity in CT patients relative to<br />
non-CT patients. Together, these data demonstrate detrimental effects of CT on brain<br />
function and biochemistry almost ten years after treatment.<br />
CTC Control<br />
Figure 1: Hypoactivation of posterior parietal cortex in breast cancer patients almost ten years after being<br />
treated with adjuvant high-dose chemotherapy (left) compared to patients not treated with chemotherapy<br />
(right) during an fMRI memory encoding task.
Animal studies exploring the underlying mechanisms of cognitive impairment<br />
(<strong>NKI</strong>-RUG) Previous experiments by our group revealed both short- and long-term,<br />
dose-dependent adverse effects of methotrexate (MTX) on learning behavior and<br />
hippocampal cell proliferation in rats. We have now investigated the potential effect<br />
of single high-dose (250 mg/kg) MTX on neuroinflammation and brain energy<br />
supply in rats using PET and immunohistochemistry. PET data showed no<br />
significant differences in neuroinflammation between control and MTX animals,<br />
while immunohistochemistry indicated clearly more activated microglia in MTX<br />
compared to control animals, one and three weeks after treatment. PET Glucose<br />
metabolism was decreased one week after treatment in MTX animals.<br />
Immunohistochemistry results showed that MTX animals had significantly less<br />
blood vessels compared to controls at both time points. Subsequent experiments will<br />
focus on the causes of microglia activation and reduced brain vascularization, the role<br />
of multidrug resistance pumps on the effect of MTX, and the potential reversing of<br />
the negative effect of MTX on learning.<br />
Late effects of chemotherapy on brain functioning in the elderly (<strong>NKI</strong>-Erasmus<br />
MC) As support is growing for the persistency of cognitive dysfunction after<br />
chemotherapy, we are investigating whether chemotherapy is associated with an<br />
increased risk for cognitive decline and dementia. Breast cancer patients who<br />
received CMF chemotherapy between 1975 and 1985 and who are currently >60 years<br />
of age are being tested with a neuropsychological examination and neuroimaging<br />
techniques. Patients are being compared with participants of the Rotterdam study, a<br />
prospective population-based cohort study investigating the prevalence and incidence<br />
of, and risk factors for chronic diseases in the elderly. 70 breast cancer survivors are<br />
currently included in the study.<br />
The influence of priming chemotherapy related cognitive problems on<br />
self-reported complaints and performance (<strong>NKI</strong>-VU University)<br />
We previously showed that pre-existing knowledge of chemotherapy-associated<br />
cognitive problems and priming the “chemo-brain” schema can increase the<br />
reporting of cognitive complaints by facilitating the accessibility of this concept. In a<br />
pilot project, we examined whether increased accessibility of a schema (in this case<br />
an age-impairs-cognition schema), also has a negative impact on cognitive test<br />
performance in healthy women (N=100). We found that increased accessibility<br />
increased self-reported cognitive complaints and also lowered performance on<br />
objective NP tests, showing that information influences participants’ mindsets which,<br />
in turn, may induce expectancy-congruent self-perceptions and performance.<br />
93<br />
PSYCHOSOCIAL RESEARCH<br />
Publications (continued)<br />
Vardy J, Wefel JS, Ahles T, Tannock IF,<br />
Schagen SB. Cancer and cancer-therapy<br />
related cognitive dysfunction: an<br />
international perspective from the Venice<br />
cognitive workshop. Ann Oncol,<br />
<strong>2008</strong>;19:623-9<br />
Schagen SB, Boogerd W, Muller MJ,<br />
Huinink WT, Moonen L, Meinhardt W,<br />
Van Dam FS. Cognitive complaints and<br />
cognitive impairment following BEP<br />
chemotherapy in patients with testicular<br />
cancer. Acta Oncol. <strong>2008</strong>;47:63-70<br />
Seigers R, Schagen SB, Beerling W,<br />
Boogerd W, Van Tellingen O, Van Dam FS,<br />
Koolhaas JM, Buwalda B. Long-lasting<br />
suppression of hippocampal cell<br />
proliferation and impaired cognitive<br />
performance by methotrexate in the rat.<br />
Behav Brain Res. <strong>2008</strong>;186:168-75<br />
Schilder CTM, Schagen SB,<br />
FSAM van Dam. Effect of hormones and<br />
hormonal treatment on cognition. In.<br />
Cognition and Cancer. Meyers & Perry eds.<br />
Cambridge University Press <strong>2008</strong>
94<br />
PSYCHOSOCIAL RESEARCH<br />
Group leader Eveline Bleiker<br />
Eveline Bleiker PhD Group leader<br />
Senno Verhoef MD Academic staff<br />
Irma Kluijt MD Academic staff<br />
Daniela Hahn MSc Academic staff<br />
Leonie Woerdeman MD Academic staff<br />
Kirsten Douma MSc PhD student<br />
Chantal Lammens MSc PhD student<br />
Willem Eijzenga MSc PhD student<br />
Chad Gundy MSc Senior statistical analyst<br />
Tanja Nagtegaal Research assistent<br />
Publications<br />
Bleiker EMA, Hendriks JHCL, Otten JDM,<br />
Verbeek ALM, Ploeg HM van der.<br />
Personality factors and breast cancer risk: a<br />
13-year follow-up. JNCI <strong>2008</strong>;100:213-218<br />
Bleiker EMA, Hahn DEE, Smets, EMA.<br />
Familiaire Tumoren Hoofdstuk. In:<br />
Psychologische patiëntenzorg in de<br />
oncologie, <strong>2008</strong> Editors JCJM de Haes, LM<br />
Gualthérie van Weezel, R Sanderman,<br />
HBM van de Wiel (Ed.) (in press)<br />
Bleiker EMA, Hahn DEE. Beslissen bij<br />
erfelijkheidsonderzoek voor kanker: kansen<br />
en keuzen. De Psycholoog, <strong>2008</strong>;43:12-19<br />
Douma KFL, Aaronson NK, Vasen HFA,<br />
Bleiker EMA. Psychosocial issues in genetic<br />
testing for familial adenomatous polyposis:<br />
a review of the literature. Psycho-Oncology<br />
<strong>2008</strong>;17:737-745<br />
Figure 2: compliance with screening by<br />
endoscopy, in 75 individuals from FAP<br />
families at high risk of developing colorectal<br />
cancer: % of individuals that reports pain<br />
and embarrassment with and without a<br />
sedative.<br />
PSYCHOSOCIAL ISSUES IN CANCER GENETICS<br />
The research line is being conducted in close collaboration with the family cancer<br />
clinic. It comprises a number of studies which are focused on two psychosocial<br />
themes in genetic counseling for cancer: 1) early detection of psychosocial problems<br />
and the development of psycho-educational interventions; and 2) the uptake and<br />
long-term psychosocial impact of risk-reducing behavior.<br />
The long term psychosocial impact of genetic testing among familial<br />
adenomatous polyposis (FAP) families This study began in 2004, in collaboration<br />
with the Netherlands Foundation for the Detection of Hereditary Tumors. The aim of<br />
the study is to evaluate the long-term (>1 yr) psychosocial impact of the diagnosis of<br />
FAP among individuals from high-risk families. In total 525 FAP-family members<br />
(response 64%) and 131 partners of individuals with a FAP-diagnosis have completed<br />
the questionnaire, and 60 respondents have been interviewed. Data collection has<br />
been completed and analyses are ongoing. Preliminary analyses indicate that: 1) 20%<br />
of individuals from FAP-families have moderate to severe levels of distress; 2) only a<br />
quarter of the moderate and severely distressed individuals received psychosocial<br />
support; 3) psychosocial variables, such as risk perception, family functioning, and<br />
knowledge about FAP, explain significantly more of the variability in distress levels<br />
than do sociodemographic and clinical variables (42% versus 7%); 4) based on<br />
medical record data, 20% of the at-risk group is less than fully compliant with<br />
surveillance advice; and 5) under-compliance is associated significantly with<br />
perceived self-efficacy, use of sedatives during surveillance, pain after surveillance<br />
and low perceived benefits of surveillance.<br />
Psychosocial aspects of genetic testing in families at high risk of multiple<br />
tumors at various sites and ages The aim of this study is to investigate the uptake<br />
of genetic testing for Li-Fraumeni Syndrome (LFS) and Von Hippel-Lindau Disease<br />
(VHL), evaluate the psychosocial consequences of (not) undergoing genetic testing,<br />
and to assess compliance with recommended surveillance programs. Data collection<br />
has been completed. In total, 243 individuals (78%) participated by completing a selfreport<br />
questionnaire. A subset of 94 respondents also participated in a semi-structured<br />
interview. First analyses of the questionnaire date indicate that: 1) 39% report moderate<br />
to high levels of distress; 2) of these, 33% had received psychosocial support; 3) of<br />
those who had not received psychosocial support, 28% indicated they would have<br />
liked to; 4) 92% of the high risk VHL family members who had received screening<br />
advice self-reported that they had been fully compliant with the advice given.<br />
Surveillance for early pancreatic neoplasia in high-risk individuals This multicenter<br />
study is evaluating the feasibility and effectiveness of surveillance for early<br />
pancreatic neoplasia in high-risk individuals. The purpose of the psychosocial arm of<br />
this study is to assess experiences and compliance with the offered surveillance<br />
program for pancreatic cancer (MRI and EUS), to evaluate the psychosocial impact of<br />
the pancreatic surveillance program, and to investigate the communication of the<br />
pancreatic risk and its preventive options to first, second and third degree relatives.<br />
Approval was obtained from the Review Board of the AMC and Erasmus MC.<br />
Experiences with nipple banking This study is being carried out in collaboration<br />
with the Department of Plastic Surgery (Leonie Woerdeman, Kalam Ahmed) and the<br />
Department of Psychosocial Counseling and Support (Daniela Hahn). During the<br />
past three years, about 300 women who had had a mastectomy directly followed by<br />
reconstruction, were offered nipple banking. Of these, about 100 women opted for<br />
nipple banking, about 160 women opted for a nipple tattoo, and about 40 women did<br />
not have any nipple reconstruction. In <strong>2008</strong>, two focus groups were conducted,<br />
including 11 women who had undergone nipple banking. These groups provided<br />
information for the development of a research proposal and a questionnaire to be used<br />
in a cross-sectional study in which experiences with three types of nipple ‘reconstruction’<br />
are compared. All 300 women will receive a psychological questionnaire, including<br />
questions about their decision about their nipple reconstruction, and their experiences<br />
with the option chosen. A prospective study is also currently being planned.
EPIDEMIOLOGY<br />
The cancer epidemiology group is currently concentrating on two principal research<br />
lines: (1) the etiology of hormone-related cancers, with a focus on gene-environment<br />
interactions; (2) the long-term health consequences of cancer treatment, particularly<br />
in terms of the risk of developing second malignancies or cardiovascular disease<br />
Risk factors for hormone-related cancer In our nationwide cohort study in<br />
families with a BRCA1/2 mutation (GEO-HEBON study), we are studying 1) whether<br />
hormonal/life-style factors modify cancer risk in BRCA1/2 families, 2) the agespecific<br />
cumulative risks of breast, ovarian and other cancers based on full pedigree<br />
information.<br />
We estimated the risks of breast and ovarian cancer in BRCA1/2 carriers, using the<br />
pedigree information of 19,668 family members in 758 families. Data were analyzed<br />
with a modified segregation analysis model adjusting for ascertainment. The<br />
increased risk of breast cancer for BRCA1- and BRCA2 carriers, as compared to the<br />
general population, was strongly age-dependent for BRCA1, but not for BRCA2.<br />
Younger BRCA1 carriers showed highest hazard ratios (HRs). No trend was found for<br />
ovarian cancer. The cumulative risk (CR) of developing breast cancer before age 70<br />
was 45% for BRCA1, and 31% for BRCA2, while the CR of ovarian cancer was 31% for<br />
BRCA1 and 7% for BRCA2. Among BRCA1 families, younger birth cohorts had<br />
much higher risks (65% for breast cancer and 45% for ovarian cancer for women<br />
born >1940), while no birth cohort effects were found for BRCA2. In addition, a<br />
higher number of family members with breast cancer strongly increased the lifetime<br />
risk among BRCA1 carriers, but not among BRCA2 carriers. The birth cohort and<br />
family history effects may partly explain the differences in estimated lifetime risks as<br />
found in the literature.<br />
We examined the association between physical activity and breast cancer risk among<br />
1,026 BRCA1/2 carriers in a retrospective cohort analysis. Survival bias was examined<br />
by comparing the results of the analysis of the entire cohort with those of a pseudoincident<br />
cohort, diagnosed/censored within ten years preceding completion of the<br />
questionnaire. The entire cohort analysis showed a non-significant breast cancer risk<br />
increase of ever sports activity lifetime (HR=1.21), while the pseudo-incident cohort<br />
analysis showed a decreased risk (HR=0.84), which suggested that survival bias was<br />
present. Among active women in the pseudo-incident cohort we observed a risk<br />
reduction for medium and high levels of intensity lifetime sports activity as<br />
compared to low levels (HR=0.59 and HR=0.77, respectively). For frequency and<br />
duration differences were smaller. Among women active in sports before age 30,<br />
dose-response associations of increasing levels of intensity, frequency, or duration<br />
with lower breast cancer risk were observed, with the strongest trends found for<br />
intensity and frequency. Ever versus never having participated in sports activity after<br />
age 30 was inversely associated with breast cancer risk (HR=0.59), but no doseresponse<br />
trends were found. These results suggest that BRCA1/2 mutation carriers<br />
can reduce their risk of breast cancer by engaging in sports activities, preferably<br />
starting at a young age.<br />
We started to coordinate the data collection of the International BRCA1/2 Carrier<br />
Cohort Study (IBCCS), in which three national studies (UK, NL, F) participate, as<br />
well as 11 (multi)center-based studies. In addition, we started to coordinate the risk<br />
factor data collection for the Consortium of Investigators of Modifiers of BRCA1/2<br />
(CIMBA), an international collaborative study on more than 10,000 BRCA1/2<br />
carriers.<br />
In our prospective cohort of 12,091 DES daughters (DESnet project), followed from<br />
December 1992 till June <strong>2008</strong> cancer incidence was assessed through linkage with<br />
the Dutch pathology database (PALGA) and the Netherlands Cancer Registry (NCR).<br />
Median age at the end of follow-up was still relatively young, 44.0 years. A total of<br />
340 incident cancers occurred. No overall increased risk of cancer was found<br />
(standardized incidence ratio (SIR)=1.0). The risk of clear cell adenocarcinoma of the<br />
vagina and cervix (CCA) was strongly increased (SIR=24.2), with the increased risk<br />
also present at ages above 40 years. Overall breast cancer risk was not increased, nor<br />
in women older than age 40. Melanoma risk diagnosed before age 40 was 60%<br />
Group leader Flora Van Leeuwen<br />
Group leader Matti Rookus<br />
95<br />
EPIDEMIOLOGY<br />
Flora Van Leeuwen PhD Group leader<br />
Matti Rookus PhD Group leader<br />
Annegien Broeks PhD Academic staff<br />
Berthe Aleman MD Academic staff<br />
Petra Nederlof PhD Academic staff<br />
Nicola Russell PhD Academic staff<br />
Emiel Rutgers MD PhD Academic staff<br />
Laura Van ’t Veer PhD Academic staff<br />
Senno Verhoef PhD Academic staff<br />
Marjanka Schmidt PhD Senior post-doc<br />
Marieke De Bruin PhD Post-doc<br />
Peggy Manders PhD Post-doc<br />
Ina Mulder PhD Post-doc<br />
Eric Vermeulen PhD Post-doc<br />
Dorien Voskuil PhD Post-doc<br />
Sandra Van den Belt-Dusebout MSc PhD student<br />
Naomi Boekel MSc PhD student<br />
Richard Brohet MSc PhD student<br />
Mathilde Cardous-Ubbink MSc PhD student<br />
Tamara Marees MSc PhD student<br />
Anouk Pijpe MSc PhD student<br />
Janneke Verloop MSc PhD student<br />
Willem Klokman MD MSc Senior statistical analyst<br />
Thea Mooij MSc Statistical analyst<br />
Wieke Heideman MSc Research assistant<br />
Esther Janssen Research assistant<br />
Kiki Jeanson MSc Research assistant<br />
Marianne Kuenen Research assistant<br />
Renée Mulder MSc Research assistant<br />
Gabey Ouwens Research assistant<br />
Renate Udo Research assistant<br />
Marie-José Blom MSc Data manager<br />
Eduard Ivanov MSc Data manager
96<br />
EPIDEMIOLOGY<br />
Publications<br />
AC Antoniou, MA Rookus, N Andrieu,<br />
RM Brohet, J Chang-Claude, S Peock,<br />
M Cook, DG Evans, R Eeles, EMBRACE,<br />
C Nogues, L Faivre, P Gesta, GENEPSO7 ,<br />
FE van Leeuwen, MGEM Ausems,<br />
GEO-HEBON, A Osorio, T Caldes,<br />
J Simard, J Lubinski, AM Gerdes, E Olah,<br />
C Fürhauser, H Olsson, B Arver, P<br />
Radice, DF Easton and DE Goldgar.<br />
Reproductive and hormonal factors, and<br />
ovarian cancer risk for BRCA1 and BRCA2<br />
mutation carriers: results from the<br />
International BRCA1/2 Carrier Cohort<br />
Study. Cancer Epidemiol Biomarkers Prev.<br />
(in press).<br />
Broeks, A., Braaf, L.M., Huseinovic, A.,<br />
Schmidt, M.K., Russel, N.S.,<br />
Van Leeuwen, F.E., Hogervorst, F.B., and<br />
‘t Veer L.J. The spectrum of ATM missense<br />
variants and their contribution to<br />
contralateral breast cancer. Breast Cancer<br />
Res. Treat. <strong>2008</strong>; 107: 243-248.<br />
Ceelen M, van Weissenbruch MM,<br />
Vermeiden JPW, van Leeuwen FE,<br />
Delemarre-van de Waal HA. Increased<br />
blood pressure in children born after in vitro<br />
fertilization. J Clin Endocrinol Metab<br />
<strong>2008</strong>: 93: 1682 – 1688.<br />
Ceelen M, van Weissenbruch MM,<br />
Roos JC, Vermeiden JP, van Leeuwen FE,<br />
Delemarre-van de Waal HA. Body<br />
composition in children and adolescents<br />
born after in vitro fertilization or<br />
spontaneous conception. J Clin Endocrinol<br />
Metab 2007;92:3417-3423<br />
De Bruin ML, Huisbrink J, Kuenen MA,<br />
Ouwens GM, Hauptmann M,<br />
van ’t Veer MB, Aleman BMP,<br />
van Leeuwen FE. Treatment-related risk<br />
factors for premature menopause<br />
following Hodgkin’s lymphoma. Blood<br />
<strong>2008</strong>; 111: 101-108<br />
De Kok IMCM, Habbema JDF,<br />
MJE Mourits, Coebergh JWW,<br />
Van Leeuwen FE. Onvoldoende gronden<br />
voor opname van vaccinatie tegen Humaan<br />
papillomavirus in het Rijksvaccinatieprogramma.<br />
Ned Tijdschr Geneeskd;<br />
<strong>2008</strong>;152:2001-2006<br />
increased, but the risk increase was no longer present at higher ages. No increased<br />
risks were found for other types of cancer. Thus, we found no increased risks for<br />
cancer except for CCA at all ages and melanoma before age 40. The increased risk of<br />
breast cancer at higher ages, as found in an US cohort of DES daughters, was not<br />
confirmed so far.<br />
Our nation-wide cohort of 25,152 women treated for subfertility was recently linked<br />
with the Dutch pathology database (PALGA), to assess the risk of breast cancer.<br />
The cohort consists of 19,145 women who received ovarian stimulation for in vitro<br />
fertilization (IVF) between 1983 and 1995 and 6,007 subfertile control women not<br />
treated with IVF. In total, we observed 378 first invasive breast cancers. Compared to<br />
the general population, the risk of breast cancer was not increased in the full cohort,<br />
nor in the IVF and control group separately (Standardized Incidence Ratio<br />
(SIR)=0.9). The risk of breast cancer among IVF-treated women was similar to that<br />
of subfertile controls (HR=1.08), adjusting for confounders like parity, age at<br />
menarche, family history of breast cancer and BMI. Ongoing analyses focus on doseresponse<br />
relations and differences in breast cancer risk according to treatment period<br />
and cause of subfertility. In <strong>2008</strong>, we started with the data-collection for the<br />
expansion of the IVF cohort (n = 13,000) through on-line questionnaires<br />
(collaboration with CW Burger, Erasmus MC Rotterdam).<br />
In part of our cohort of breast cancer patients aged
Late effects of cancer treatment Now that curative treatment is available for a<br />
substantial group of cancer patients, it is increasingly important to evaluate how the<br />
occurrence of late complications of treatment affects their long-term survival. We aim<br />
to evaluate the risk of second cancers and cardiovascular disease (CVD) after radio-<br />
and chemotherapy for Hodgkin’s lymphoma (n=3,400), testicular cancer (n=2,707)<br />
and breast cancer (n=50,000) over a period of up to 30 years after primary treatment.<br />
After our previous findings of increased risk for cardiovascular diseases among<br />
patients treated for Hodgkin lymphoma, we further investigated the excess risk for<br />
cerebrovascular disease in this group of patients. Among 2201 5-year survivors,<br />
treated for Hodgkin lymphoma before age 51 (median follow-up time of 17.5 years),<br />
we observed 96 patients developing cerebrovascular disease (55 strokes, 31 transient<br />
ischemic attacks (TIA), 10 patients with TIA and stroke; median age 52 years). Most<br />
ischemic events were of large-artery atherosclerotic (36%) or cardioembolic subtype<br />
(24%). The 30-year cumulative incidence accounting for death as a competing risk<br />
for ischemic stroke or TIA 30 years after Hodgkin’s lymphoma treatment was 7%<br />
(figure 1). Compared to the general population Hodgkin lymphoma survivors<br />
experience a 2-fold increased risk for stroke and a 3-fold increased risk for TIA.<br />
Radiation to the neck and mediastinum was an independent risk factor for ischemic<br />
cerebrovascular disease (HR=2.5). Treatment with chemotherapy was not associated<br />
with an increased risk. Hypertension, diabetes mellitus and hypercholesterolemia<br />
were associated with the occurrence of ischemic cerebrovascular disease, whereas<br />
smoking and overweight were not. Patients treated for Hodgkin’s lymphoma<br />
experience a substantially increased risk of stroke and TIA, associated with radiation<br />
to the neck and mediastinum. Physicians should consider appropriate risk-reducing<br />
strategies.<br />
With regard to second cancer risk among survivors of Hodgkin lymphoma we<br />
focused this year on pleural malignancies and more detailed analyses of breast cancer<br />
risk. Malignant mesothelioma is a relatively uncommon malignancy. Although the<br />
pathogenesis is primarily related to asbestos, malignant mesothelioma may be<br />
associated with radiation exposure. Recently, increased risks for mesothelioma<br />
following radiation for lymphoma have been reported. Because these findings are<br />
based on small numbers of patients, confirmation is needed. We examined<br />
malignant mesothelioma risk in 2567 5-year survivors of Hodgkin lymphoma.<br />
Overall, the risk was almost 26-fold increased compared to the general population<br />
leading to an absolute excess risk of 3.5 cases per 10,000 patients per year. Histology<br />
and survival of the mesothelioma cases following Hodgkin lymphoma were<br />
comparable to mesothelioma cases from the general population, but the proportions<br />
of males and asbestos-exposed individuals were lower than expected. The evidence<br />
for radiotherapy as a cause of mesothelioma independent of exposure to asbestos is<br />
expanding, and the diagnosis ‘mesothelioma’ should be kept in mind whenever<br />
related symptoms arise in previously irradiated patients.<br />
Figure 3. The cumulative incidence of ischemic stroke and TIA following Hodgkin’s lymphona treatment,<br />
accounting for death as a competing risk.<br />
Publications (continued)<br />
97<br />
EPIDEMIOLOGY<br />
Hoogendoorn WE, Hollema H,<br />
van Boven HH, Bergman E,<br />
de Leeuw-Mantel G, Platteel I, Fles R,<br />
Nederlof PM, Mourits MJE,<br />
van Leeuwen FE, and the Comprehensive<br />
Cancer Centers’ TAMARISK-group.<br />
Prognosis of uterine corpus cancer after<br />
tamoxifen treatment for breast cancer.<br />
Breast Cancer Res Treat <strong>2008</strong>;112:99-108<br />
De Jong D, Vasmel WLE, De Boer JP,<br />
Verhave G, Barbé E, Caparie MK,<br />
van Leeuwen FE. Risk of anaplastic large<br />
cell lymphoma in women with breast<br />
implants. JAMA <strong>2008</strong>;300/17: 2030<br />
Gronwald J, Pijpe A, Byrski T, Huzarski T,<br />
Stawicka M, Cybulski C, van Leeuwen Fe,<br />
Lubínski J, Narod SA. Early radiation<br />
exposures and BRCA1-associated breast<br />
cancer in young women from Poland.<br />
Breast Cancer Res Treat <strong>2008</strong>;112:581-4<br />
Heideman WH, Russell NS, Gundy C,<br />
Rookus MA, Voskuil DW. The frequency,<br />
magnitude and timing of post-diagnosis<br />
body weight gain in Dutch breast cancer<br />
survivors. Eur J Cancer (in press)<br />
Hooning MJ, Aleman B, Baaijens MHA,<br />
Hauptmann M, Klijn JGM, Noyon R,<br />
Stovall M, van Leeuwen FE. Roles of<br />
radiotherapy and chemotherapy in the<br />
development of contralateral breast cancer.<br />
J Clin Oncol <strong>2008</strong>;26:5561-8<br />
JM Korse, JMG Bonfrer, M van Beurden,<br />
RHM Verheijen, MA Rookus. Estradiol<br />
and testosterone levels are lower after an<br />
oophorectomy than after a natural<br />
menopause. Tumor Biology (in press)<br />
Marees T, Moll AC, Imhof SM,<br />
de Boer MR, Ringens PJ, van Leeuwen FE.<br />
Risk of Second Malignancies in Survivors of<br />
Retinoblastoma: More Than 40 Years of<br />
Follow-Up. J Natl Cancer Inst. 2009 Dec 9<br />
Schaapveld M, Visser O, Louwman MJ,<br />
Willemse PHB, de Vries EGE,<br />
van der Graaf WT, Otter R, Coebergh JW,<br />
van Leeuwen FE. The impact of adjuvant<br />
therapy on controlateral breast cancer risk<br />
and the prognostic significance of<br />
controlateral breast cancer: a populationbased<br />
study in the Netherlands. Breast<br />
Cancer Res Treat <strong>2008</strong>;110:189-197
98<br />
EPIDEMIOLOGY<br />
Publications (continued)<br />
Schaapveld M, Visser O, Louwman MJ,<br />
de Vries EGE, Willemse PHB, Otter R,<br />
van der Graaf WTA, Coebergh JWW,<br />
van Leeuwen FE. Risk of new primary nonbreast<br />
cancers following breast cancer<br />
treatment: A Dutch population-based study.<br />
J Clin Oncol <strong>2008</strong>;8:1239-1246<br />
Travis LB, Hodgson D, Allen J,<br />
Van Leeuwen FE. Second cancers. In:<br />
DeVita VT Jr, Hellman S, Rosenberg SA<br />
(eds). Cancer: principles and practice of<br />
oncology (ed 8). Philadelphia, PA:<br />
Lippincott <strong>2008</strong>:2718-2742<br />
Van Leeuwen FE, Swerdlow AJ, Travis LB.<br />
Second cancers after treatment of Hodgkin<br />
lymphoma. In: Hoppe RT, Mauch PM,<br />
Armitage JO, Diehl V, Weiss LM. (Eds.).<br />
Hodgkin Lymphoma, second edition.<br />
Philadelphia: Lippincott Williams &<br />
Wilkins. 2007;347-370<br />
Vermeulen E, Schmidt MK, Aaronson NK,<br />
Kuenen M, van Leeuwen FE. Obtaining<br />
‘fresh’ consent for genetic research with<br />
biological samples archived ten years ago.<br />
EJC (in press)<br />
Voskuil DW, Vrieling A, Korse CM,<br />
Beijnen JH, Bonfrer JM, van Doorn J,<br />
Kaas R, Oldenburg HS, Russell NS,<br />
Rutgers EJ, Verhoef S, van Leeuwen FE,<br />
van’t Veer LJ, Rookus MA. Effects of<br />
lycopene on the insulin-like growth factor<br />
(IGF) system in premenopausal breast<br />
cancer survivors and women at high<br />
familial breast cancer risk. Nutr. Cancer<br />
<strong>2008</strong>;60:342 – 353<br />
Vrieling A, Voskuil DW, Bonfrer JM,<br />
Korse CM, van Doorn J, Cats A,<br />
Depla AC, Timmer R, Witteman BJ,<br />
van Leeuwen FE, Van’t Veer LJ,<br />
Rookus MA, Kampman E. Lycopene<br />
supplementation elevates circulating<br />
insulin-like growth factor binding protein-1<br />
and -2 concentrations in persons at greater<br />
risk of colorectal cancer. Am J Clin Nutr.<br />
2007;86:1456-1462<br />
Vrieling A, Rookus MA, Kampman E,<br />
Bonfrer JMG, Bosma A, Cats A,<br />
van Doorn J, Korse CM, Witteman BJM,<br />
van Leeuwen FE, van ‘t Veer LJ, Voskuil DW.<br />
No effect of red clover-derived isoflavone<br />
intervention on the Insulin-like Growth Factor<br />
(IGF) system; a randomized cross-over trial in<br />
women. Cancer Epidemiol Biomarkers Prev.<br />
Oct. <strong>2008</strong>;17:2585-2593<br />
The strongly elevated risk of breast cancer following treatment for Hodgkin<br />
lymphoma has become a major concern for female survivors of this disease. Within<br />
our Dutch Hodgkin lymphoma survivors cohort, we examined the risk of breast<br />
cancer up to more than 30 years after treatment of Hodgkin lymphoma, focusing on<br />
the effect of radiation fields. Among 1122 female 5-year survivors, treated for<br />
Hodgkin lymphoma before age 51 (median follow-up time of 17.8 years), we<br />
identified 120 cases of breast cancer. The 30-year cumulative incidence of breast<br />
cancer accounting for death as a competing risk was 19%. Importantly, mantle field<br />
irradiation was associated with a 2.7-fold significantly increased risk of breast cancer<br />
compared to similarly dosed radiation to the mediastinum alone. This finding is<br />
reassuring since present-day radiotherapy for Hodgkin lymphoma employs smaller<br />
radiation volumes.<br />
To further study the effects of radiotherapy, chemotherapy, reproductive and genetic<br />
factors on the risk of breast cancer after Hodgkin lymphoma, we are performing a<br />
nationwide case-control study (collaboration with Division Experimental Therapy,<br />
A. Broeks, L. van ‘t.Veer). So far we have identified 138 case patients, who were<br />
individually matched to 398 controls. All women still alive receive a questionnaire on<br />
lifestyle factors and hormone use and are asked to provide a blood sample for genetic<br />
analyses.<br />
In collaboration with division of Experimental Therapy we also examined tumor<br />
characteristics and gene expression profiles of 22 breast cancers following<br />
radiotherapy for Hodgkin lymphoma from our larger series, and compared these<br />
with age-matched sporadic breast cancer without prior radiation exposures. The<br />
results point to a distinct molecular signature of radiation-associated breast cancer.<br />
We also assessed the risk of secondary non-breast cancers in a recently treated<br />
population-based cohort of breast cancer patients focused on the association with<br />
treatment and prognostic implications. The cohort comprised 58,068 Dutch patients<br />
diagnosed with invasive breast cancer between 1989 and 2003. After a median<br />
follow-up of 5.4 years, 2,578 second non-breast cancers had occurred. Compared with<br />
the Dutch female population at large, in this cohort, the SIR of second non-breast<br />
cancers was significantly increased (SIR=1.22). The absolute excess risk was 13.6 per<br />
10,000 person-years. SIRs were elevated for cancers of the esophagus, stomach,<br />
colon, rectum, lung, uterus, ovary, kidney, and bladder cancers, and for soft tissue<br />
sarcomas, melanoma, non-Hodgkin’s lymphoma, and acute myeloid leukemia. The<br />
10-year cumulative incidence of second non-breast cancers (adjusting for competing<br />
risks) was 5.4%. Among patients younger than 50 years, radiotherapy was associated<br />
with an increased lung cancer risk (HR=2.31) and chemotherapy with decreased risk<br />
for all second non-breast cancers (HR=0.78) and for colon and lung cancer. Among<br />
patients age 50 years and older, radiotherapy was associated with raised risk of soft<br />
tissue sarcoma (HR=3.43); chemotherapy with increased risks of melanoma, uterine<br />
cancer, and acute myeloid leukemia; and hormonal therapy with uterine cancer<br />
(HR=1.78). A second non-breast cancer significantly affected the patient’s prognosis<br />
(HR=3.98 for overall mortality).
EARLY STAGE TECHNOLOGY ASSESSMENT<br />
Constructive Technology Assessment of the introduction of a 70-gene micro<br />
array prognostic test in breast cancer treatment From 2003 until end 2006 a<br />
technology assessment study was conducted on the introduction of a 70-gene micro<br />
array test as a prognostic tool in the treatment of node negative breast cancer. As the<br />
diffusion of this technology is in an early stage and the course of development not<br />
easy to predict, an evaluation approach is chosen that takes the technology dynamics<br />
into account, constructive technology assessment (CTA).<br />
The project proceeds well: a paper on the overall results on CTA was accepted, an<br />
internal guideline on patient rights concerning banked tissue finalised and a scenario<br />
session was conducted together with the breast group international (BIG). The<br />
results of these scenarios will be used for the Cost Effectiveness Analysis of which<br />
the preliminary (promising) results will be send to the Dutch Health Insurance<br />
Board by end of <strong>2008</strong>.<br />
In 2009 finalising the CEA modelling, a CEA of the 70-gene test in France and a<br />
comparison between cost-effectiveness in France and the Netherlands are foreseen.<br />
Building on the experiences of early stage technology assessment, funds were<br />
granted to conduct scenario analysis and cost effectiveness modelling on the most<br />
promising results of the CTMM project focusing on the use of markers in the<br />
treatment of breast cancer.<br />
In preparation of the establishment of a surgical center for minimal invasive<br />
interventions, a preparatory study is being conducted into the appropriate<br />
methodology for early stage technology assessment for surgical innovations, using<br />
the operation robot as a case.<br />
Operations improvement in oncology Translating operations management and<br />
-research (OM/OR) principles into oncologic care is likely to improve both quality<br />
and efficiency of hospital processes.<br />
In order to define optimal performance levels a series of international benchmarking<br />
projects has been performed on comparing performance of 3 Comprehensive Cancer<br />
Centers, 3 ambulatory chemotherapy treatment centers (ACT), 6 fundamental research<br />
organisations and in <strong>2008</strong> a study on 4 Radiotherapy departments was concluded.<br />
Applying operations research methods have resulted in improvements in efficiency<br />
of the AVL ACT of 25-30% and after after acceptance of a paper on this topic, a<br />
multicenter proposal will be drafted.<br />
Using the experience of earlier OR simulation techniques (such as a verification of<br />
the efficiency of the operation room planning and scheduling), in <strong>2008</strong> a project was<br />
concluded on the use of simulation to attain a high degree of open access in the<br />
radiology department; reduction of the length of the diagnostic track, including CT,<br />
form three to one week and reducing the number of hospital visits was proven to be<br />
possible and resulted in implementation proposals.<br />
In cooperation with the UT PhD student Peter van Berkel started halftime on<br />
mathematic analysis of care pathways within the oncologic hospital setting and<br />
measuring the effect of increased focus on efficient capacity use.<br />
Rehabilitation, physical activity and cancer Survivorship care and rehabilitation<br />
are important elements of a cancer center’s program.In <strong>2008</strong> we have focused on<br />
strengthening and expanding the infrastructure in which studies in this area can be<br />
conducted. A multidisciplinary rehab program was started for breast cancer survivors<br />
receiving adjuvant treatment in cooperation concerning Herstel and and an<br />
agreement was reached to start a formal head and neck rehab program, approved by<br />
health insurance companies. A retrospective needs-assessment study was performed<br />
under 109 breast cancer survivors. Between 20 and 30% indicated need for care or<br />
rehabilitation in both physical and psychological health domains.<br />
Apart from cooperation in the drafting of a meanwhile funded RCT on physical<br />
exercise during chemotherapy, WvH was requested to act as scientific coordinator of<br />
a program proposal within Alpe d’Huzes focussing on ‘patient empowerment, return<br />
to work, tele-monitoring and implementation of relevant findings and programs all<br />
related to physical exercise and supported by innovative IT’. It is foreseen that this<br />
large action research program will be submitted early 2009.<br />
99<br />
PSYCHOSOCIAL RESEARCH<br />
Group leader Wim Van Harten<br />
Wim Van Harten MD PhD Group leader<br />
Valesca Retel MSc Research staff<br />
Jolien Bueno de Mesquita MD Research staff<br />
Marian Hummel Academic staff<br />
Wineke Van Lent Research staff<br />
Peter Van Berkel Research staff<br />
Rozan Gilles Research assistant<br />
Miranda Van Duijn MSc Academic staff<br />
Rutger Dahmen MD Academic staff<br />
Inge Haenen Research assistant<br />
Joost Deetman Research assistant<br />
Gijs Hesselink Research assistant<br />
Ingrid Nota Research assistant<br />
Publications<br />
Retèl VP, Hummel JM, Van Harten WH.<br />
Early phase Technology Assessment of<br />
Nanotechnology in Oncology, Tumori, 94:<br />
284-290, <strong>2008</strong> Genova<br />
Van der Ploeg HP, Streppel KR,<br />
van der Beek AJ, Van der Woude LH,<br />
Vollenbroek-Hutten MMR, Van Harten WH,<br />
Van Mechelen W. Underlying Mechanisms of<br />
Improving Physical Activity Behavior after<br />
Rehabilitation. Int. J. Behav. Med.<br />
<strong>2008</strong>;15:101-108<br />
Retèl VP, Bueno de Mesquita JM,<br />
Hummel MJM, Van de Vijver MJ,<br />
Douma KFL, Karsenberg K, Van Dam FSAM,<br />
Van Krimpen C, Bellot FE, Roumen RMH,<br />
Linn SC, Van Harten WH. Constructive<br />
Technology Assessment (CTA) as a tool in<br />
coverage with evidence development: the case of<br />
the 70-gene prognosis signature for breast cancer<br />
diagnostics. Int. J. Techn. Ass. in Health Care.<br />
2009;25:73-83<br />
Klopper AHJ, Meerding NM,<br />
Van Harten WH, Wilderom CPM.<br />
Measuring Stereotypical Images between<br />
Medical Doctors and Managers in Hospitals.<br />
J. Health Org. and Manag (in press)<br />
Van Lent WAM, Goedbloed N,<br />
Van Harten WH. Improving the efficiency<br />
of a chemotherapy day unit; applying a<br />
business approach to oncology. Eur. J.<br />
Cancer (in press)
100<br />
DIAGNOSTIC ONCOLOGY<br />
Division head Laura van ’t Veer (at interim)<br />
DEPARTMENT OF CLINICAL CHEMISTRY<br />
Willem Nooijen PhD Head<br />
Hans Bonfrer PhD Academic staff<br />
Olaf van Tellingen PhD Academic staff<br />
Nienke van den Brink - de Vries Graduate<br />
student<br />
Tessa Buckle Technical staff<br />
Tiny Korse Technical staff<br />
Dorothé Linders Technical staff<br />
Marian Buning - Kager Technical staff<br />
Arifa Fazalalikhan Technical staff in training<br />
DEPARTMENT OF NUCLEAR MEDICINE<br />
Cornelis Hoefnagel MD PhD Head<br />
Philippe Baars MD Academic staff<br />
Fijs van Leeuwen PhD Academic staff<br />
Saar Muller PhD Academic staff<br />
Michiel Sinaasappel PhD Academic staff<br />
Ferida Sivro - Prndelj MD Academic staff<br />
Renato Valdés Olmos MD PhD Academic staff<br />
Wouter Vogel MD PhD Academic staff<br />
Tjeerd Aukema MD Clinical research fellow<br />
Lenka Vermeeren MD Clinical research fellow<br />
Jaap Teunissen MD Registrar<br />
Saskia Baank Technical staff<br />
Martine Bakker Technical staff<br />
Carolien Beers - Bauhuis Technical staff<br />
Natascha Bruin Technical staff<br />
Petra Doodeman Technical staff<br />
Christel Feenstra Technical staff<br />
Rick Muusers Technical staff<br />
Bert Pool Technical staff<br />
Lyandra Rooze Technical staff<br />
Mariska Sonneborn Technical staff<br />
Colinda Vroonland Technical staff<br />
DEPARTMENT OF PATHOLOGY<br />
Hester van Boven MD PhD Head<br />
Olga Balague Ponz MD PhD Academic staff<br />
Frans Hogervorst PhD Academic staff<br />
Daphne de Jong MD PhD Academic staff<br />
Petra Nederlof PhD Academic staff<br />
Renée van Pel MD Academic staff<br />
Efraim Rosenberg PhD Academic staff<br />
Marc van de Vijver MD PhD Academic staff<br />
Loes van Velthuysen MD PhD Academic staff<br />
Laura van ’t Veer PhD Academic staff<br />
Jelle Wesseling MD PhD Academic staff<br />
Douwe Atsma Technical staff<br />
Lucie Boerrigter- Barendsen Technical staff<br />
Michael Knauer MD Academic staff<br />
Jolien Bueno de Mesquita Graduate student<br />
DIVISION OF<br />
DIAGNOSTIC ONCOLOGY<br />
DEPARTMENT OF CLINICAL CHEMISTRY<br />
Pharmacological studies in mice<br />
Nienke van den Brink-de Vries, Tessa Buckle, Roos Oostendorp, Arifa Fazalalikhan,<br />
Olaf van Tellingen<br />
High-grade gliomas, in particular glioblastoma multiforme (GBM), are among the<br />
deadliest and most devastating of human cancers. Current treatment involves a<br />
combination of surgery, radiotherapy and temozolomide chemotherapy but this<br />
offers only a very modest survival benefit. A major part of our work is dedicated to<br />
define the role of the BBB in the chemo-resistance of GBM with special emphasis on<br />
the role of drug transporting proteins. The expression of several ABC transporters<br />
(e.g. ABCB1, ABCG2, ABCC1-5) has been reported and we are investigating their<br />
functional importance using knockout mouse models. Because of overlapping<br />
substrate specificities of these transporters the use of compound knockouts is<br />
essential. Recently, we have been able to create Abcb1a/b;abcg2;abcc4 and<br />
abcg2,abcc4; abcc5 compound knockout strains that will be used for our<br />
pharmacokinetics studies. We have now also created abcb1a/b;Abcg2 knockout nude<br />
mice and have used these to demonstrate that the efficacy of temozolomide against<br />
intracranial tumors is higher than in wildtype nude mice, which is in line with the<br />
50% higher brain penetration of this agent in abcb1a/b;abcg2 knockout mice. We<br />
have also created new cell lines which over-express EGFRvIII and that will be<br />
characterized and used to determine the efficacy of agents that target the EGFR-PI3K-<br />
AKT-mTor pathway. Many of these agents are substrates of ABC transporters.<br />
The second part of our research involves the development of murine brain tumor<br />
models using Cre/LoxP conditional mice. This platform is suited to generate<br />
spontaneous high-grade gliomas in adult mice. Grade III and IV tumors arise in<br />
conditional Kras V12 ;Ink4a/Arf, Kras V12 ;Ink4a/Arf;P53 and Kras V12 ;Ink4a/Arf;Pten<br />
mice after stereotactic intracranial injection of a self-deleting lentivirus mediating<br />
astrocyte-specific expression of Cre (GFAP-Cre lentivirus). Unfortunately, we have not<br />
been successful in creating tumors by injecting a mixture of the GFAP-Cre virus and<br />
a lentivirus containing EGFRvIII into conditional Ink4a/Arf and Ink4a/Arf;Pten<br />
mice. Most likely, the expression of EGFRvIII in infected cells is too low. We expect<br />
that our models will be very useful to supporting the selection of the most promising<br />
combinations of molecularly-targeted agents and cytotoxic drugs for clinical trial.<br />
Besides protecting the brain, ABC transporters (e.g. ABCB1, ABCG2) are considered<br />
to be markers of stem cells and are thought to protect them from xenotoxic stress.<br />
We have now undertaken in vivo studies to establish the protective role of ABC<br />
transporters in bone marrow progenitor cells using the chemotherapeutic agent<br />
topotecan TPT), which is a substrate of several ABC transporters. Wildtype (WT)<br />
mice were sub-lethally irradiated (9 gy) and, subsequently, received donor bone<br />
marrow from WT or ABC transporter knockout mice the next day. After a recovery<br />
period of 6 – 8 weeks, these chimeric mice were exposed to TPT and hematological<br />
parameters (WBC, Hb, RBC) were assessed prior (baseline), during and after<br />
treatment. Thus, each animal served as it own reference. Following the exposure to<br />
0.5 mg TPT/kg/day x5 for three consecutive weeks, only the abcg2 and abcb1a/b;abcg2<br />
chimeras fell ill with Hb levels declining to less than 40% of the baseline values.<br />
WBC values also declined during the first 2 wks but stabilized thereafter, suggesting<br />
that this regimen did not deplete the most primitive stem cell population. 51-Cr<br />
labeling confirmed that the more marked decline of Hb was not due to a higher loss<br />
of mature RBC’s in abcg2 chimeras. Since we had previously found that Abcc1 was<br />
involved in the protection of the WBC lineage, we also checked abcg2;abcb1a/b;abcc1<br />
chimeras but Hb and WBC values were similar as in abcg2 and abcg2;abcb1a/b<br />
chimeras. In conclusion, when abcg2 is absent, TPT causes significant toxicity in the<br />
erythroid lineage, but does not cause a depletion of the more primitive bone marrow<br />
stem cell compartment. Further experiments involving abcc4 are ongoing, to address<br />
recent reports that this ABC transporter is also expressed in bone marrow cells.
Neuroendocrine tumours<br />
Tiny Korse, Hans Bonfrer, Otto Visser, Babs Taal<br />
In collaboration with the Division of Medical Oncology and Department of<br />
Cardiology of the Slotervaart Hospital, we investigated the role of NT-proBNP as<br />
diagnostic marker for carcinoid heart disease and as prognostic marker in patients<br />
with neuroendocrine tumours. From 102 NET-patients serum samples were obtained<br />
and cardiac ultrasound studies was performed from 1999 thru 2007. The criterion<br />
for CHD was tricuspidal regurgitation (TR) stage III/IV. We found that NT-proBNP is<br />
a promising diagnostic marker for CHD. Elevated NT-proBNP in addition to elevated<br />
CgA levels showed worse survival than elevated CgA only. In association with the<br />
Comprehensive Cancer Centre we evaluate epidemiological data of neuroendocrine<br />
tumours from 1990 until 2006. The total number of patients is 9126.<br />
The Department of Clinical Chemistry is also involved in a study of the Division of<br />
Molecular Biology (Marieke Vollebergh, Sabine Linn group). Amphiregulin, TGFa,<br />
IGF-1 and IGFBP-3 were measured in serum and pleura effusion of patients with<br />
non-small cell lung cancer to investigate their predictive value for Erlotinib response.<br />
In collaboration with the Department of Internal Medicine of the AMC, we measured<br />
S100B and NSE in elderly patients with hip fracture to investigate the possible<br />
associations with different subtypes of delirium. We found that S100B levels were<br />
higher during delirium from 1 day before up until 8 days after surgery. No significant<br />
difference in S100B or NSE levels was seen between the hyperactive, hypoactive and<br />
mixed subtype of delirium.<br />
Biomarker discovery using MALDI-MS based proteomics tools We are involved<br />
in a pilot study of the OncoProteomics Laboratory of the VUmc. In this pilot we will<br />
compare exoprotease activities in serum samples of diffent storage time. These<br />
exoprotease activities are measured by using MALDI-TOF mass spectrometric<br />
analysis of the degradation peptides of exogenous labeled substrates. Each labeled<br />
degradation peptide is quantitated by an internal standard peptide.<br />
DEPARTMENT OF NUCLEAR MEDICINE<br />
Clinical Nuclear Medicine<br />
Joke Baars, Axel Bex, Jose Belderbos, Jan Paul de Boer, Sjaak Burgers, Paul Baas, Kenneth Gilhuijs,<br />
Niels Graafland, Michel van den Heuvel, Simon Horenblas, Ingrid Kappers, Houke Klomp, Bin Kroon,<br />
Charlotte Lange, Joost Leijte, Wim Meinhardt, Omgo Nieweg, Hester Oldenburg, Iris van der Ploeg,<br />
Henk van der Poel, Emiel Rutgers, Marieke Straver, Marcel Verheij, Marie-Jeanne Vrancken-Peters,<br />
Babs Taal, Jelle Teertstra, Ly Tran, Michel Wouters, Tjeerd Aukema, Philippe Baars, Cornelis Hoefnagel,<br />
Marina Kartachova, Saar Muller, Ferida Sivro, Michiel Sinaasappel, Renato Valdés Olmos,<br />
Lenka Vermeeren, Wouter Vogel<br />
The extension of the hybrid imaging activities has led to new studies using<br />
SPECT-CT and PET-CT. The majority of current studies are based on the evaluation<br />
of the sentinel node procedure and the use of 18F-FDG PET-CT. New studies using<br />
PET tracers for imaging of hypoxia, apoptosis, and other tumor cell processes are<br />
ongoing. The pioneer activities with a portable gamma camera for real time<br />
intraoperative radioguided imaging will be complemented with a mini PET ring<br />
device for dedicated breast molecular imaging. Finally, radioinmunotherapy with<br />
131I-rituximab in non-Hodgkin’s lymphoma is in progress.<br />
SPECT-CT for sentinel node identification SPECT-CT, which was introduced at<br />
the <strong>NKI</strong>-AVL in 2006, evolved from two-dimensional to three-dimensional display<br />
(figure 1) and was further incorporated for the anatomical localization of sentinel<br />
nodes in various malignancies.<br />
In 30 out of 85 melanoma patients (35%) with conventional lymphoscintigrams that<br />
were difficult to interpret or showed unusual lymphatic drainage patterns or did not<br />
depict a sentinel node SPECT-CT led to significant more information for the surgeon;<br />
this resulted in a different incision in 17 patients, an incision at another site in 8 and<br />
101<br />
DIAGNOSTIC ONCOLOGY<br />
Stella Mook MD Graduate student<br />
Aafke Wieringa - Ariaens Technical staff<br />
Henrique Ruijter - Schippers Technical staff<br />
Esther Scheerman Technical staff<br />
Marjanka Schmidt PhD Post-doc<br />
Carla Schippers - Gillissen Technical staff<br />
Ivon Tielen Technical staff<br />
THE NETHERLANDS CANCER INSTITUTE<br />
FAMILY CANCER CLINIC<br />
Senno Verhoef MD PhD Head<br />
Frans Hogervorst PhD Academic staff, head<br />
Diagnostic Laboratory<br />
Laura van ’t Veer PhD Academic staff<br />
Irma Kluijt MD Academic staff<br />
Priscilla Axwijk MD Academic staff<br />
Eveline Bleiker Academic staff<br />
Daniela Hahn Academic staff<br />
Petra Nederlof PhD Academic staff<br />
Efraim Rosenberg Academic staff<br />
Sophie van der Velden Genetic associate<br />
Anja van Rens Genetic associate<br />
Gea Wigbout Genetic associate<br />
Marije Olsthoorn - Ooms Research assistant<br />
Daoud Ait Moha Research assistant<br />
Mohamed Achachah Technical staff<br />
Rob Plug Technical staff - Quality staff<br />
Roelof Pruntel Technical staff<br />
Majella Boutmy - de Lange Technical staff<br />
Esther Scheerman Technical staff<br />
Mobien Kasiem Technical staff<br />
Abderrahim Ajouaou Technical staff<br />
DEPARTMENT OF RADIOLOGY<br />
Jelle Teertstra MD Head<br />
Peter Besnard MD Academic staff<br />
Kenneth Gilhuijs PhD Academic staff<br />
Wim Koops MD Academic staff<br />
Charlotte Lange Academic staff<br />
Robert Kröger MD Academic staff<br />
Claudette Loo MD Academic staff<br />
Saar Muller PhD Academic staff<br />
Frank Pameijer MD PhD Academic staff<br />
Warner Prevoo MD Academic staff<br />
Michiel Sinaasappel PhD Academic staff<br />
Tanja Alderliesten PhD Post-doc<br />
Fijs van Leeuwen PhD Post-doc<br />
Christian Siedschlag PhD Post-doc<br />
Anita Paape Technical staff<br />
Kenneth Pengel Technical staff<br />
Lukas Batteau Technical staff<br />
Patrick Chin Post-doc<br />
Tessa Buckle Technical staff<br />
Anne van Leeuwen Graduate student<br />
Alexander Schmitz Graduate student<br />
Annemarie Schmitz Graduate student<br />
Jincey Sriram Post-doc
102<br />
DIAGNOSTIC ONCOLOGY<br />
Publications<br />
Ackerstaff AH, Balm AJ, Rasch CR,<br />
de Boer JP, Wiggenraad R, Rietveld DH,<br />
Gregor RT, Kröger R, Hilgers FJ. First-year<br />
quality of life assessment of an intra-arterial<br />
(RADPLAT) versus intravenous<br />
chemoradiation phase III trial. Head Neck.<br />
<strong>2008</strong> Oct 28 (epub ahead of print)<br />
Antoniou AC, Spurdle AB, Sinilnikova OM,<br />
Healey S, Pooley KA, Schmutzler RK,<br />
Versmold B, Engel C, Meindl A, Arnold N,<br />
Hofmann W, Sutter C, Niederacher D,<br />
Deissler H, Caldes T, Kämpjärvi K,<br />
Nevanlinna H, Simard J, Beesley J, Chen X;<br />
Kathleen Cuningham Consortium for<br />
Research into Familial Breast Cancer,<br />
Neuhausen SL, Rebbeck TR, Wagner T,<br />
Lynch HT, Isaacs C, Weitzel J, Ganz PA,<br />
Daly MB, Tomlinson G, Olopade OI, Blum JL,<br />
Couch FJ, Peterlongo P, Manoukian S,<br />
Barile M, Radice P, Szabo CI, Pereira LH,<br />
Greene MH, Rennert G, Lejbkowicz F,<br />
Barnett-Griness O, Andrulis IL, Ozcelik H;<br />
OCGN, Gerdes AM, Caligo MA, Laitman Y,<br />
Kaufman B, Milgrom R, Friedman E;<br />
Swedish BRCA1 and BRCA2 study<br />
collaborators, Domchek SM, Nathanson KL,<br />
Osorio A, Llort G, Milne RL, Benítez J,<br />
Hamann U, Hogervorst FB, Manders P,<br />
Ligtenberg MJ, van den Ouweland AM;<br />
DNA-HEBON collaborators, Peock S, Cook M,<br />
Platte R, Evans DG, Eeles R, Pichert G,<br />
Chu C, Eccles D, Davidson R, Douglas F;<br />
EMBRACE, Godwin AK, Barjhoux L,<br />
Mazoyer S, Sobol H, Bourdon V, Eisinger F,<br />
Chompret A, Capoulade C, Bressac-de<br />
Paillerets B, Lenoir GM, Gauthier-Villars M,<br />
Houdayer C, Stoppa-Lyonnet D; GEMO,<br />
Chenevix-Trench G, Easton DF; CIMBA.<br />
Common breast cancer-predisposition alleles<br />
are associated with breast cancer risk in<br />
BRCA1 and BRCA2 mutation carriers.<br />
Am J Hum Genet. <strong>2008</strong>;82:937-48<br />
Booman M, Szuhai K, Rosenwald A,<br />
Hartmann E, Kluin-Nelemans H, de Jong D,<br />
Schuuring E, Kluin P. Genomic alterations<br />
and gene expression in primary diffuse large<br />
B-cell lymphomas of immune-privileged sites:<br />
the importance of apoptosis and<br />
immunomodulatory pathways. J Pathol.<br />
<strong>2008</strong>;216:209-17<br />
Borgemeester MC, van den Brekel MW,<br />
van Tinteren H, Smeele LE, Pameijer FA,<br />
van Velthuysen ML, Balm AJ. Ultrasoundguided<br />
aspiration cytology for the assessment<br />
of the clinically N0 neck: Factors influencing<br />
its accuracy. Head Neck. <strong>2008</strong> Aug 14<br />
Figure 1: Three-dimensional anatomical display of fused SPECT-CT images using volume rendering<br />
showing drainage from the tumor injection site (T) to sentinel nodes (arrows) in oral cavity malignancy<br />
(A), melanoma of the trunk (B), breast cancer (C) and penile cancer (D).<br />
an extra incision in 5. In another 19 patients sentinel nodes were more clearly<br />
visualized by SPECT-CT than by conventional lymphoscintigraphy, but the surgical<br />
approach remained unchanged.<br />
In 134 breast cancer patients with unusual lymphatic drainage or a pattern that was<br />
difficult to interpret or non-visualization on conventional lymphoscintigraphy<br />
SPECT-CT was additionally performed. Fused SPECT-CT images showed additional<br />
sentinel nodes in 15 patients (11%) and enabled an accurate incision in 102 patients<br />
(76%), an extra incision in 6, and avoided an incision in two patients with spots due<br />
to skin contamination of the tracer.<br />
Besides two-dimensional display fused SPECT-CT images can be displayed using<br />
volume rendering which enables depiction of sentinel nodes in relation to<br />
surrounding muscle and bone structures in a 3D image. This modality was evaluated<br />
as an additional tool in 23 patients with melanoma and 7 with breast cancer. In 23<br />
patients (77%) a more precise anatomical location was accomplished leading to an<br />
adjustment of the surgical incision or exploration route in 15 (50%).<br />
Both two- and three-dimensional fused SPECT-CT were used to analyze the drainage<br />
of the groin in 50 melanoma patients according to the Daseler anatomical<br />
classification of the inguinal areas. Sentinel nodes were depicted in the superior<br />
lateral zone (10%), superior medial (13%), inferior medial (42%), central (26%), and<br />
external iliac (8%). Majority of second-tier nodes were located in the superior lateral<br />
zone (9%), inferior medial (5%), central (13%), external iliac (54%), common iliac<br />
(2%) and obturator fossa (6%). Tumor-positive sentinel nodes were found in the<br />
inferior medial (83%) and central (17%) zones. No lymphatic drainage was seen in<br />
the inferior lateral zone which suggests that this zone could be excluded from a<br />
complete dissection.<br />
SPECT-CT was also used to visualize tumor-blockage and rerouting of lymphatic<br />
drainage in 17 patients with penile cancer and proven unilateral inguinal lymph node<br />
metastasis. SPECT-CT showed tracer uptake in 4 of the 17 palpable metastastic<br />
nodes, no uptake in three, and rerouting to a neo-sentinel node in 10. These findings<br />
support the observation that metastatic involvement of a sentinel node can lead to<br />
blocked inflow and rerouting to a new sentinel node causing a false-negative<br />
procedure.<br />
In 45 out of 46 patients (98%) with prostate cancer, SPECT-CT led to anatomical<br />
sentinel node localization after transrectal ultrasound guided tracer administration.<br />
Sentinel nodes outside the area of extended pelvic lymphadenectomy were found at<br />
laparoscopy in 16 patients (35%). According to the extra anatomical information for
laparoscopy and additional visualized sentinel nodes SPECT-CT had a mayor contribu<br />
tion in 28 patients (61%). Sentinel node metastases were found in 15 patients (33%).<br />
Real time imaging for intraoperative sentinel node detection The use of a<br />
portable CsI(Na) gamma camera using an innovative device for simultaneous<br />
99m Tc/ 125 I detection for intraoperative laparoscopic sentinel node localization was<br />
evaluated in 16 patients with prostate cancer, 2 with testicular cancer and 2 with renal<br />
cell carcinoma. Six hours after tracer administration laparoscopy was performed and<br />
the portable camera equipped with a 4 mm opening pinhole collimator set to display<br />
both 99m Tc for SN visualisation and 125 I as a pointer for SN seeking. The matching of<br />
both signals, seen on screen during laparoscopy, indicated the site of the sentinel<br />
node, which was subsequently removed and measured (figure 2). The portable<br />
camera contributed to directly identify sentinel nodes in 18 patients (90%).<br />
This procedure was also useful to retrieve para-aortic sentinel nodes. In 12 out of<br />
14 patients with different malignancies and para-aortic drainage preoperative sentinel<br />
node identification by SPECT-CT and real time intraoperative imaging using the<br />
portable gamma camera led to surgical localization and removing of the sentinel<br />
nodes.<br />
More recently this approach was introduced for malignancies of the oral cavity<br />
contributing to identify 20 sentinel nodes in 4 patients. In all patients drainage was<br />
bilateral and all visualized sentinel nodes were found in levels I, II and III of the<br />
neck. Both SPECT-CT and the portable gamma camera were able to detect more<br />
sentinel nodes in the proximity of the injection area.<br />
Figure 2: Axial fused SPECT-CT (A) showing a left para-aortic sentinel node (arrow) which is<br />
subsequently localized at laparoscopy using intraoperative imaging with a portable gamma camera (B).<br />
This sentinel node is real time displayed (C) on screen (arrow) and after resection is controlled for<br />
radioactivity (D) by ex-vivo imaging by means of the gamma camera (arrows).<br />
PET-CT in lung cancer The role of 18 F-FDG PET-CT in the diagnosis of lung cancer<br />
in an approach of out-patient fast track assessment was evaluated in 114 patients with<br />
the suspicion of a malignancy on chest X-ray referred for further analysis to the <strong>NKI</strong>-<br />
AVL. A malignancy was diagnosed in 94 patients (82%) and in 91 of them PECT-CT<br />
was positive. In 10 out 20 patients with benign pulmonary lesions PET-CT was also<br />
positive. PET-CT led to the diagnosis of disseminated disease in 14 patients. It was<br />
concluded that PET-CT provides acceptable sensitivity and accuracy for rapid<br />
diagnosing lung cancer in new patients with in a 90% of the hypermetabolic lesions<br />
malignancy as outcome.<br />
In a multicenter trial 1059 FDG PET-CT scans of patients with suspected or proven<br />
lung cancer were retrospectively evaluated in order to evaluate the feasibility of FDG<br />
PET-CT limited to the thoracic range. Not scanning above the shoulders and below<br />
the caudal tip of the liver leads to missed proved second primaries in 0.7% and<br />
103<br />
DIAGNOSTIC ONCOLOGY<br />
Publications (continued)<br />
Borgemeester MC, van den Brekel MW,<br />
van Tinteren H, Smeele LE, Pameijer FA,<br />
van Velthuysen ML, Balm AJ. Ultrasoundguided<br />
aspiration cytology for the<br />
assessment of the clinically N0 neck: factors<br />
influencing its accuracy. Head Neck.<br />
<strong>2008</strong>;30:1505-13<br />
Boss DS, Valdés Olmos RA,<br />
Sinaasappel M, Beijnen JH, Schellens JH.<br />
Application of PET/CT in the development<br />
of novel anticancer drugs. Oncologist.<br />
<strong>2008</strong>;13:25-38<br />
Broeks A, Braaf LM, Huseinovic A,<br />
Schmidt MK, Russell NS, van Leeuwen FE,<br />
Hogervorst FB, Van ‘t Veer LJ. The<br />
spectrum of ATM missense variants and<br />
their contribution to contralateral breast<br />
cancer. Breast Cancer Res Treat.<br />
<strong>2008</strong>;107:243-8<br />
Bueno-de-Mesquita JM, Linn SC, Keijzer R,<br />
Wesseling J, Nuyten DS, van Krimpen C,<br />
Meijers C, de Graaf PW, Bos MM, Hart AA,<br />
Rutgers EJ, Peterse JL, Halfwerk H,<br />
de Groot R, Pronk A, Floore AN, Glas AM,<br />
Van ‘t Veer LJ, van de Vijver MJ.<br />
Validation of 70-gene prognosis signature in<br />
node-negative breast cancer. Breast Cancer<br />
Res Treat. <strong>2008</strong> Sep 26 (epub ahead of<br />
print)<br />
Claes A, Gambarota G, Hamans B,<br />
van Tellingen O, Wesseling P, Maass C,<br />
Heerschap A, Leenders W. Magnetic<br />
resonance imaging-based detection of glial<br />
brain tumors in mice after antiangiogenic<br />
treatment. Int J Cancer <strong>2008</strong>;122:1981-6<br />
de Boer JP, Hiddink RF, Raderer M,<br />
Antonini N, Aleman BM, Boot H,<br />
de Jong D. Dissemination patterns in nongastric<br />
MALT lymphoma. Haematologica.<br />
<strong>2008</strong>;93:201-6<br />
de Jong D, Enblad G. Inflammatory cells<br />
and immune microenvironment in<br />
malignant lymphoma. J Intern Med.<br />
<strong>2008</strong>;264:528-36<br />
de Jong D, Vasmel WL, de Boer JP,<br />
Verhave G, Barbé E, Casparie MK,<br />
van Leeuwen FE. Anaplastic large-cell<br />
lymphoma in women with breast implants.<br />
JAMA. <strong>2008</strong>;300:2030-5
104<br />
DIAGNOSTIC ONCOLOGY<br />
Publications (continued)<br />
de Jong D, Xie W, Rosenwald A,<br />
Chhanabhai M, Gaulard P, Klapper W,<br />
Lee A, Sander B, Thorns C, Campo E,<br />
Molina T, Hagenbeek A, Horning S,<br />
Lister A, Raemaekers J, Salles G,<br />
Gascoyne R, Weller E.<br />
Immunohistochemical prognostic markers in<br />
diffuse large B-cell lymphoma: validation of<br />
tissue microarray as a prerequisite for broad<br />
clinical applications. A study from the<br />
Lunenburg Lymphoma Biomarker<br />
Consortium (LLBC). J Clin Pathol. <strong>2008</strong><br />
Sep 15 (epub ahead of print)<br />
de Vreeze RS, de Jong D, Haas RL,<br />
Stewart F, van Coevorden F. Effectiveness<br />
of radiotherapy in myxoid sarcomas is<br />
associated with a dense vascular pattern.<br />
Int J Radiat Oncol Biol Phys.<br />
<strong>2008</strong>;72:1480-7<br />
de Vreeze RS, de Jong D, Tielen IH,<br />
Ruijter HJ, Nederlof PM, Haas RL,<br />
van Coevorden F. Primary retroperitoneal<br />
myxoid/round cell liposarcoma is a<br />
nonexisting disease: an immunohistochemical<br />
and molecular biological<br />
analysis. Mod Pathol. <strong>2008</strong> Sep 26<br />
(epub ahead of print)<br />
de Vreeze RS, Koops W, Haas RL,<br />
van Coevorden F. An unusual case of<br />
hemosiderotic fibrohistiocytic lipomatous<br />
lesion: correlation of MRI and pathologic<br />
findings. Sarcoma. <strong>2008</strong>;<strong>2008</strong>:893918<br />
de Vries NA, Zhao J, Kroon E, Buckle T,<br />
Beijnen JH, van Tellingen O.<br />
P-glycoprotein and breast cancer resistance<br />
protein: two dominant transporters working<br />
together in limiting the brain penetration of<br />
topotecan. Clin Cancer Res 200713:6440-9<br />
Gambarota G, Leenders W, Maass C,<br />
Wesseling P, van der Kogel B,<br />
van Tellingen O, Heerschap A.<br />
Characterisation of tumour vasculature in<br />
mouse brain by USPIO contrast-enhanced<br />
MRI. Br J Cancer <strong>2008</strong>;98:1784-9<br />
Garcia-Herrera A, Colomo L, Camós M,<br />
Carreras J, Balague O, Martinez A,<br />
Lopéz-Guillermo A, Estrach T, Campo E.<br />
Primary cutaneous small/medium CD4+<br />
T-cell lymphomas: a heterogeneous group of<br />
tumors with different clinicopathologic<br />
features and outcome. J Clin Oncol.<br />
<strong>2008</strong>;26:3364-71<br />
incorrect staging with therapeutic consequences in 1.2% of the cases, while reducing<br />
the number of irrelevant findings in 20%, and reducing additional investigations,<br />
stress, and cost in 10%. Limited range of PET-CT scanning may be a feasible<br />
alternative for initial staging of lung cancer, allowing faster patient throughput on<br />
scarce PET resources.<br />
Sequential 18 F-FDG PET-CT was used to evaluate metabolic tumor response in<br />
operable stage I/II non-small cell lung cancer patients receiving preoperative<br />
neoadjuvant erlotinib daily for a period of 3 weeks. In 3 out of 12 patients PET-CT<br />
revealed >50% decrease in tumour uptake, while pathology showed a significant<br />
response in 5 patients. In another study 18 F-FDG PET-CT was used to assess tumor<br />
response in locally advanced non-small cell lung carcinoma patients receiving<br />
Cetuximab, in combination with concurrent chemo- / radiotherapy. In 3 out of 12<br />
patients near or complete metabolic tumor response while in another patient more<br />
than 50% tumor uptake reduction was observed 8 weeks after treatment.<br />
PET-CT in breast cancer In 19 consecutive T2N1 breast cancer patients PET-CT<br />
studies in prone position were evaluated in the context of the adjustment of the<br />
design of a new dedicated mini-PET ring camera aimed for screening and biopsy of<br />
FDG avid lesions (European project MAMMI EU-037555). The hanging breast<br />
diameters varied from 7 to 15 cm and the length from 5 to 14 cm. In 17 out of 19<br />
patients (89%) lesions were measurable for FDG uptake with an average SUVmax of<br />
5.7 (range 2.6-14.4) and tumor/background ratio of 14.2 (4-58). For normal breast<br />
SUVmax was 0.4 (0.2-1.4). Prone position allowed better tumor delineation than<br />
supine position. The dedicated mini-PET detector-ring system, which will be<br />
clinically validated the next year, will be allocated inside a device for hanging the<br />
breast and is expected to reach a spatial resolution near 1.5 mm.<br />
SPECT-CT for tumor imaging SPECT-CT acquired 1 hr after injecting 99m Tc-MIBI<br />
was evaluated in the prediction of early chemotherapy response in 11 patients with<br />
stage IIIB or IV NSCLC. MIBI studies were performed within 24 hours before<br />
starting platinum-containing chemotherapy. The relationship between prechemotherapy<br />
lesion uptake and tumor size change at 4 weeks after chemotherapy<br />
completion was analyzed. The intensity of 99m Tc-MIBI lesion uptake correlated<br />
significantly with reduction in tumor size suggesting that the tracer can be used to<br />
predict early treatment efficacy and disease progression in advanced lung cancer.<br />
The visual and quantitative evaluation of tumor uptake of 99m Tc Hynic-rh-Annexin V<br />
was evaluated in 38 patients with various malignancies. SPECT was acquired before<br />
and within two days after the start of therapy and maximal counts per pixel were<br />
calculated in the tumor volume for every target lesion. Visual evaluation of tumor<br />
uptake was based on a 4-grade score. Good intra-observer and inter-observer<br />
reproducibility were found with significant correlation between changes in tumor<br />
uptake and therapy outcome.<br />
Radionuclide therapy Radionuclide therapy using 131 I-MIBG was evaluated in 12<br />
patients with malignant pheochromocytoma and 7 with paraganglioma. Median<br />
cumulative dose was 22.2 GBq (6.8-81.4). Objective tumor response, assessed by<br />
monitoring of bidirectional tumor size reduction on CT or MRI, was achieved in 47%<br />
of the patients. Biochemical response rate was 67% and symptomatic response 89%.<br />
Overall median follow-up was 29 months and hematological side effects were<br />
observed seen in 26% of the patients.<br />
Treatment of differentiated thyroid carcinoma was modernized by the introduction of<br />
recombinant human TSH (rhTSH) for selected patient groups. In low-risk patients<br />
stimulation with rhTSH allows 131 I ablation of functional thyroid remnants after<br />
thyroidectomy without withdrawal of thyroid hormone suppletion, thus improving<br />
quality of life. In high-risk patients with a relative contraindication for a deep<br />
hypothyroidic state, rhTSH allows high-dose 131 I treatment with a low risk on<br />
complications.
Molecular Imaging Nuclear Medicine<br />
Fijs van Leeuwen, Tessa Buckle, Anne van Leeuwen, Patrick Chin, Henk van de Poel, Emiel Rutgers,<br />
Michiel Sinaasappel, Jos Jonkers, Renato Valdés Olmos<br />
New molecular imaging approaches for tumor staging and response<br />
monitoring One of the main challenges in surgical oncology is intraoperative<br />
detection of preoperatively defined lesions. At present (molecular) imaging is a<br />
rapidly growing research field aimed at non-invasive, but accurately, diagnosing<br />
disease. Consequently, it becomes of increasing importance that the surgical<br />
procedures stay ‘up to date’ and enable the incorporation of such novel imaging<br />
approaches. To achieve such incorporation, we are developing intraoperative imaging<br />
approaches that are complementary to radioactivity based preoperative surgical<br />
planning procedures.<br />
Intraoperative visualization of the sentinel lymph node In mouse models<br />
representative for metastatic breast cancer and prostate cancer, we have investigated<br />
the value of intraoperative fluorescence imaging during sentinel lymph node<br />
dissection procedures. For these studies, we have used the current clinical standard<br />
99m Tc-NanoColl and visible blue dye as reference for near infra red fluorescence<br />
procedures with indocyanine green. Intraoperatively the latter shows great potential<br />
in both breast and prostate cancer. In both breast and prostate models the<br />
fluorescence guidance is significantly better than the one obtained with visible blue<br />
dyes only. Data from our preclinical studies have already prompted application for<br />
clinical pilot studies.<br />
Development of targeted imaging agents for surgical guidance Targeted<br />
intraoperative imaging procedures require imaging agents with a high tumor<br />
specificity. For this purpose we target a common marker for metastatic cancer, the<br />
chemokine receptor CXCR4. In mammary mouse tumor models we investigated the<br />
diagnostic potential of a radiolabeled peptide antagonists agents CXCR4 (figure 3).<br />
Furthermore, nanotechnology is exploited for its potential in high intensity<br />
fluorescence imaging. Nanoparticles such as quantum dots have a bright and narrow<br />
photoemission making them suitable for intraoperative imaging studies. Usually, the<br />
use of heavy metals like Cd and Pb in quantum dots limits their use in vivo. As a<br />
consequence, we are currently investigating alterative ‘non toxic’ quantum dots.<br />
Alternatively we are making use of multivalent dendritic structures in order to<br />
improve the fluorescence intensity and tumor specificity.<br />
Figure 3: CXCR4 targeted imaging agents for surgical guidance: a) Overlay of X-ray and bioluminescent<br />
images accurately depict the location of the primary tumor and lymphatic metastases, B) Distribution (iv<br />
injection) of a 125I-labeled cyclic peptide that serves as an antagonist to CXCR4 revealing large amounts of<br />
unspecific staining, and C) The same imaging agent after locoregional injection, demonstrating a higher<br />
tumor specificity.<br />
105<br />
DIAGNOSTIC ONCOLOGY<br />
Publications (continued)<br />
Gedik GK, Hoefnagel CA, Bais E,<br />
Valdés Olmos RA. 131I-MIBG therapy in<br />
metastatic phaeochromocytoma and<br />
paraganglioma. Eur J Nucl Med Mol<br />
Imaging. <strong>2008</strong> 35:725-33<br />
Geurts TW, Balm AJ, van Velthuysen ML,<br />
van Tinteren H, Burgers JA, vanZandwijk N,<br />
Klomp HM. Survival after surgical<br />
resection of pulmonary metastases and<br />
second primary squamous cell lung<br />
carcinomas in head and neck cancer. Head<br />
Neck. <strong>2008</strong> Oct 28 (epub ahead of print)<br />
Geurts TW, van Velthuysen MLF,<br />
Broekman F, Hooft van Huysduynen T,<br />
van den Brekel MWM, van Zandwijk N,<br />
van Tinteren H, Nederlof PM, Balm AJM,<br />
Brakenhoff RH. Clin Cancer Res <strong>2008</strong> (in<br />
press)<br />
Gunn SR, Mohammed MS, Gorre ME,<br />
Cotter PD, Kim J, Bahler DW,<br />
Preobrazhensky SN, Higgins RA,<br />
Bolla AR, Ismail SH, de Jong D, Eldering<br />
E, van Oers MH, Mellink CH, Keating MJ,<br />
Schlette EJ, Abruzzo LV, Robetorye RS.<br />
Whole-genome scanning by array<br />
comparative genomic hybridization as a<br />
clinical tool for risk assessment in chronic<br />
lymphocytic leukemia. J Mol Diagn.<br />
<strong>2008</strong>;10:442-51<br />
Hartog H, van der Graaf WT, Wesseling J,<br />
van der Veer E, Boezen HM. Measurement<br />
of insulin-like growth factor-1 and insulinlike<br />
growth factor binding protein-3 after<br />
delayed separation of whole blood samples.<br />
Clin Biochem. <strong>2008</strong>;41:636-9<br />
Hoebers FJ, Kartachova M, De Bois J,<br />
Van den Brekel MW, Van Tinteren, H,<br />
Van Herk M, Rasch CR, Valdés Olmos RA,<br />
Verheij M. 99mTc Hynic-rh-Annexin V<br />
scintigraphy for in vivo imaging of apoptosis<br />
in patients with head and neck cancer<br />
treated with chemoradiotherapy. Eur J Nucl<br />
Med Mol Imaging. <strong>2008</strong>;35:509-18<br />
Hoebers FJ, Pameijer FA, de Bois J,<br />
Heemsbergen W, Balm AJ, Schornagel JH,<br />
Rasch CR. Prognostic value of primary<br />
tumor volume after concurrent<br />
chemoradiation with daily low-dose<br />
cisplatin for advanced-stage head and neck<br />
carcinoma. Head Neck. <strong>2008</strong>;30:1216-23
106<br />
DIAGNOSTIC ONCOLOGY<br />
Publications (continued)<br />
Hoefnagel CA, Sivro F. Hoofdstuk<br />
Radionucliden therapie. In:<br />
Van den Broek W, Barneveld P, Lemstra<br />
C, Van Urk P: Leerboek Nucleaire<br />
Geneeskunde, Elsevier <strong>2008</strong>:559-577<br />
Hofstra RM, Spurdle AB, Eccles D,<br />
Foulkes WD, de Wind N, Hoogerbrugge N,<br />
Hogervorst FB; for the IARC Unclassified<br />
Genetic Variants Working GroupThe<br />
members of the IARC Working Group are<br />
listed in the Appendix. The International<br />
Agency for Research on Cancer (IARC),<br />
based in Lyon, France, is part of the World<br />
Health Organization. Tumor characteristics<br />
as an analytic tool for classifying genetic<br />
variants of uncertain clinical significance.<br />
Hum Mutat. <strong>2008</strong>;29:1292-1303<br />
Holm C, Kok M, Michalides R, Fles R,<br />
Koornstra R, Wesseling J, Hauptmann M,<br />
Neefjes J, Peterse J, Stål O, Landberg G,<br />
Linn S. Phosphorylation of the oestrogen<br />
receptor alpha at serine 305 and prediction<br />
of tamoxifen resistance in breast cancer.<br />
J Pathol. <strong>2008</strong> Sep 23 (epub ahead of print)<br />
Holstege H, Joosse SA, van Oostrom CTM,<br />
Nederlof PM, de Vries A3, and Jonkers J.<br />
Increased frequency of p53 truncating<br />
mutations in BRCA1-associated breast<br />
cancer. Submitted.<br />
Hoogendoorn WE, Hollema H,<br />
van Boven HH, Bergman E,<br />
de Leeuw-Mantel G, Platteel I, Fles R,<br />
Nederlof PM, Mourits MJ, van Leeuwen FE.<br />
Comprehensive Cancer Centers TAMARISKgroup.<br />
Prognosis of uterine corpus cancer after<br />
tamoxifen treatment for breast cancer. Breast<br />
Cancer Res Treat. <strong>2008</strong>;112:99-108<br />
Horlings HM, van Laar RK, Kerst JM,<br />
Helgason HH, Wesseling J,<br />
van der Hoeven JJ, Warmoes MO, Floore A,<br />
Witteveen A, Lahti-Domenici J, Glas AM,<br />
Van ‘t Veer LJ, de Jong D. Gene expression<br />
profiling to identify the histogenetic origin of<br />
metastatic adenocarcinomas of unknown<br />
primary. J Clin Oncol. <strong>2008</strong>;26:4435-41<br />
Jansen MC, van Wanrooy S,<br />
van Hillegersberg R, Rijken AM,<br />
van Coevorden F, Prevoo W, van Gulik TM.<br />
Assessment of systemic inflammatory<br />
response (SIR) in patients undergoing<br />
radiofrequency ablation or partial liver<br />
resection for liver tumors. Eur J Surg Oncol.<br />
<strong>2008</strong>;34:662-7<br />
DEPARTMENT OF PATHOLOGY<br />
(Part of the research of the Pathology Department is carried out within Division of<br />
Experimental Therapy and is described there)<br />
MOLECULAR PATHOLOGY<br />
EGFR mutation in lung carcinomas and response to Iressa therapy<br />
Daphne de Jong, Petra Nederlof, Erik Thunissen, Michel van den Heuvel, Marieke Vollebergh,<br />
Henrique Ruijter, Ivon Tielen, Lucie Boerrigter, Aafke Wieringa-Ariaens<br />
Gefitinib (Iressa, ZD1839), an inhibitor of epidermal growth factor receptor-tyrosine<br />
kinase (EGFR-TK), has shown potent anti-tumor effects and improved symptom and<br />
quality-of-life of a subset of patients with advanced NSCLC. However, a large portion<br />
of the patients does not respond to this agent. Recent publications show a correlation<br />
between the presence of a mutation (small in-frame deletion or point mutation) in<br />
exon 18, 19, 20 or 21 of the EGFR gene and response to therapy. Currently we are<br />
investigating tumour characteristics such as pathological parameters, EGFR and<br />
KRAS mutation status and EGFR gene amplification (CISH) of a large series of<br />
NSCLC from our institute and the VU medical centre (Dr Erik Thunissen) and<br />
correlation with response to Iressa therapy.<br />
c-KIT mutation in Gastrointestinal Stromal Tumors (GIST) and response to<br />
imatinib therapy<br />
Both activating c-KIT mutations and mutations resulting in resistance to imatinib<br />
therapy have been described in literature. Mutation analysis of c-KIT and PDGFRA is<br />
performed on small tumor biopsies and preliminary results indeed reveal single or<br />
multiple mutations in primary tumors and resistant recurrences.<br />
HNSCC 2nd tumor versus metastasis by LOH and p53 mutation analysis<br />
T.W. Geurts1 , Loes van Velthuysen, F. Broekman2 , T. Hooft van Huysduynen2 , Michiel van den Brekel,<br />
Nico van Zandwijk, Harm van Tinteren, Petra Nederlof, Fons Balm, Ruud Brakenhoff2 Patients treated for head and neck cancer often develop a lung tumor that can be<br />
diagnosed as distant metastasis (DM) or second primary tumor (SPT). Clinical and<br />
histopathological criteria are unreliable for differentiation, while patterns of allelic<br />
loss (LOH) might be more suited. In this study we use TP53 mutation analysis for<br />
validation of an allelic loss marker panel and a decision algorithm that we have been<br />
using to distinguish DM and SPT in curatively treated head and neck squamous cell<br />
carcinoma (HNSCC) patients with newly diagnosed pulmonary squamous cell<br />
carcinoma.<br />
The TP53 mutation data validate the suitability of the LOH marker panel and decision<br />
algorithm for differential diagnosis of DM and SPT in the lung. LOH analysis can<br />
theoretically be exploited in all cases and is less laborious than mutation analysis.<br />
Gene Expression Profiling to identify the histogenetic origin of metastatic<br />
adenocarcinomas of unknown primary<br />
Hugo Horlings, Ryan van Laar1 , Martijn Kerst, Helgi Helgason, Jelle Wesseling, Annuska Glas3 ,<br />
Laura van ’t Veer, Daphne de Jong<br />
Patients with adenocarcinoma of unknown primary origin (ACUP) constitute<br />
approximately 4% of all malignancies. For effective treatment of these patients, it is<br />
considered optimal to identify the primary tumor origins. Currently, the success rate<br />
of the diagnostic work-up is only 20% to 30%.<br />
Our goal was to evaluate the contribution of gene expression profiling for routine<br />
clinical practice in patients with ACUP. Formalin-fixed, paraffin-embedded (FFPE)<br />
samples for a validation series of 84 patients with a known primary adenocarcinoma<br />
and a testing series of 38 patients with ACUP was collected. An extensive<br />
immunohistochemical panel classified 16 of the patients with ACUP, whereas 22<br />
1Academic Medical Center<br />
2VU University Medical Center, Amsterdam<br />
3Agendia BV, Amsterdam
patients remained unclassified for their histogenetic origin. Information about<br />
staging procedures and clinical follow-up were available in all patient cases. The gene<br />
expression–based assay classified the primary site correctly in 70 (83%) of 84 patient<br />
cases of primary and metastatic tumors of known origin, with good sensitivity for the<br />
majority of the tumor classes and relatively poor sensitivity for primary lung<br />
adenocarcinoma. Gene expression profiling identified 15 (94%) of 16 patients with<br />
initial ACUP who were classified by immunohistochemistry, and it made a valuable<br />
contribution to a potential site of origin in 14 of the 22 patients with ACUP.<br />
Therefore it can be concluded that the gene expression platform can classify correctly<br />
from FFPE samples the majority of tumor classes both in patients with known<br />
primary and in patients with ACUP and this method may be an attractive additional<br />
analytic approach to assist with the histogenetic diagnosis of patients with ACUP.<br />
These findings are now prospectively explored in a prospective, diagnostic<br />
multicenter trial (EORTC CU-003-CR).<br />
Molecularly supported differentiation models in liposarcoma with<br />
consequences for diagnostic and clinical management<br />
Ronald de Vreeze, Henrique Ruijter, Ivon Tielen, Lucie Boerrigter, Aafke Ariaens, Petra Nederlof,<br />
Rick Haas, Frits van Coevorden, Daphne de Jong<br />
Liposarcoma can be distinguished in well-defined disease entities, characterized by<br />
morphology and in part by genetic alterations as well-differentiated, de-differentiated,<br />
myxoid, round cell (MRLS), and pleomorphic. Well-differentiated (WLS) and dedifferentiated<br />
liposarcoma is characterized by amplification of the 12q13-15 region<br />
including MDM2 and CDK4 genes. MRLS is characterized by a t(12;16) or t(12;22)<br />
translocation. These classifying markers have been used to explore several clinical<br />
and biological aspects of these diseases and their interrelations. The metastatic or<br />
second primary nature of MRLS, defined as tumor presentation in at least two<br />
separate sites before manifestation in the lungs, is a matter of debate with essential<br />
clinical consequences. Using the detailed molecular composition of the translocation<br />
and loss-of-heterozygozity (LOH) analysis, we were able to show that apparent<br />
multifocality actually represents metastatic disease in all instances. Moreover, LOH<br />
patterns showed that MRLS is characterized by extensive and ongoing mutation over<br />
time, resulting in a relatively large spectrum of coexisting subclones as compared to<br />
other solid tumors.<br />
In rare instances, mixed-type liposarcomas with components of MRLS and WDL are<br />
seen. On the basis of MRI features, these cases were collected from the files of the<br />
<strong>NKI</strong>-AVL. Molecular analysis showed that these mixed type liposarcomas should not<br />
be regarded as collision tumors, but as an extreme variant of the morphological<br />
spectrum within a single biological entity. These findings support a stem cell model<br />
with different levels of maturation within specific disease entities and expands the<br />
morphological spectrum of MRLS. This stem cell model is currently further explored<br />
in a large series of lipomatous tumors encompassing the complete spectrum from<br />
undifferentiated mesenchymal proliferations to terminally differentiated tumors.<br />
Tumor microenvironment in malignant lymphoma<br />
Jan Paul de Boer, Martijn Kerst, Joke Baars, Colette van de Bogaard, Dennis Veldhuizen,<br />
Martine van Glabbeke, John Raemaekers, Lunenburg Lymphoma Biomarker Consortium,<br />
Daphne de Jong<br />
Over the past few years, it has become clear that the biological and clinical behavior<br />
of malignant lymphoma is not only determined by the properties of the tumor cells<br />
themselves, but are also largely by the interaction of the tumor cells with their nonmalignant<br />
microenvironment. The composition and functional status of the tumor<br />
microenvironment is highly variable between different classes of malignant<br />
lymphoma and may provide both growth-supportive and growth suppressive signals<br />
via components of the adaptive and innate immune response. We have been mostly<br />
involved in studies in this field in follicular lymphoma (FL) and gastric marginal<br />
zone lymphoma, MALT-type. The precise nature of the cell populations in FL is still<br />
unclear and published data on their prognostic significance are highly conflicting.<br />
This may be partly due to heterogeneous composition of both patient series and<br />
107<br />
DIAGNOSTIC ONCOLOGY<br />
Publications (continued)<br />
Joosse SA, van Beers EH, Tielen IH,<br />
Horlings H, Peterse JL, Hoogerbrugge N,<br />
Ligtenberg MJ, Wessels LF, Axwijk P,<br />
Verhoef S, Hogervorst FB, Nederlof PM.<br />
Prediction of BRCA1-association in hereditary<br />
non-BRCA1/2 breast carcinomas with<br />
array-CGH. Breast Cancer Res Treat. <strong>2008</strong><br />
Joosse SA, van Beers EH, Tielen IH,<br />
Horlings H, Peterse JL, Hoogerbrugge N,<br />
Ligtenberg MJ, Wessels LF, Axwijk P,<br />
Verhoef S, Hogervorst FB, Nederlof PM.<br />
Prediction of BRCA1-association in<br />
hereditary non-BRCA1/2 breast carcinomas<br />
with array-CGH. Breast Cancer Res Treat.<br />
<strong>2008</strong> Aug 14. (epub ahead of print)<br />
Kappers I, Klomp HM, Burgers JA,<br />
Van Zandwijk N, Haas RL, van Pel R.<br />
Neoadjuvant (induction) erlotinib response<br />
in stage IIIA non-small-cell lung cancer.<br />
J Clin Oncol. <strong>2008</strong>;26:4205-7<br />
Kartachova MS, Valdés Olmos RA, Haas,<br />
Hoebers FJ, Van Herk M, Verheij M.<br />
99mTc-HYNIC-rh-annexin-V scintigraphy;<br />
visual and quantitative evaluation of early<br />
treatment-induced apoptosis to predict<br />
treatment outcome. Nucl Med Commun.<br />
<strong>2008</strong>;29:39-44<br />
Kartachova MS, Verheij M, van Eck BL,<br />
Hoefnagel CA, Valdés Olmos RA.<br />
Radionuclide Imaging of apoptosis in<br />
maliganancies: promise and pitfalls of<br />
99mTc-Hynic-rh-annexin V Imaging. Clin<br />
Med Oncol <strong>2008</strong>;1:319-25<br />
Knoops L, de Jong D. The role of the p53<br />
pathway in the treatment of follicular<br />
lymphoma. Cell Cycle. <strong>2008</strong>;7:436-9<br />
Korse CM, Bonfrer JM, Aaronson NK,<br />
Hart AA, Taal BG. Chromogranin A as an<br />
Alternative to 5-Hydroxyindoleacetic Acid in<br />
the Evaluation of Symptoms during<br />
Treatment of Patients with Neuroendocrine<br />
Tumors. Neuroendocrinology <strong>2008</strong> October 7<br />
Kroon BB, Hoefnagel CA,<br />
Valdés Olmos RA, Nieweg OE. Regional<br />
lymph nodes at a distance Ned Tijdschr<br />
Geneeskd. <strong>2008</strong>;152:1997-2000<br />
Kroon BK, Leijte JA, van Boven H,<br />
Wessels LF, Velds A, Horenblas S,<br />
Van ‘t Veer LJ. Microarray gene-expression<br />
profiling to predict lymph node metastasis<br />
in penile carcinoma. BJU Int. <strong>2008</strong>;<br />
102:510-5
108<br />
DIAGNOSTIC ONCOLOGY<br />
Publications (continued)<br />
Leijte JA, Valdés Olmos RA, Nieweg OE,<br />
Horenblas S. Anatomical Mapping of<br />
lymphatic Drainage in Penile Carcinoma<br />
with SPECT-CT: Implications for the<br />
Extent of Inguinal Lymph Node Dissection.<br />
Eur Urol. <strong>2008</strong>;54:885-92<br />
Loo CE, Teertstra HJ, Rodenhuis S,<br />
van de Vijver MJ, Hannemann J, Muller SH,<br />
Peeters MJ, Gilhuijs KG. Dynamic contrastenhanced<br />
MRI for prediction of breast cancer<br />
response to neoadjuvant chemotherapy: initial<br />
results.AJR Am J Roentgenol. <strong>2008</strong>;191:1331-8<br />
Lukas A, van der Weide M, Boogerd W,<br />
Prevoo W, Zuurmond WW, Sanders M.<br />
Adhesive rachnoiditis following<br />
percutaneous cervical cordotomy--may we<br />
still use lipiodol? J Pain Symptom Manage.<br />
<strong>2008</strong>;36:e1-4<br />
Marchetti S, de Vries NA, Buckle T, Bolijn MJ,<br />
van Eijndhoven MA, Beijnen JH, Mazzanti R,<br />
van Tellingen O, Schellens JH. Effect of the<br />
ATP-binding cassette drug transporters ABCB1,<br />
ABCG2, and ABCC2 on erlotinib hydrochloride<br />
(Tarceva) disposition in in vitro and in vivo<br />
pharmacokinetic studies employing Bcrp1-/-/<br />
Mdr1a/1b-/- (triple-knockout) and wild-type<br />
mice. Mol Cancer Ther <strong>2008</strong>;7:2280-7<br />
Meijnen P, Gilhuijs KGA, Rutgers EJ.Th.<br />
The effect of margins of the clinical<br />
management of ductal carcinoma in situ of<br />
the breast, J. surgical oncology, <strong>2008</strong> (in<br />
press). van Leeuwen FWB, Buckle T.,<br />
Kersbergen A., Rottenberg S., Gilhuijs KGA.<br />
Non-invasive functional imaging of<br />
P-glycoprotein-mediated doxorubicin<br />
resistance in a mouse model for hereditary<br />
breast cancer to predict response, and assign<br />
P-gp inhibitor sensitivity, Eur. J. Nucl.<br />
Med. Mol. Im., <strong>2008</strong> (in press).<br />
Meinhardt W, Valdés Olmos RA,<br />
Van der Poel HG, Bex A, Horenblas S.<br />
Laparoscopic sentinel node dissection for<br />
prostate carcinoma: technical and<br />
anatomical observations. BJU Int.<br />
<strong>2008</strong>;102:714-7<br />
Monzo M, Navarro A, Bandres E, Artells R,<br />
Moreno I, Gel B, Ibeas R, Moreno J,<br />
Martinez F, Diaz T, Martinez A, Balagué O,<br />
Garcia-Foncillas J. Overlapping expression of<br />
microRNAs in human embryonic colon and<br />
colorectal cancer. Cell Res. <strong>2008</strong>;18:823-33<br />
treatments. Therefore, we explored this feature in an immunohistochemicl study in<br />
patients treated in an EORTC/BNLI trial comparing fludarabine to CVP<br />
chemotherapy. Most importantly, some T-cell and accessory cell populations showed<br />
an homogeneous prognostic impact, while others had a different and sometimes<br />
opposite effect in the treatment arms. FoxP3 regulatory T-cells and CD68 positive<br />
macrophages seem to be key players in the growth regulation of FL cells. Within the<br />
<strong>NKI</strong>-AVL, this feature is now further explored by FACS analysis on pre- and post<br />
treatment cytological aspirates in FL patients.<br />
Our department is the pathology coordinator of a large international consortium of<br />
lymphoma trial organizations (including a.o. EORTC, HOVON, BNLI, GELA, ECOG)<br />
who jointly validate and study the prognostic impact of (immunohistochemical)<br />
biomarkers in large cohorts of uniformly treated lymphoma patients, concentrating<br />
on FL and diffuse large B-cell lymphoma (DLBCL). Validation studies for DLBCL<br />
were concluded and a series of over 1200 cases of DLBCL has been evaluated for 8<br />
potential prognostic markers. Biological prognostic indices (BIPI) has been<br />
developed for these patients that stratify specific groups of DLBCL with high<br />
accuracy. Similar studies in FL have been started, including a validation study.<br />
Development of a human in mouse model of breast carcinoma to optimize<br />
alkylating or targeted treatment<br />
Petra ter Brugge, Petra Kristel, Jos Jonkers, Jelle Wesseling<br />
In recent years, significant progress has been made in identifying the various types of<br />
breast cancer by histogenetic criteria. The best known classification concerning<br />
invasive ductal carcinoma not otherwise specified (IDC NOS) comprises ER-positive<br />
luminal A and B tumors, HER2-positive tumors, and basal-like tumors (Sorlie et al.,<br />
PNAS 2001; 98:10869). Such classifications have far reaching consequences for the<br />
prognosis and response to therapy in breast cancer. The basal-like, for which no<br />
adequate targeted therapy is available yet, and the HER2-positive tumors have a<br />
worse prognosis and occur at a younger age. Moreover, these tumors tend to develop<br />
resistance to conventional and targeted therapy very rapidly. Therefore, more efficient<br />
therapy is warranted to improve patient survival.<br />
In the development of new therapies, the availability of preclinical in vivo models is of<br />
great importance. Ideally, these in vivo models would recapitulate the heterogeneity<br />
in histogenetic characteristics, expression profiles, signaling pathways, and response<br />
to therapy found in human breast cancers. Xenograft models, in which human tumor<br />
tissue is engrafted directly into recipient mice, could be very suitable for this purpose.<br />
However, only a limited number of breast tumor xenografts have been established so<br />
far (Marangoni et al., Clin. Cancer Res. (2007) 13: 3989).<br />
We therefore decided to set up a panel of xenografts in which human breast<br />
carcinomas can be studied in a dynamic and reproducible manner. These xenograft<br />
models can than be used to analyse e.g. tumor progression, invasion, response to<br />
therapy, and acquisition of drug resistance. Furthermore, these models could also be<br />
used to test (combinations of) new targeted therapeutics. We will use our panel of<br />
breast cancer xenografts to analyse the response of homologous recombination<br />
deficient tumors on alkylating agents and PARP inhibitors. Second, we will study<br />
factors that determine sensitivity and resistance to SERMs in estrogen receptor (ER)<br />
positive breast cancer xenografts.<br />
We have recently started to set up the xenograft models. For this, fresh breast cancer<br />
fragments are obtained from the Department of Pathology. Fragments are cut into<br />
small pieces and implanted into mammary fat pads of immunocompromised mice.<br />
So far, pieces from 27 different breast tumors have been implanted. Of these, 13 were<br />
found to be ER-positive. Five of the tumors were positive for HER2 amplification,<br />
including 3 of the ER positive tumors. Of the remaining tumors, 7 showed no<br />
expression of ER, PR of HER2, whereas 5 tumors still have to be classified.<br />
Tumor outgrowth is expected to occur 5 to 8 months after implantation. After<br />
outgrowth, tumors will be serially transplanted followed by analysis of cytogenetic<br />
features, gene expression profile and histology from both the original tumor and the<br />
xenografts. Tumors that can be stably passaged will be used for therapeutic<br />
intervention studies.
In addition to developing our own xenograft panel, we will also use already<br />
established xenograft models that are made available to us by the group of Marie-<br />
France Poupon in the Preclinical Investigation Unit at the Institute Curie in Paris,<br />
France.<br />
Genomic profiling, molecular markers and therapy response in breast cancer<br />
Juliane Hannemann, Jorma de Ronde, Lennart Mulder, Marc van de Vijver, Laura van ’t Veer,<br />
Marieke Straver, Marie-Jeanne Vrancken Peters, Sjoerd Rodenhuis, Jelle Wesseling, Petra Nederlof<br />
The objective of the clinical and pathology part of the project supported by the Center<br />
for Translational Molecular Medicine (CTMM) is to develop clinically useful and<br />
feasible tests (using molecular markers) to predict the response or resistance of<br />
primary breast cancers to specific chemotherapeutic agents or combinations of these.<br />
This is important, since preoperative (neoadjuvant) chemotherapy may lead to<br />
excellent responses in some tumors and to no response at all in others. The tests to<br />
be developed will be based on DNA, mRNA and protein analysis. DNA and mRNAbased<br />
tests should ideally be developed into MLPA tests, in collaboration with MRC<br />
Holland Currently, a collection of pre-chemotherapy biopsies and post-chemotherapy<br />
tissue bank samples have been procured of over 300 patients with primary breast<br />
cancer who received preoperative treatment. The preoperative samples have been<br />
analyzed by the pathology department (Marc van de Vijver, Jelle Wesseling) and<br />
mRNA and DNA have been isolated. Furthermore, 200 mRNA expression arrays<br />
(microarray facility <strong>NKI</strong> and Agendia BV) have been run for over 100 samples,<br />
homologous recombination deficiency tests have been done. These include singlegene<br />
expression estimates (BRCA1) using quantitative rtPCR, aCGH signature<br />
derived BRCA1-ness classification (Nederlof et al), BRCA1 methylation status and<br />
EMSY amplification detection using MLPA and other techniques and for selected<br />
samples partial sequencing of the BRCA1 gene to detect common mutations. The<br />
analysis of these data is ongoing.<br />
In addition, the gene expression of pre-chemotherapy biopsies has revealed that <strong>NKI</strong><br />
70 gene prognosis profile low risk tumors have an extremely low rate of pathological<br />
complete remission, where similarly classified high-risk tumors include all chemoresponsive<br />
tumors (p
110<br />
DIAGNOSTIC ONCOLOGY<br />
Publications (continued)<br />
Plon SE, Eccles DM, Easton D, Foulkes WD,<br />
Genuardi M, Greenblatt MS, Hogervorst FB,<br />
Hoogerbrugge N, Spurdle AB, Tavtigian SV;<br />
for the IARC Unclassified Genetic Variants<br />
Working GroupThe members of the<br />
Working Group are listed in the Appendix..<br />
Sequence variant classification and<br />
reporting: recommendations for improving<br />
the interpretation of cancer susceptibility<br />
genetic test results. Hum Mutat.<br />
<strong>2008</strong>;29:1282-1291<br />
Prevoo W, van den Bosch MA, Horenblas S.<br />
Guideline ‘Renal cell carcinoma’ Ned Tijdschr<br />
Geneeskd. <strong>2008</strong>;152:1350; author reply 1350.<br />
(Dutch)<br />
Prevoo W, van den Bosch MA, Horenblas S.<br />
Radiofrequency ablation for treatment of<br />
sporadic angiomyolipoma. Urology.<br />
<strong>2008</strong>;72:188-91<br />
Schiltz PM, Dillman RO, Korse CM,<br />
Cubellis JM, Lee GJ, De Gast GC. Lack of<br />
elevation of serum S100B in patients with<br />
metastatic melanoma as a predictor of<br />
outcome after induction with an autologous<br />
vaccine of proliferating tumor cells and<br />
dendritic cells. Cancer Biother Radiopharm<br />
<strong>2008</strong>;23:214-21<br />
Seigers R, Schagen SB, Beerling W,<br />
Boogerd W, van Tellingen O, van Dam FS,<br />
Koolhaas JM, Buwalda B. Long-lasting<br />
suppression of hippocampal cell proliferation<br />
and impaired cognitive performance by<br />
methotrexate in the rat. Behav Brain Res<br />
<strong>2008</strong>;186:168-75<br />
Smeenk RM, Bruin SC, van Velthuysen ML,<br />
Verwaal VJ. Pseudomyxoma peritonei.<br />
Curr Probl Surg. <strong>2008</strong>;45:527-75 (review)<br />
Smeenk RM, van Velthuysen ML, Verwaal VJ,<br />
Zoetmulder FA. Appendiceal neoplasms and<br />
pseudomyxoma peritonei: a population based<br />
study. Eur J Surg Oncol. <strong>2008</strong>;34:196-201<br />
Smit LH, Nieweg OE, Mooi WJ, Bonfrer JM,<br />
Haanen JB, Kroon BB, De Gast GC. Value of<br />
serum S-100B for prediction of distant relapse<br />
and survival in stage III B/C melanoma.<br />
Anticancer Res <strong>2008</strong>;28:2297-302<br />
Sonke GS, Ludwig I, van Oosten H,<br />
Baars JW, Meijer E, Kater AP, de Jong D.<br />
Poor outcomes of chronic active Epstein-<br />
Barr virus infection and hemophagocytic<br />
lymphohistiocytosis in non-Japanese adult<br />
patients. Clin Infect Dis. <strong>2008</strong>;47:105-8<br />
Integration of DNA copy number alterations and prognostic gene expression<br />
signatures in breast cancer patients<br />
Hugo Horlings, Carmen Lai, Dimitry Nuyten, Hans Halfwerk, Petra Kristel, Erik van Beers,<br />
Simon Joosse, Marcel Reinders, Petra Nederlof, Lodewijk Wessels, Laura van ’t Veer,<br />
Marc van de Vijver<br />
Several prognostic gene expression profiles have been identified in breast cancer. In<br />
spite of this progress in prognostic classification, the underlying mechanisms that<br />
drive these gene expression patterns remain unknown. Specific genetic alterations<br />
that can partly be studied by analyzing DNA copy number changes are an important<br />
mechanism of tumor development and progression and are also associated with<br />
changes in gene expression. We carried out array Comparative Genomic<br />
Hybridization (aCGH) in 68 human breast carcinomas were part of the previous<br />
published <strong>NKI</strong>-295 series, for which gene expression and clinical data were available.<br />
We used a two-class supervised algorithm for the identification of regions of<br />
chromosomal alteration (SIRAC). We identified genes in the altered regions for<br />
which the expression level is significantly correlated with the copy number. The<br />
previously identified poor prognosis 70-gene signature (<strong>NKI</strong>) is associated with gain<br />
of 3q26-27, 8q22-24 and 17q24-25 and a loss of 14q31; the 70-gene good prognosis<br />
profile is associated with loss at 16q11-24; basal-like tumors with gain of 6p12-24,<br />
10p12-14, 12p13 and losses at 4p15, 5q11-34, 8q24, 10q23-24, 12q13-21 14q12-23,<br />
15q14-25; HER2+ breast tumors have an amplification at 17q12 (where the HER2<br />
gene is located) ((figure 4). These findings help to establish a link between genetic<br />
changes and gene expression signatures that determine tumor behavior in breast<br />
cancer, enabling a better understanding of the tumor biology that causes poor<br />
prognosis.<br />
Figure 4: A summary of the results of the integration of SIRAC algorithm results (DNA copy number<br />
changes) with corresponding gene expression data. We show the aberrations per chromosomal arm with<br />
respect to prognostic gene signatures. Regions of aberration detected have been color-coded based on the<br />
direction of the aberration. Green was used for gain, red for losses and for example HER2, the HER2<br />
positive samples are the class of interest and these tumors are characterized by a gain on 17q, hence the<br />
green block in cell 17q.
Validation of the 70-gene prognosis-signature (MammaPrint) in breast<br />
cancer subpopulations<br />
Stella Mook, Michael Knauer, Marjanka Schmidt, Inge Eekhout, Jelle Wesseling, Sabine Linn,<br />
Marc van de Vijver, Emiel Rutgers, Laura van ’t Veer<br />
Previous studies of 70-gene signature demonstrated that women with cancerous<br />
lymph node involvement and a ‘low-risk’ genomic signature had a considerably better<br />
prognosis than their ‘high-risk’ -signature counterparts. We undertook a validation<br />
project to test the prognostic value of the 70-gene signature in 241 patients with 1-3<br />
involved lymph nodes. The 70-gene signature was the strongest predictor of survival<br />
eight years after an initial diagnosis, even after taking into account other important<br />
considerations, such as whether or not these women received adjuvant<br />
chemotherapy. As a result of this work, the MINDACT trial has been amended so<br />
that women with 1-3 involved lymph nodes can be included in the trial.<br />
The 70-gene prognosis-signature has been validated in various series, but so far not<br />
exclusively in postmenopausal patients. However, the majority of breast cancer<br />
patients are older, postmenopausal women who are increasingly being offered<br />
adjuvant chemotherapy despite the more favorable biological characteristics of their<br />
tumors, such as a lower proliferation rate and a high endocrine sensitivity.<br />
Therefore, we validated the signature in 148 patients aged between 55 and 71 years.<br />
This validation study provides evidence that the 70-gene prognosis-signature<br />
(MammaPrint), is also a predictor of disease outcome in postmenopausal breast<br />
cancer patients, especially for early breast cancer-related events. Since the beneficial<br />
effect of chemotherapy in older women predominantly occurs in the first 5 years, the<br />
signature can be of clinical use in selecting postmenopausal women for adjuvant<br />
chemotherapy.<br />
In total for about 1700 <strong>NKI</strong> diagnosed patients a retrospective 70-gene prognosis<br />
signature was assessed and published in various publications. We have now compiled<br />
all in one database and are evaluating risk assessment by the prognosis signature in<br />
much larger patient groups, thus increasing the power. Ongoing separate analyses<br />
include, her2postive, endocrine responsiveness, tumor diameter, age selected<br />
patients groups. Moreover, the predictiveness of signature subgroups for therapy is<br />
evaluated, e.g., the higher likelihood of 70 gene poor prognostic patients to respond<br />
to chemotherapy.<br />
Validation of Adjuvant! Online<br />
Stella Mook, Marjanka Schmidt, Peter Ravdin, Tony van der Velde, Jelle Wesseling, Sterre Rutgers,<br />
Emiel Rutgers, Laura van ’t Veer<br />
Nowadays, adjuvant treatment recommendations for early stage breast cancer<br />
patients are based on the estimated risk of breast cancer relapse and death and the<br />
expected benefit of adjuvant treatment. Several tool have been developed to make<br />
these estimates for the individual patient. One of these tools is Adjuvant! Online<br />
(www.adjuvantonline.com), which calculates a 10-year survival probability based on<br />
the patient’s age, tumor size, grade and estrogen receptor. In addition, the computer<br />
program also calculates the likely benefit to be expected for adjuvant systemic<br />
treatment. In 2005 Olivotto et al. validated Adjuvant! Online in 4083 patients using<br />
the British Columbia Breast Cancer Outcomes Unit database. They compared<br />
observed outcomes with expected outcomes calculated by Adjuvant!. The 10-year<br />
predicted and observed outcomes were within 1% for the whole group and for most<br />
subgroups predicted and observed outcomes were within 2%.<br />
Adjuvant! Online is already being used by physicians. In addition, in the MINDACT<br />
trial the clinical risk is calculated by Adjuvant! Online. To assess the validity of<br />
Adjuvant! Online for Dutch breast cancer patients we will compare predicted and<br />
observed disease outcome in 2 Dutch cohorts of approximately 10.000 patients in<br />
total.<br />
111<br />
DIAGNOSTIC ONCOLOGY<br />
Publications (continued)<br />
Spurdle AB, Couch FJ, Hogervorst FB,<br />
Radice P, Sinilnikova OM; for the IARC<br />
Unclassified Genetic Variants Working<br />
Group The members of the IARC<br />
Unclassified Genetic Variants Working<br />
Group are listed in the Appendix..<br />
Prediction and assessment of splicing<br />
alterations: implications for clinical testing.<br />
Hum Mutat. <strong>2008</strong>;29:1304-1313<br />
Straver ME, van Adrichen JC, Rutgers EJTh,<br />
Rodenhuis S, Linn SC, Loo C,<br />
Gilhuijs KGA, Oldenburg HSA,<br />
Wesseling J, Russel NS, Ontonini N,<br />
Vrancken Peeters MTFD, Neoadjuvante<br />
systemische therapie bij het primair<br />
operabel mammcarcinoom: meer voordelen<br />
dan alleen borstsparende behandeling, Ned<br />
Tijdschrift Geneesk <strong>2008</strong>;152:2519-2525<br />
Thieblemont C, Rolland D, Baseggio L,<br />
Felman P, Gazzo S, Callet-Bauchu E,<br />
Traverse-Glehen A, Houlgatte R, Fu K,<br />
Weisenburger D, De Jong D, Jaffe ES,<br />
Rosenwald A, Ott G, Coiffier B, Berger F.<br />
Comprehensive analysis of GST-pi<br />
expression in B-cell lymphomas: Correlation<br />
with histological subtypes and survival.<br />
Leuk Lymphoma. <strong>2008</strong>;49:1403-6<br />
Tischkowitz M, Hamel N, Carvalho MA,<br />
Birrane G, Soni A, van Beers EH,<br />
Joosse SA, Wong N, Novak D,<br />
Quenneville LA, Grist SA; kConFab,<br />
Nederlof PM, Goldgar DE, Tavtigian SV,<br />
Monteiro AN, Ladias JA, Foulkes WD.<br />
Pathogenicity of the BRCA1 missense<br />
variant M1775K is determined by the<br />
disruption of the BRCT phosphopeptidebinding<br />
pocket: a multi-modal approach.<br />
Eur J Hum Genet. <strong>2008</strong>;16:820-32<br />
Valdés Olmos R.A, Vidal-Sicart,<br />
Nieweg OE. SPECT -CT and real-time<br />
intraoperatieve imaging: new tools for<br />
sentinel node localization and radioguided<br />
surgery? Eur J Nucl Med Mol Imaging.<br />
<strong>2008</strong> (epub ahead of print)<br />
Valdés Olmos RA, Kroon BB,<br />
Hoefnagel CA, Horenblas S. Penile<br />
carcinoma – Radionuclide Imaging and<br />
PET. In: de la Rosette JJMCH et al:<br />
Imaging in oncological Urology, Springer-<br />
Verlag <strong>2008</strong>:345-350
112<br />
DIAGNOSTIC ONCOLOGY<br />
Publications (continued)<br />
Valdés Olmos RA, Van der Ploeg I,<br />
Rutgers E, Nieweg OE. Sentinel nodes<br />
outside the axilla: more accurate staging or<br />
unnecessary morbidity? Rev Senologia<br />
Patol Mam <strong>2008</strong>;21:22-26<br />
Valdés Olmos RA, Vogel WV, Hoefnagel CA.<br />
Hoofdstuk Tumoren. In: Van den Broek W,<br />
Barneveld P, Lemstra C, Van Urk P:<br />
Leerboek Nucleaire Geneeskunde, Elsevier<br />
<strong>2008</strong>:505-557<br />
van den Berg RM, Teertstra HJ,<br />
van Zandwijk N, van Tinteren H, Visser C,<br />
Pasic A, Sutedja TG, Baas P, Golding RP,<br />
Postmus PE, Smit EF. CT detected<br />
indeterminate pulmonary nodules in a<br />
chemoprevention trial of fluticasone. Lung<br />
Cancer. <strong>2008</strong>;60:57-61<br />
Van der Ploeg IM, Kroon BB, Antonini N,<br />
Valdés Olmos RA, Rutgers EJ, Nieweg OE.<br />
Axillary and extra-axillary lymph node<br />
recurrences after a tumor-negative sentinel<br />
node biopsy for breast cancer using<br />
intralesional tracer administration. Ann<br />
Surg Oncol. <strong>2008</strong>;15:1025-31<br />
Van der Ploeg IM, Nieweg OE, van Rijk MC,<br />
Valdés Olmos RA, Kroon BB. Axillary<br />
recurrence after a tumour-negative sentinel<br />
node biopsy in breast cancer patients; A<br />
systemic review and meta-analysis of the<br />
literature. Eur J Surg Oncol. <strong>2008</strong> (epub<br />
ahead of print)<br />
Van der Ploeg IM, Tanis PJ, Valdés Olmos RA,<br />
Kroon BB, Rutgers EJ, Nieweg OE. Breast<br />
Cancer Patient with Extra-Axillary<br />
Sentinel Nodes Only may be Spared<br />
Axillary Lymph Node Dissection. Ann Surg<br />
oncol. <strong>2008</strong> (epub ahead of print)<br />
Van der Ploeg IM, Valdés Olmos RA,<br />
Kroon BB, Nieweg OE. The Hybrid<br />
SPECT/CT as an additional lymphatic<br />
mapping tool in patients with breast cancer.<br />
World J Surg. <strong>2008</strong>;32:1930-4<br />
Van der Ploeg IM, Valdés Olmos RA,<br />
Kroon BB, Nieweg OE. Tumor-positive<br />
sentinel node biopsy of the groin in<br />
clinically node-negative melanoma patients;<br />
superficial or superficial and deep lymph<br />
node dissection? Ann Surg Oncol.<br />
<strong>2008</strong>;5:1485-91<br />
THE NETHERLANDS CANCER INSTITUTE FAMILY CANCER<br />
CLINIC<br />
Mohamed Achachah, Abderrahim Ajouaou, Priscilla Axwijk, Majella Boutmy - de Lange, Daniela Hahn,<br />
Frans Hogervorst, Irma Kluijt, Mobien Kasiem, Petra Nederlof, Rob Plug, Roelof Pruntel, Anja van Rens,<br />
Efraim Rosenberg, Esther Scheerman, Laura van ’t Veer, Sophie van de Velden, Senno Verhoef,<br />
Gea Wigbout<br />
In <strong>2008</strong> the number of families who have sought genetic advice through our hospital<br />
since the start of the Family Cancer Clinic in 1995 has increased again by more than<br />
10%. Unfortunately the number of clients that can be accepted by the counseling and<br />
laboratory remains maximized as a consequence of limits imposed by the insurance<br />
companies. In <strong>2008</strong> 583 new patients were seen for complex genetic counseling.<br />
Most families seen at our family cancer clinic present with a possible genetic<br />
predisposition for breast and/or ovarian cancer. The DNA-diagnostic laboratory has<br />
screened more than 2600 families for germ line mutations in the BRCA1/2 genes<br />
since the start. These families are obtained through our Family Cancer Clinic and the<br />
oncogenetic section of the Department of Clinical Genetics of the Academic Medical<br />
Center. In 270 families a BRCA1 mutation and in 146 families a BRCA2 mutation<br />
was identified. In more than 1300 family members predictive, presymptomatic<br />
testing for these genes was performed and 508 persons were shown to be carrier of<br />
the predisposing mutation. Sequence variants which may or may be not the familial<br />
disposition are a major challenge for the laboratory but with national and<br />
international effort and collaboration we are able to resolve each year a substantial<br />
part of them. Unfortunately, we still have 127 families with unresolved sequence<br />
variants in one of the two genes.<br />
The laboratory has been testing new techniques in order to shorten the through put<br />
time of samples. We started using Conformation Sensitive Capillary Electrophoresis<br />
combined with automated pipetting and analysis. After two years of testing we<br />
shifted to high resolution melting curve analysis which proves to be less complex in<br />
handling and implementation.<br />
Using Array CGH for hereditary breast/ovarian cancer we study whether the<br />
genomic aberrations in breast tumors are similar to the specific aberrations found in<br />
BRCA1 tumors (in collaboration with Simon Joosse and Petra Nederlof, Division of<br />
Experimental Therapy and Diagnostic Oncology). More than 100 a-CGH analyses<br />
have been completed, for high risk families and unclassified variant analysis mostly.<br />
A BRCA2 classifier has been validated at this very moment and the implementation<br />
of this classifier into the diagnostics will hopefully being carried out in 2009.<br />
For families with hereditary non polyposis colorectal cancer we have mutation<br />
scanning tests for mismatch repair genes hMLH1, hMSH2 and hMSH6 and the<br />
MutY (MYH) gene. Microsatellite instability tests and immunohistochemistry for the<br />
mismatch repair genes is carried out in collaboration with the pathologist Daphne de<br />
Jong. Furthermore methylation of the hMLH1 promoter and the presence of a<br />
specific somatic mutation in the BRAF gene (V600E) are being assessed. About 50%<br />
of the micro satellite instable(MSI-high) tumors with absent staining of hMLH1 have<br />
a methylated promoter. This result has direct consequences for the clinical<br />
interpretation of the family history data.<br />
The Family Cancer Clinic contributes data to several multi-center national and<br />
international research projects, e.g. GEO-HEBON (gene environment interactions in<br />
hereditary breast and ovarian cancer, see Division Psychosocial Research and<br />
Epidemiology), a national study of families with Li Fraumeni and variant Li<br />
Fraumeni(-like) syndrome, DNA-profiling by classic cGH and array cGH of breast<br />
and ovarian cancer patients (see Division of Experimental Therapy), the Breast<br />
Cancer Linkage Consortium, the BCAC and CIMBA consortiums, a study into the<br />
biological significance of so called unclassified variants (DNA changes of which it is<br />
uncertain whether they be pathogenic mutations or polymorphisms) in a national<br />
collaboration with other DNA-diagnostic labs, coordinated from LUMC by Dr Peter<br />
Devilee cs and by participating in the IARC Unclassified Genetic Variants Working<br />
Group (Frans Hogervorst), psychosocial studies, in collaboration with the department
of Psychosocial Research and Epidemiology and clinical and genetic research in<br />
families with gastrointestinal cancer, including stomach cancer and pancreatic cancer<br />
(Annemieke Cats, Division of Medical Oncology).<br />
The guidelines for hereditary breast cancer management and hereditary colon<br />
carcinoma have been formulated and are being implemented. In collaboration with<br />
the Academic Medical Centre families are included in studies of familial stomach<br />
cancer and pancreatic cancer, for whom periodic screening programs are being<br />
developed, tried out and evaluated.<br />
In <strong>2008</strong> a PhD student started a project on the implementation of genetic counseling<br />
and where necessary rapid DNA-testing in recently diagnosed breast cancer patients:<br />
the TIME- study. In this multi-centre study in collaboration with the Division of<br />
Medical Genetics of the Utrecht Medical Centre, patients from 14 different hospitals<br />
will be enrolled for a period of 18 months.<br />
DEPARTMENT OF RADIOLOGY<br />
Laboratory for Diagnostic Image Research<br />
Kenneth Gilhuijs, Tanja Alderliesten, Christian Siedschlag, Lukas Batteau, Anita Paape,<br />
Kenneth Pengel, Angelique Schlief, Jincey Sriram, Alexander Schmitz, Annemarie Schmitz,<br />
Claudette Loo, Saar Muller, Jelle Teertstra, Wouter Vogel, Fijs van Leeuwen, Patrick Chin,<br />
Tessa Buckle, Anne van Leeuwen<br />
The diagnostic-imaging laboratory at the department of Radiology pursues new<br />
imaging techniques, image processing, multi-modality registration, pattern<br />
recognition and molecular imaging to improve the sensitivity and specificity of<br />
cancer diagnosis, pre-treatment assessment of tumor extent, and image-guided<br />
therapy.<br />
Automated fusion of MRI and PET to monitor response to neoadjuvant<br />
chemotherapy Contrast-enhanced (CE) MRI and PET/CT of the breast provide<br />
complementary information on neovascularization and tumor cell metabolism.<br />
This information may advance the individualized therapy of patients treated with<br />
neoadjuvant chemotherapy. In a pilot study we assessed the feasibility of fusing<br />
CE MRI and FDG-PET/CT.<br />
Nine patients with breast cancer (ø 23 mm - 58 mm) underwent CE MRI in prone<br />
position using a bilateral MRI breast coil. Wash-in and wash-out kinetics were<br />
established at 90 s and at 450 s, respectively. FDG-PET/CT in prone orientation was<br />
obtained using a replica of the same MRI breast coil. MRI and PET/CT were<br />
automatically aligned by deformable registration using: (a) fiducial control point<br />
alignment constrained by local conservation of volume (FCP) or (b) particle-swarm<br />
optimization using optimization of mutual information (PSO). Accuracy was<br />
assessed by residual misalignment of fiducial landmarks on fat-parenchyma<br />
transitions. SUV and perfusion were correlated.<br />
Average residual misalignment was 5 mm (FCP) and 8 mm (PSO), respectively.<br />
In all cases, asymmetric FDG uptake between left and right breast corresponded with<br />
increased wash-in at MRI. In 4/9 tumors (>40 mm) inhomogeneous FDG uptake<br />
occurred in homogeneous regions of contrast wash-in at MRI. These regions<br />
correlated with increased contrast wash-out.<br />
Figure 5: Left: MRI maximum intensity projection of an invasive ductal cancer, visualizing the<br />
neovascular network. The circles indicate protrusions wihtout aberrant behavior. Right: PET fused with<br />
MRI showing predominant metabolic activity in the protrusions (red), thus complementing MRI.<br />
113<br />
DIAGNOSTIC ONCOLOGY<br />
Publications (continued)<br />
Van der Ploeg IM, Valdés Olmos RA,<br />
Kroon BB, Rutgers EJ, Nieweg OE. The<br />
hidden sentinel node and SPECT/CT in<br />
breast cancer patients. Eur J Nucl Med Mol<br />
Imaging. <strong>2008</strong> (epub ahead of print)<br />
Van der Ploeg IMC, Nieweg OE,<br />
Valdés Olmos RA. De sentinel node<br />
procedure bij het mammacarcinoom.<br />
Gamma Professional <strong>2008</strong>;58:20-23<br />
van der Vegt B, de Bock GH, Hollema H,<br />
Wesseling J. Microarray methods to<br />
identify factors determining breast cancer<br />
progression: Potentials, limitations, and<br />
challenges. Crit Rev Oncol Hematol. <strong>2008</strong><br />
Oct 8 (epub ahead of print)<br />
van ‘t Veer MB, de Jong D, Mackenzie M,<br />
Kluin-Nelemans HC, van Oers MH,<br />
Zijlstra J, Hagenbeek A, van Putten WL.<br />
High-dose Ara-C and beam with autograft<br />
rescue in R-CHOP responsive mantle cell<br />
lymphoma patients.Br J Haematol. <strong>2008</strong><br />
Nov 13 (epub ahead of print)<br />
Veltkamp SA, Pluim D, van Tellingen O,<br />
Beijnen JH, Schellens JH. Extensive<br />
metabolism and hepatic accumulation of<br />
gemcitabine after multiple oral and<br />
intravenous administration in mice. Drug<br />
Metab Dispos <strong>2008</strong>;36:1606-15<br />
Vogel WV. Hoofdstuk Beeldfusie. In:<br />
Van de Broek W, Barneveld P, Lemstra C,<br />
Van Urk P: Leerboek Nucleaire<br />
Geneeskunde, Elsevier <strong>2008</strong>: 207-222<br />
Vollebergh MA, Nederlof PM, Wessels LF,<br />
Schmidt MK, Joosse SA, van Beers E,<br />
Froklage F, Holtkamp M, Schrama JG,<br />
Wesseling J, Hauptmann M, de Bruin M,<br />
Rodenhuis S and Linn SC Predicting<br />
response to alkylating chemotherapy in<br />
breast cancer patients using array<br />
comparative genomic hybridization<br />
(submitted)<br />
Voskuil DW, Vrieling A, Korse CM,<br />
Beijnen JH, Bonfrer JM, van Doorn J,<br />
Kaas R, Oldenburg HS, Russell NS,<br />
Rutgers EJ, Verhoef S, van Leeuwen FE,<br />
Van ‘t Veer LJ, Rookus MA. Effects of<br />
lycopene on the insulin-like growth factor<br />
(IGF) system in premenopausal breast<br />
cancer survivors and women at high<br />
familial breast cancer risk. Nutr Cancer<br />
<strong>2008</strong>;60:342-53
114<br />
DIAGNOSTIC ONCOLOGY<br />
Publications (continued)<br />
Vrieling A, Rookus MA, Kampman E,<br />
Bonfrer JM, Bosma A, Cats A, van Doorn J,<br />
Korse CM,Witteman BJ, van Leeuwen FE,<br />
Van ‘t Veer LJ, Voskuil DW. No effect of red<br />
clover-derived isoflavone intervention on the<br />
insulin-like growth factor system in women<br />
at increased risk of colorectal cancer.<br />
Cancer Epidemiol Biomarkers Prev<br />
<strong>2008</strong>;17:2585-93<br />
Vrieling A, Voskuil DW, Bosma A, Majoor DM,<br />
van Doorn J, Cats A, Depla AC, Timmer R,<br />
Witteman BJ, Wesseling J, Kampman E,<br />
Van ‘t Veer LJ. Expression of insulin-like<br />
growth factor system components in<br />
colorectal tissue and its relation with serum<br />
IGF levels. Growth Horm IGF Res. <strong>2008</strong><br />
Sep 16 (epub ahead of print)<br />
Vrieling C, de Jong D, Boot H, de Boer JP,<br />
Wegman F, Aleman BM. Long-term results<br />
of stomach-conserving therapy in gastric<br />
MALT lymphoma. Radiother Oncol.<br />
<strong>2008</strong>;87:405-11<br />
Weigelt B, Horlings HM, Kreike B,<br />
Hayes MM, Hauptmann M, Wessels LF,<br />
de Jong D, Van de Vijver MJ, Van ‘t Veer LJ,<br />
Peterse JL. Refinement of breast cancer<br />
classification by molecular characterization<br />
of histological special types. J Pathol.<br />
<strong>2008</strong>;216:141-50<br />
Zeleniuch-Jacquotte A, Gu Y, Bruning PF,<br />
Bonfrer JM, Koenig KL, Arslan AA,<br />
Toniolo P, Shore RE. Re: C-reactive protein<br />
and risk of breast cancer. J Natl Cancer Inst<br />
<strong>2008</strong>;100:443-4<br />
Zuur JK, Muller SH, Vincent A,<br />
Sinaasappel M, de Jongh FH, Hilgers FJ.<br />
Assessment of tracheal temperature and<br />
humidity in laryngectomized individuals<br />
and the influence of a heat and moisture<br />
exchanger on tracheal climate. Head Neck.<br />
<strong>2008</strong>;30:1072-82<br />
From this pilot study we conclude that automatic deformable co-registration of FDG-<br />
PET/CT and CE MRI is clinically feasible. CE MRI enhances the conspicuity of<br />
asymmetric FDG uptake in left and right breast. Increased wash-out at MRI is<br />
correlated with high SUV in the tumor. Automated PET/MR deformable registration<br />
and multi-modality computer-aided assessment of therapy response will be further<br />
investigated in the CTMM CaRe project, initiated in <strong>2008</strong>.<br />
The impact of preoperative MRI on breast-conserving surgery of invasive<br />
cancer: a comparative cohort study This research aimed to assess whether<br />
preoperative contrast-enhanced magnetic resonance imaging (MRI) of the breast<br />
influences the rate of incomplete tumor excision. Methods In a cohort of 349 women<br />
with invasive breast cancer, patients eligible for breast-conserving therapy (BCT) on<br />
the basis of conventional imaging and palpation only (N = 176) were compared to<br />
those who had an additional preoperative MRI (N = 173). Multivariate analysis was<br />
applied to explore associations with incomplete tumor excision. Results MRI detected<br />
larger extent of breast cancer in 19 women (11.0%), leading to treatment change:<br />
mastectomy (8.7%) or wider excision (2.3%). Tumor excision was incomplete in<br />
22/159 (13.8%) wide local excisions in the MRI group and in 35/180 (19.4%) in the<br />
non-MRI group (P = 0.17). Stratified to tumor type, incompletely excised infiltrating<br />
ductal carcinoma (IDC) was significantly associated with absence of MRI: 11/136<br />
(8.1%) versus 2/126 (1.6%) (MRI present) (P = 0.02). No significant factors explained<br />
incomplete excision of other tumor types. Conclusion Preoperative MRI did not<br />
significantly affect the overall rate of incomplete tumor excision, but it yielded<br />
significantly lower rate of incompletely excised IDC. The reduction of incomplete<br />
excisions after MRI was smaller than the rate of a prior treatment change incurred<br />
by MRI.<br />
Accurate definition of the treatment target for non-small cell lung cancer<br />
using PET/CT-pathology matching In ongoing efforts to improve the treatment<br />
target for non-small cell lung cancer (NSCLC) by correlating pretreatment diagnostic<br />
imaging with histopathology, we developed a model to predict the extent of the gross<br />
tumor volume (GTV, primary tumor) and the clinical target volume (CTV, surrounding<br />
microscopic disease) at pathology from PET/CT images.<br />
High-resolution multi-slice CT scans and 18F-FDG-PET scans were obtained from 34<br />
patients with NSCLC prior to lobectomy. The GTVCT was manually delineated and<br />
GTVPET was automatically extracted from PET images by taking the 42% isocontours<br />
of the maximum SUV. These volumes were compared with the reference<br />
pathology volumes (GTVpath). At pathology, microscopic thin sections covering the<br />
tumor and surrounding area of about 2 cm were obtained and their images overlaid<br />
onto the macroscopic images. GTVpath and surrounding microscopic disease were<br />
delineated.<br />
To develop a multivariate model that predicts the histopathology-proven target based<br />
on CT and PET data, we extracted image features such as GTVCT and GTVPET, Max<br />
SUV, and tumor circularity at CT. These features, together with other characteristics<br />
such as tumor type and patient age, were correlated with the histopathology findings<br />
in order to predict the volume of the GTVpath and the presence and extension of<br />
microscopic spread.<br />
The radius of the delineated GTVCT was on average 6 mm larger than the GTVpath<br />
radius. On the other hand, the GTVPET corresponded to GTVpath within 1 mm. We<br />
found microscopic spread in about 50% of our patients. In order to capture all<br />
microscopic spread in 90% of patients, a CTV margin of 15 mm would be required.<br />
This is considerably larger than most other studies have reported.<br />
We conclude that the true GTV for NSCLC patients can be obtained from 18F-FDG-<br />
PET images but is overestimated by CT. Taking lung deformations into account, a 15<br />
mm CTV margin would be required to cover the microscopic spread in 90% of<br />
patients. However, in half the number of patients no surrounding microscopic spread<br />
was found.
Molecular imaging for tumor staging and response monitoring<br />
Fijs van Leeuwen, Tessa Buckle, Ariena Kersbergen, Sven Rottenberg, Jos Jonkers, Saar Muller,<br />
Kenneth Gilhuijs<br />
Functional information provided by molecular imaging can be used to reveal the<br />
molecular basis of a tumor in a non-invasive manner. This can, in turn, be used to<br />
stage tumor progression and monitor tailored therapy response. Studies are currently<br />
performed at the departments of Radiology/Nuclear Medicine and the <strong>NKI</strong> ‘mouse<br />
cancer clinic’ and use the (advanced) mouse models present at the <strong>NKI</strong>.<br />
Staging an detection DCIS In a mouse model representative for ductal carcinoma<br />
in situ (DCIS), diagnostic approaches are being studied that can be used to identify<br />
tumor progression from an initial preinvasive to invasive lesions. For these studies<br />
we use a combination of PET and MRI. PET can be used to visualize changes in the<br />
molecular biology of the tumor e.g. increase in proliferation (18F-FLT) or metabolism<br />
(18F-FDG). Contrast enhanced MRI provides complementary information regarding<br />
changes in tumor size and vascular physiology. Finally we wish to exploit such a<br />
complementary imaging approach in concurrent PET/MRI imaging.<br />
Analysis of drug resistance and response Using ‘spontaneous’ mouse models for<br />
hereditary breast cancer with a known response to therapy we investigate<br />
endogenous and non-invasive markers for response. Here computer aided diagnosis<br />
of resistance induced by Mdr1 drug transporter activity has been used to noninvasively<br />
predict therapeutic resistance to doxorubicin. Since this tumor model also<br />
has a well documented sensitivity/resistance pattern (sensitive and resistant) against<br />
docetaxel, it has also been used in docetaxel response investigations based on<br />
apoptosis imaging with 99mTc Annexin V. Unfortunately, the latter approach proved<br />
not to be response-specific and does not allow non-invasive prediction of<br />
chemotherapeutic response. Present we are investigating other endogenous markers<br />
that may allow early prediction of the therapeutic efficacy.<br />
115<br />
DIAGNOSTIC ONCOLOGY
116<br />
MEDICAL ONCOLOGY<br />
Division head, Sjoerd Rodenhuis<br />
Sjoerd Rodenhuis MD PhD Head<br />
Joke Baars MD PhD Academic staff<br />
Paul Baas MD PhD Academic staff<br />
André Bergman MD PhD Academic staff<br />
Jos Beijnen PhD Academic staff<br />
Christian Blank MD PhD Academic staff<br />
Willem Boogerd MD PhD Academic staff<br />
Henk Boot MD PhD Academic staff<br />
Wieneke Buikhuisen MD PhD Academic staff<br />
Sjaak Burgers MD PhD Academic staff<br />
Annemieke Cats MD PhD Academic staff<br />
Jan Paul De Boer MD PhD Academic staff<br />
Dieta Brandsma MD PhD Academic staff<br />
Elke Brouwers PhD Academic staff<br />
John Haanen MD PhD Academic staff<br />
Alwin Huitema PhD Academic staff<br />
Martijn Kerst MD PhD Academic staff<br />
Marjolein Klous PhD Academic staff<br />
Sabine Linn MD PhD Academic staff<br />
Anne Lukas MD PhD Academic staff<br />
Bastiaan Nuyen PhD Academic staff<br />
Hilde Rosing PhD Academic staff<br />
Jan Schellens MD PhD Academic staff<br />
Jan Schornagel MD PhD Academic staff<br />
Marianne Smits MD PhD Academic staff<br />
Gabe Sonke MD Academic staff<br />
Babs Taal MD PhD Academic staff<br />
Wim Ten Bokkel Huinink MD PhD<br />
Academic staff<br />
Margot Tesselaar MD Academic staff<br />
Michel Van Den Heuvel MD PhD<br />
Academic staff<br />
Marchien Van Der Weide MD PhD<br />
Academic staff<br />
Roel Van Gijn PhD Academic stafff<br />
Nico Van Zandwijk MD PhD Academic staff<br />
Karin Beelen Temporary staff<br />
Emile Kerver MD PhD Temporary staff<br />
Peter Kunst MD PhD Temporary staff<br />
Alet Mager Temporary staff<br />
Lidwine Tick MD PhD Temporary staff<br />
Catrien Tromp-van Driel Temporary staff<br />
David Boss PhD student<br />
Carola Damen PhD student<br />
Maarten Deenen PhD student<br />
Thomas Dorlo PhD student<br />
Anne Charlotte Dubbelman PhD student<br />
Claudia Heijens PhD student<br />
Marie-Christine Gast PhD student<br />
Robert Jansen PhD student<br />
Ron Keizer PhD student<br />
Stijn Koolen PhD student<br />
DIVISION OF MEDICAL ONCOLOGY<br />
CLINICAL PHARMACOLOGY OF ANTICANCER DRUGS<br />
Jan Schellens, Jos Beijnen, Henk Boot, Annemieke Cats, Alwin Huitema, Serena Marchetti,<br />
Bastiaan Nuijen, Hilde Rosing<br />
Background and objectives<br />
The division of Clinical Pharmacology remains involved in research in different<br />
phases of anticancer drug development. This concerns: I) Pharmaceutical<br />
formulation, II) Bioanalytical method development and implementation in clinical<br />
pharmacological studies, III) Early clinical phase I/II studies and IV) supportive care<br />
studies in patients with breast cancer.<br />
Research activities of the division of Clinical Pharmacology of the department of<br />
Medical Oncology, the department of Pharmacy & Pharmacology and the department<br />
of Experimental Therapy (group Schellens) are closely integrated.<br />
In <strong>2008</strong>, we continued clinical research fully compliant with ICH-GCP guidelines,<br />
previously certified by the Dutch Ministry of Health. The department of Pharmacy &<br />
Pharmacology successfully continued its official governmental GLP (in the field of<br />
analytical chemistry), GMP (formulation and manufacturing of investigational<br />
cytotoxic drugs) and GDP (ISO9002 for worldwide distribution of clinical supplies)<br />
licenses.<br />
Research performed in the year <strong>2008</strong><br />
I Pharmaceutical formulation<br />
Formulation research and clinical manufacture of small molecule experimental<br />
anticancer agents has continued with the anti-metastatic compound NAMI-A for<br />
which a suitable pharmaceutical formulation was developed and the first batches for<br />
clinical trial use were manufactured.<br />
Clinical manufacture (Thalidomide tablets) of oral formulations of anticancer agents<br />
has continued. A pharmaceutical development program of oral taxanes was initiated<br />
and the first formulation composed of a solid dispersion of docetaxel has entered the<br />
clinic. Optimization of this formulation will continue in 2009. Currently a similar<br />
formulation of paclitaxel is in development which is expected to be ready for clinical<br />
testing in the course of 2009.<br />
A first formulation project aimed at the GMP-manufacture of ready-to-label<br />
molecular imaging agents has been started with bevacizumab as its first product.<br />
The Amsterdam BioTherapeutics Unit (AmBTU) is now fully operational for the<br />
manufacture of GMP-grade DNA-plasmids. A lyophilized pharmaceutical<br />
formulation of pDERMATT (plasmid DNA Encoding Recombinant Mart-1 and<br />
Tetanus Toxin fragment-c) for the intradermal administration by tattooing was<br />
developed for a phase I clinical trial which started by the end of <strong>2008</strong> in our institute.<br />
The next project, the pharmaceutical development and manufacture of HPV E6 and<br />
E7 pDNA vaccines for clinical trial use initiated. Clean room facilities are in place to<br />
enable GMP-preparation of T-lymphocytes for TIL and T-cell receptor gene therapy,<br />
for which the development of the preparation processes is ongoing. The research<br />
program in collaboration with Utrecht University aiming at the pharmaceutical<br />
development of non-viral vectors (e.g. lipoplexes and polyplexes) for improvement of<br />
DNA-plasmid transfection is ongoing. An ex vivo human skin screening model is in<br />
place and has helped to identify important aspects of intradermal pDNA<br />
administration and non-viral vector composition. The ultimate aim of this program<br />
is, of course, to take promising vectors to the clinic.<br />
II Bioanalytical method development and implementation in PK studies<br />
A new linear ion trap Mass Spectrometer (MS) has been purchased for our laboratory.<br />
The new instrument (LTQ XL) offers multiple dissociation techniques generating<br />
more fragments and extending the low mass range. The high resolution enables<br />
isolation of isobaric mass peaks for cleaner MSn spectra. Furthermore, the ultra fast
polarity switching allows high sensitivity analysis of unknowns. The instrument will<br />
be used for quantitative analysis of multiple analytes, metabolic profiling and<br />
proteomic research. We have also decided to extend our triple quadruple MS<br />
equipments with the purchase of a highly sensitive API 4000 (Sciex) MS detector.<br />
E7080 is a new experimental tyrosine kinase inhibitor and assays have been validated<br />
in whole blood, plasma, urine, and feces for the quantification of the parent drug and<br />
its four metabolites. These assays will be used to support a mass balance study with<br />
the drug early next year. For bendamustine, a bifunctional mechlorethamine<br />
alkylating drug, we are in the process of setting up assays for the quantitative analysis<br />
of the drug and its known metabolites in several biomatrices for the analysis of<br />
samples to be collected in a mass balance study. For both mass balance studies, urine<br />
and fecal samples will be used to generate structural information of potential new<br />
metabolites using the novel LTQ XL MS instrument.<br />
New formulations for apaziquone (EO9) have been developed and we were involved<br />
in stability studies and the bioanalysis of EO9 and its metabolite EO5a. Porous<br />
graphitic carbon material as stationary phase has been used to quantify capecitabine<br />
and four metabolites (5]-deoxy-5-fluorocytidine, 5]-deoxy-5-fluorouracil, 5-fluorouracil<br />
and dihydro-5-fluorouracil) in plasma to support clinical studies of the drug. The<br />
same inert material was used to separate mono-, di-, and tri-phosphates of<br />
gemcitabine and dFdU in human PBMCs and the method has been successfully<br />
validated. For these assays, stable isotopic-labeled internal standards were<br />
synthesized from the labeled nucleosides.<br />
Other new bioanalytical methods for anti-cancer drugs that were developed and<br />
validated are: vincristine and actinomycine D, 2-amino-1-methyl-6phenylimidazo[4,5-b]pyridine<br />
(PhIP), tamoxifen, dasatinib, sunitinib and sorafenib.<br />
Sample analysis were performed for clinical trials within and outside the institute<br />
and included topotecan, irinotecan and its active metabolite SN-38, trabectedin,<br />
indibulin, paclitaxel, docetaxel, gemcitabine and dFdU, gemcitabine triphosphate,<br />
and cyclophosphamide in combination with fludarabine.<br />
For trastuzumab, a humanized monoclonal antibody (MAb), an assay has been<br />
developed to quantify the intact MAb using liquid chromatography coupled to<br />
fluorescence detection. For the isolation of trastuzumab from the plasma matrix, an<br />
anti-idiotype MAb generated after rabbit immunization was used.<br />
During the bioanalysis of Miltefosine, a drug used in the treatment of the parasitic<br />
disease Leishmaniasis, it became clear that the capsules given to the patients<br />
contained no active ingredients. Based on our findings the Bangladeshi government<br />
has started an investigation.<br />
III Early clinical phase I/II studies<br />
III-1 Novel approaches to improve oral bioavailability<br />
We have demonstrated that the oral applicability of commonly used anticancer drugs<br />
is limited by the affinity for one or more of the ABC transporters expressed in the<br />
epithelial layer of the gastro-intestinal tract, in particular P-glycoprotein (Pgp;<br />
ABCB1), Breast Cancer Resistance Protein (BCRP; ABCG2), MRP2 (ABCC2) and/or<br />
affinity for cytochrome P450 3A (CYP3A). Based on our proof of concept trial<br />
demonstrating high systemic exposure to orally applied docetaxel as a drinking<br />
solution in combination with the ‘boosting’ agent ritonavir, an efficacious inhibitor of<br />
gut wall and hepatic CYP3A4, we tested an oral capsule formulation of docetaxel,<br />
denoted ModraDoc001, in patients administered together with ritonavir (figure 1).<br />
The systemic exposure to oral docetaxel was on average 75% of that achieved after<br />
intake of the drinking solution of docetaxel that we applied previously. Importantly<br />
however, the interpatient variation of systemic exposure to docetaxel was substantially<br />
lower after intake of the capsule (%CV of 93% after intake of the drinking solution<br />
and 36% after intake of the capsules). In addition, the intrapatient variability in<br />
systemic exposure to docetaxel after intake of ModraDoc001 plus ritonavir was low<br />
(%CV 20%). This represents an excellent basis for the swift further clinical<br />
development of this concept. Currently, other boosting agents are being tested in<br />
combination with ModraDoc001, such as ketoconazole and grapefruit juice. We have<br />
also continued studies with oral paclitaxel (ModraPac).<br />
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Nienke Lankheet PhD student<br />
Jelte Meulenaar PhD student<br />
Johannes Moes PhD student<br />
Roos Oostendorp PhD student<br />
Susanne Quaak PhD student<br />
Rob Ter Heine PhD student<br />
Bas Teunissen PhD student<br />
Ly Tran PhD student<br />
Liia Vainchtein PhD student<br />
Joost Van Den Berg PhD student<br />
Jolanda Van Den Hoven PhD student<br />
Annemieke Van Winden PhD student<br />
Carolien Alderden Technical staff<br />
Abadi Gebretensae Technical staff<br />
Michel Hillebrand Technical staff<br />
Ciska Koopman-Kroon Technical staff<br />
Luc Lucas Technical staff<br />
Lianda Nan-Offeringa Technical staff<br />
Joke Schol Technical staff<br />
Bas Thijssen Technical staff<br />
Matthijs Tibben Technical staff<br />
Marja Roelvink Clinical trial manager<br />
Sandra Adriaansz Nurse practitioner<br />
Annelies Boekhout Nurse practitioner<br />
Ria Dubbelman Nurse practitioner<br />
Joke Foekema Nurse practitioner<br />
Emmy Harms Nurse practitioner<br />
Marjo Holtkamp Nurse practitioner<br />
Marianne Keessen Nurse practitioner<br />
Maria Kuiper Nurse practitioner<br />
Henk Mallo Nurse practitioner<br />
Annemarie Nol Nurse practitioner<br />
Albert Olijve Nurse practitioner<br />
Margaret Schot Nurse practitioner<br />
Wilma Uyterlinde Nurse practitioner<br />
Jana Van Der Sar Nurse practitioner
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Figure 1: Plasma concentration-time profiles of a representative patient who was treated with oral<br />
docetaxel formulated as capsules ModraDoc001 (denoted as MODEA) on two occasions and with an oral<br />
drinking solution on a third occasion, on all accasions taken together with one single dose of 100 mg of the<br />
boosting agent ritonavir.<br />
III-2 Pharmacology (PK/PD, ADME, mass balance) of novel anticancer drugs<br />
Currently, we are performing twenty-eight phase I/II, pharmacological and proof of<br />
concept studies. We recruited over 220 new patients in these studies in <strong>2008</strong>. Sixteen<br />
of these studies are two- or multicenter studies. Representative examples are<br />
described below.<br />
a. Studies with the poly-ADP-ribose polymerase (PARP1) inhibitor AZD2281<br />
In a two-center phase I trial we test the safety, optimal dose and schedule of<br />
AZD2281. The drug is given daily BID in combination with three-weekly carboplatin<br />
and paclitaxel, or in combination with weekly paclitaxel. This study is based on our<br />
recent experience with AZD2281 given as single agent. AZD2281 is taken orally daily<br />
without interruption at doses of 100-200 mg BID. We have demonstrated promising<br />
anticancer activity of the combination of carboplatin and AZD2281. However,<br />
prolonged mild bone marrow suppression made 3-weekly dosing unfeasible. For this<br />
reason the ‘bone marrow sparing’ agent paclitaxel was added to the three weekly<br />
administration of carboplatin. Preliminary results reveal that this strategy is feasible<br />
and active. Weekly paclitaxel at 80 mg/m 2 is feasible in combination with AZD2281<br />
and was found to be active. Current studies aim to assess the activity in a subgroup of<br />
patients with the genotype and phenotype of homologous recombination deficiency.<br />
b. Phase I study with an oral gemcitabine prodrug<br />
Gemcitabine is an attractive candidate for oral application. Based on the experience<br />
obtained in a phase I study with oral gemcitabine, applied every other day for 21 days<br />
followed by one week of rest, we started a new phase I trial with a gemcitabine<br />
prodrug. Since oral gemcitabine is swiftly deanimated presystemically, a prodrug was<br />
developed in which the amino group of gemcitabine is protected from deamination<br />
by the linker valproic acid. Clinical and laboratory studies with this novel prodrug<br />
formulation of gemcitabine have revealed excellent safety and relevant systemic<br />
exposure to gemcitabine after oral administration. Significant activity was observed in<br />
two patients with advanced mesothelioma who are now on study for more than 10<br />
months and who clearly showed disease progression prior to start of intake of oral<br />
gemcitabine prodrug. Complete and persistent PSA normalization has been observed<br />
in advanced and hormonal and docetaxel refractory prostate cancer. In this patient,<br />
the oral gemcitabine prodrug is given in combination with erlotinib.<br />
c. Studies with angiogenesis inhibitors<br />
Two phase I studies with the orally available angiogenesis inhibitors E7080 and Bay<br />
57-9352 are ongoing. The main mechanism of action of these three molecules is<br />
inhibition of VEGFR 1 and 2. In dose-escalating studies, E7080 is applied as a single<br />
agent and Bay 57-9352 is given in combination with irinotecan and capecitabine.<br />
Hypertension and proteinuria are the main toxicities of these agents. We started a<br />
third trial with an angiogenesis agent this year. It is pazopanib given in combination<br />
with topotecan. Of interest, significant antitumor activity has been observed in all
three studies. In several heavily pretreated patients with advanced mesothelioma,<br />
melanoma, rectal cancer, osteosarcoma, soft tissue sarcoma and head and neck<br />
cancer, significant antitumor activity including long lasting partial remissions were<br />
noted.<br />
d. Other phase I studies<br />
We finalized this year the three-center phase I study with the orally available and<br />
irreversible HER1, -2 and -4 inhibitor PFS-00299804. This is a first-in-man study<br />
initiated in our institute. The optimal dose-level for further testing is 40 mg/day for<br />
21 days q day 28. Moderate to significant but overall manageable skin and gastrointestinal<br />
toxicity was observed. Other phase I studies are being performed with the<br />
aurora kinase B inhibitor AZD1152, showing neutropenia as dose-limiting activity.<br />
Significant antitumor activity was observed in carcinoma of unknown primary site.<br />
III -3 Pharmacokinetic and pharmacodynamic modelling and simulation<br />
Pharmacokinetic and pharmacodynamic (PK/PD) modeling and simulation were<br />
used to study the influence of polymorphisms in drug metabolizing enzymes on the<br />
pharmacokinetics and toxicity of high-dose chemotherapy with cyclophosphamide,<br />
thiotepa and carboplatin. Polymorphisms in glutathione-S-transferases (GST) play a<br />
role in the variability in thiotepa pharmacokinetics. However, the allele frequencies<br />
were low in the population. Polymorphisms in genes encoding for aldehyde<br />
dehydrogenase (ALDH) were related to the occurrence of severe liver toxicity and<br />
hemorrhagic cystitis, suggesting a role for genotyping in preventing toxicity after<br />
intensive alkylating chemotherapy.<br />
Data from a phase I dose escalating trial of the experimental oral anticancer agent<br />
E7820 were analyzed using PK/PD modeling. The influence of food on the PK of this<br />
agent was assessed. The relationship between exposure to E7820 and alfa-2-integrin<br />
expression on platelets (a possible biomarker for E7820) was described by an indirect<br />
effect model, enabling the assessment of different dosing schedules on the levels of<br />
this biomarker.<br />
The program of oral docetaxel was supported by the development of a population<br />
pharmacokinetic (PK) model to evaluate and quantify the influence of ritonavir on<br />
the PK of docetaxel. Thirty-six patients were included in two studies, and PK data<br />
were collected. The absolute bioavailability of docetaxel increased 2-fold from 14 to<br />
29% by ritonavir co-administration. The inhibition and re-activation of CYP3A4 by<br />
ritonavir occurred instantaneously and was best described by an equilibrium constant<br />
(K eq) of 0.068 mL/µg. A maximum inhibition of docetaxel clearance of more than<br />
90% was reached. A PK model describing both the PK of orally and iv. administered<br />
docetaxel in combination with ritonavir was successfully developed. Coadministration<br />
of ritonavir leads to increased oral absorption and to a reduced<br />
elimination rate of docetaxel.<br />
III-4 Pharmacogenomics to identify patients at risk for toxicity or undertreatment<br />
Pharmacogenetic screening of patients treated with 5-FU/Capecitabine<br />
The genotype of the DPYD gene in patients treated with 5-FU or capecitabine was<br />
determined, in order to relate the genotype and phenotype to severe 5-FU-induced<br />
toxicity. The previously developed diagnostic real-time PCR assay was applied in<br />
routine clinical practice demonstrating the feasibility of daily assessment of the<br />
DYPD*2 genotype confirming the results of high-throughput screening of this<br />
mutation in order to test the prevention of severe 5-FU-related toxicity. To date, a<br />
population of more than 600 patients has been genotyped prior to start of<br />
capecitabine therapy, amongst which seven heterozygotes have been identified.<br />
III-5 Proteomics<br />
We finalized recruitment in proteomics studies aimed to identify and prospectively<br />
validate unique protein profiles in serum of patients with breast, gastric, colorectal<br />
and renal cell cancer. The analysis employing the ProteinChip SELDI-TOF MS<br />
system (Ciphergen) demonstrated unique protein profiles discriminating patients<br />
with advanced disease from normal controls in all these diseases. In line with all<br />
other proteomics studies published to date we did not identify unique oncoproteins.<br />
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IV Supportive care studies in patients with breast cancer<br />
We continued the originally single center study to identify the best therapeutic<br />
intervention in female patients with menopausal complaints induced by treatment<br />
for breast cancer. This year, we opened the study also in the Albert Schweitzer<br />
Hospital in Dordrecht to increase the recruitment rate in this trial. Currently, 75 of<br />
the planned 100 patients have been recruited. It concerns a prospective double-blind<br />
randomized placebo-controlled intervention study with placebo, venlafaxine (Efexor ® )<br />
and clonidine hydrochloride.<br />
We continued the multicenter cardiac protection trial with the angiotensine II (ATI)<br />
receptor antagonist candesartan in patients with Her 2 positive breast cancer who<br />
received adjuvant treatment with trastuzumab. This is a double blind randomized<br />
trial versus placebo. The study is open in 18 Dutch hospitals and 40 of the 200<br />
planned patients have been registered.<br />
CLINICAL IMMUNOTHERAPY AND TARGETED THERAPY<br />
John Haanen, Christian Blank, Sandra Adriaansz, Willem Boogerd, Henk Mallo<br />
The clinical immuno- and targeted therapy group is responsible for the treatment of<br />
melanoma and renal cell carcinoma patients. Translational immunotherapy research<br />
focuses on adoptive cellular therapies, such as T-cell receptor gene therapy and<br />
treatment with tumor-infiltrating lymphocytes (TIL) for melanoma and DNA and<br />
peptide vaccination studies for HPV-related squamous cell cancers. For renal cell<br />
cancer our group is leading in the development of investigator-initiated phase II/III<br />
trials to improve the treatment with small molecule receptor tyrosine kinase<br />
inhibitors (RTKI), mTOR kinase inhibitors and combinations of cytokines and antiangiogenesis<br />
drugs.<br />
Melanoma<br />
Participation in phase III clinical studies:<br />
We participated in two phase III clinical trials in which a fully human monoclonal<br />
antibodies against ‘cytotoxic lymphocyte antigen-4’ (CTLA4) is the study drug.<br />
Blockade of CTLA4, a molecule expressed on activated T lymphocytes with<br />
immunomodulating effects on the immune response (safe guard against<br />
overstimulation) has been shown to increase cancer-specific immune responses in a<br />
number of malignancies. Only HLA-A*0201-positive patients can be enrolled since<br />
the treatment comprises two gp100 peptide vaccines that have binding affinity only<br />
for HLA-A2 molecules. Patients are randomized between peptide vaccine only, MDX-<br />
010 or ipilimumab only or the combination. The <strong>NKI</strong>-AVL has enrolled 11 patients in<br />
this study so far.<br />
Translational research with DNA vaccination in melanoma patients:<br />
This year we received approval from both the CCMO and the Ministry of<br />
Environmental affairs for the start of a phase I gene therapy study with a DNA<br />
vaccine that was produced within our own GMP facility (Amsterdam Biotherapeutics<br />
Unit (Am-BTU). The vaccine will be delivered into the epidermis of patients with<br />
stage IV melanoma using a tattoo or permanent make-up device (see Division of<br />
Immunology). We plan to enroll the first patient in <strong>2008</strong>.<br />
Translational research with tumor infiltrating lymphocytes in melanoma patients:<br />
We are preparing for a clinical phase II study using tumor-infiltrating lymphocytes<br />
for patients with metastatic melanoma. This is based on results from a single arm,<br />
single institution trial performed at the Surgery Branch of the NIH, Bethesda, USA,<br />
which showed a 50% objective response rate in heavily pretreated stage IV melanoma<br />
patients. Ours will be the first trial in Europe comparing TIL treatment to standard<br />
Dacarbazine chemotherapy. As of September <strong>2008</strong> the Division of Immunology and<br />
Medical Oncology is working closely together to prepare for this high impact trial.<br />
Renal cell carcinoma<br />
In the renal cell carcinoma program we closely collaborate with Dr Axel Bex from<br />
the urology-oncology group and with Dr Florry Vyth-Dreese who supervises the
monitoring of clinical immunotherapy studies (in collaboration with the Clinical<br />
Laboratory, headed by Dr. Wim Nooijen).<br />
We participated in a treatment-use program of the small molecule sunitinib, a<br />
multiple receptor tyrosine kinase inhibitor with high affinity for VEGF-R, PDGF-R,<br />
c-KIT and FLT3. Since VEGF and PDGF play prominent roles in the pathogenesis of<br />
sporadic clear cell renal cell cancer, inhibition of kinase activity of their receptors<br />
appeared to be a rational therapy in this patient population.<br />
The overall results in a group of more than 82 patients has been analyzed in<br />
collaboration with VUMC (Van den Veldt et al. Br J Cancer). The main finding of this<br />
study was that also in a unselected patient population suntinib treatment results in<br />
progression-free survival of about 9 months. Importantly, half of the patients<br />
required dose reductions. Based on these results a Dutch study is being designed that<br />
will base sunitinib dosing on pharmacokinetics in order to optimize both in terms of<br />
efficacy and toxicity sunitinib treatment.<br />
An investigator-initiated study to define the optimal time point for tumor<br />
nephrectomy in primary metastatic renal cell carcinoma patients is ongoing. This<br />
study is has now also been approved by the EORTC executive board as a randomized<br />
phase III study that will start in 2009. Patients will be randomized to receive either 3<br />
cycles of sunitinib treatment prior to tumor nephrectomy or immediate nephrectomy.<br />
Post surgery both groups will (re)start sunitinib treatment until disease progression.<br />
Study endpoint: Progression-free and overall survival.<br />
In addition, we participated in two phase I studies: sunitinib + IL-21 as first line<br />
treatment and sorafenib + IL-2 as second line treatment for metastatic clear cell renal<br />
cell cancer.<br />
BREAST CANCER THERAPY AND RESPONSE PREDICTION<br />
Sjoerd Rodenhuis, Sabine Linn, Gabe Sonke, Jos Beijnen, Corine Ekhart, Juliane Hannemann,<br />
Marjo Holtkamp, Alwin Huitema, Ingrid Mandjes, Paula Nederlof, Margaret Schot,<br />
Marc Van de Vijver, Laura Van ‘t Veer, Jelle Wesseling.<br />
The objective of this program is to develop and improve potentially curative therapy<br />
for patients with locoregional or oligometastatic breast cancer. For all these studies,<br />
close collaborations are maintained with many other clinical departments and<br />
research divisions. The Molecular Pathology Department is central in the response<br />
prediction studies. In <strong>2008</strong>, about 175 patients were entered in 8 ongoing adjuvant<br />
medical studies in breast cancer, and in six studies in advanced disease.<br />
Preoperative chemotherapy<br />
The preoperative chemotherapy program continues to accrue approximately 75<br />
patients per year and now includes data and tissues of over 300 patients. This<br />
program is the core of an expanding multidisciplinary research effort to optimize<br />
response prediction (e.g. with mRNA expression arrays) and to improve response<br />
monitoring (e.g., with contrast-enhanced MRI) to allow a change in chemotherapy<br />
regimen when the development of a pathologic complete remission appears to be<br />
unlikely. A CTMM project based on this clinical work, but with many contributions<br />
of basic research laboratories, began in November 2007. It involves a collaboration of<br />
the institute with 2 academic partners (the Academic Medical Center in Amsterdam<br />
and the Erasmus Medical Center in Rotterdam) and three industrial partners<br />
(Organon, Philips and MRC Holland).<br />
A distinct group of breast cancers lack the ability to repair double-strand DNA breaks<br />
through homologous recombination (HR). For these tumors, bifunctional alkylating<br />
agents function as targeted toxins (as do PARP inhibitors), since tumor cells are<br />
highly sensitive while other cells are not. This situation is present in breast cancers of<br />
patients who carry a BRCA1 or BRCA2 mutation, but has also been reported in about<br />
30% of sporadic breast cancers. Tumors of patients in the neoadjuvant program are<br />
tested for HR defects, and if present, are eligible for treatment in a randomized phase<br />
II study of intermediate dose alkylator therapy. Preliminary findings indicate that part<br />
of the HR deficient tumors can be identified through aGCH analysis.<br />
HER2 positive breast cancers strongly benefit from Trastuzumab-based systemic<br />
treatment, but this is not yet standard in the neo-adjuvant setting. Up-front use of<br />
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Trastuzumab may also allow the avoidance of anthracyclines at least for a subgroup<br />
of tumors. A multi-centre phase II study investigating the response to nonanthracycline<br />
based chemotherapy incorporating Trastuzumab is currently recruiting<br />
patients and collecting tissues. Preliminary results from our center show promising<br />
pathologic complete response rates (figure 2).<br />
Figure 2: Pathological complete response rate of breast and axilla following preoperative chemotherapy<br />
with trastuzumab, carboplatin and paclitaxel in HER2-positive locally advanced breast cancer.<br />
Abbreviations: CR (b+a): pathological complete response of both breast and axilla; CR (breast):<br />
pathological complete response of the primary tumor; ER: estrogen receptor.<br />
Microarrays, prognosis and response prediction Microarray technology is<br />
gaining credibility as an important diagnostic tool in guiding treatment decisions in<br />
breast cancer. Our institute has developed a 70-gene prognostic signature that<br />
outperformed classic clinico-pathologic prognostic factors and remained a strong<br />
independent prognostic factor in multivariate analysis. In collaboration with<br />
AGENDIA, we compared the performance of the signature to predict response to<br />
neoadjuvant chemotherapy. All pathologic complete remissions were observed in<br />
tumors with a high-risk profile, confirming the expectation that the more slowly<br />
proliferating low-risk tumors may be less sensitive to chemotherapy.<br />
In 2004, the MATADOR study was initiated and this study is currently open in 31<br />
centers in the Netherlands; ~240 patients have been included. The primary objective<br />
of this study is to identify predictive gene expression profiles for a disease-free<br />
survival benefit of either a docetaxel-containing regimen (docetaxel-doxorubicincyclophosphamide)<br />
or an accelerated doxorubicin-cyclophosphamide regimen.<br />
In collaboration with Laura Van ‘t Veer and the Erasmus University (Rotterdam) we<br />
have validated and refined a predictive profile for tamoxifen resistance in the<br />
metastatic setting (see also under Division of Experimental Therapy). We are<br />
currently investigating the utility of this profile for the adjuvant setting.<br />
We have recently started a preoperative single centre trial to study responsiveness of<br />
hormone-receptor positive breast cancers to Tamoxifen, Anastrozole or Anastrozole<br />
in combination with Fulvestrant in postmenopausal patients. Prior to primary<br />
surgery, patients are exposed at least two weeks to hormonal agents. Blood and tumor<br />
tissue is collected before and after treatment for molecular studies, including gene<br />
expression profiling. Changes in Ki67 expression, in combination with changes in<br />
TUNEL labeling as a marker for apoptosis are used as a read-out for hormonal<br />
therapy responsiveness.<br />
In collaboration with Leiden University Medical Center and the TEAM study group,<br />
the TEAM II study has been initiated to investigate the optimal duration of<br />
neoadjuvant Exemestane therapy and to study the benefit of three years adjuvant oral<br />
Ibandronate in addition to standard adjuvant systemic therapy in postmenopausal<br />
patients with hormone-receptor positive early breast cancer.
THORACIC ONCOLOGY<br />
Paul Baas, Sjaak Burgers, Michel Van den Heuvel, Jose Belderbos, Wieneke Buikhuisen,<br />
Houke Klomp, Peter Kunst, Petra Nederlof, Jan Schellens, Michel Wouters<br />
The department’s clinical research focuses on immunological studies, combined<br />
modality approaches and mesothelioma treatment.<br />
Non-Small Cell Lung Cancer (NSCLC) Patients with early stage NSCLC and who<br />
are candidates for surgical resection are included in a neo-adjuvant study with<br />
erlotinib, which is given for 3 weeks. This study is led by the department of thoracic<br />
surgery (Houke Klomp). Preliminary findings indicate that such a short course of<br />
targeted therapy causes a significant degree of apoptosis at pathological examination.<br />
In patients with advanced NSCLC, a pharmacogenomics study has now included 100<br />
patients who have received cisplatin and gemcitabine as first-line chemotherapy. The<br />
correlation between plasma levels and metabolism drugs and the presence of Single<br />
Nucleotide Polymorphisms (SNP’s) is currently being analyzed. In patients with<br />
unexpected metastasis in N2 lymph nodes during surgery, the effect of mediastinal<br />
RT is under evaluation in the European phase III LUNGART study. A total of 700<br />
patients will be required.<br />
Two vaccination studies have been initiated in <strong>2008</strong> for patients with advanced stages<br />
of NSCLC: A phase IB study of maintenance therapy with Selectikine after a short<br />
course of RT; and a randomized phase II study of Stimuvax with or without<br />
radiotherapy in HLA-A2 positive patients (figure 3). Both studies build on the<br />
postulated synergetic effects of simultaneous radiotherapy and immunotherapy.<br />
In addition, patients will be enrolled in a phase III vaccination study with Lucanix.<br />
Figure 3: Working mechanism of Stimuvax.<br />
In second line NSCLC, we have participated in a randomized study of erlotinib and<br />
placebo vs. erlotinib and sunitinib, a tyrosine kinase inhibitor which blocks both the<br />
VEGF receptor and the EGF receptor.<br />
Small Cell Lung Cancer (SCLC) In patients with Limited Stage disease, we<br />
participate in the European phase III CONVERT study, which investigates the effects<br />
of concurrent twice daily radiotherapy and chemotherapy vs. sequential chemotherapy<br />
and radiotherapy. For patients with extensive stage disease, a phase II study with<br />
sunitinib induction therapy is planned and will start beginning of 2009. In the first<br />
six weeks after registration, single agent Sunitinib is given and patients are closely<br />
monitored for response/progression with PET-CT scans.<br />
Pleural disease In 2007, the pleural fluid bank study was initiated to allow the<br />
development of innovative diagnostic and treatment algorithms. All patients<br />
presenting with a pleural effusion, who are candidates for drainage are asked to<br />
participate and to consent with biomarker research employing both frozen serum<br />
and pleural fluid samples. Currently we have patient data and matched samples in<br />
over 300 patients.<br />
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Malignant Pleural Mesothelioma (MPM) The national randomized phase III<br />
maintenance study of thalidomide after standard chemotherapy in MPM has now<br />
accrued 80% of the target population. A subgroup of patients in both arms will be<br />
analyzed for predictive and prognostic factors of serial serum samples including<br />
VEGF, bFGF, IL-6 and Cytokeratin markers.<br />
The EORTC phase II study 08931 tested the toxicity of induction chemotherapy<br />
followed by extra-pleural-pneumonectomy and hemi-thoracic irradiation. The toxicity<br />
data will be analyzed by the EORTC in the first quarter of 2009.<br />
For second and third line therapy we offer patients a randomized phase III study of<br />
Vorinostat vs. placebo. This international study has passed its first endpoint at 220<br />
out of 660 planned patients and since no toxicity was observed the second stage has<br />
been initiated.<br />
For 2009, a randomized phase I-II study will examine the effect of Axitinib (a potent<br />
oral anti-vascular drug) to standard chemotherapy in patients with MPM. Biopsies<br />
and dynamic MRI examination before and after 3 courses of chemotherapy will be<br />
performed to evaluate the effect of the addition of Axitinib. Vascular staining,<br />
apoptosis and circulating endothelial/tumor cells will also be measured, in addition<br />
to the changes in vascular flow analysis by MRI.<br />
GASTROENTEROLOGY<br />
Henk Boot, Annemieke Cats, Marianne Smits, Babs Taal, Hans Bonfrer, Maarten Deenen,<br />
Edwin Jansen, Tiny Korse, Jan Schellens, Marcel Verheij<br />
Neuroendocrine tumours (NET) Urinary excretion of 5-HIAA, the degradation<br />
product of serotonin, is widely available as a marker in NET, but has a low sensitivity<br />
and the 24-hour collection is inconvenient for patients with diarrhea. The serum<br />
marker Chromogranin A (CgA) has the advantage of being positive in NETs of<br />
various origin, but a thorough evaluation during follow-up is lacking. In our study<br />
gastrointestinal NET patients (n=39) were monitored 3-monthly for one year during<br />
treatment. Significant correlations were found between CgA and symptoms such as<br />
physical functioning (p=0.01) and overall quality of life (p=0.03), in contrast to<br />
5-HIAA. Similarly, Cox regression showed an association between CgA levels and<br />
survival time (p=0.02), and not for 5-HIAA levels.<br />
In NET fibrosis of the tricuspid heart valve (carcinoid heart disease or CHD) is a well<br />
known complication probably as a result of exposure to serotonin produced by liver<br />
metastases. This complication develops in 20-70% and is a major cause of death in<br />
patients with carcinoid syndrome. To detect this in an early phase and to select<br />
patients for heart valve surgery we evaluated the role of NT-proBNP, a natriuretic<br />
peptide available as a marker of left ventricular dysfunction, and CgA.<br />
Figure 4: ROC-curve demonstrating the accuracy of<br />
CgA and NT-proBNP distinguishing patients with<br />
severe CHD from patients without or with mild CHD.<br />
Among 102 NET-patients, CHD was present at cardiac ultrasound in 14 patients<br />
(14%). In 12 of these patients (86%) NT-proBNP was elevated, with a specificity of<br />
80% (figure 4). In the univariate logistic regression analysis NT-proBNP (p=
survival (p
126<br />
RADIOTHERAPY<br />
Division head, group leader, Marcel Verheij<br />
Berthe Aleman MD PhD Academic staff<br />
Harry Bartelink MD PhD Academic staff<br />
Jose Belderbos MD PhD Academic staff<br />
Eugène Damen PhD Academic staff<br />
Roel De Boer PhD Academic staff<br />
Luc Dewit MD PhD Academic staff<br />
Paula Elkuizen PhD Academic staff<br />
Rick Haas MD PhD Academic staff<br />
Olga Hamming-Vrieze MD Academic staff<br />
Guus Hart MSc Academic staff<br />
Wilma Heemsbergen MSc Academic staff<br />
Frank Hoebers MD PhD Academic staff<br />
Edwin Jansen MD Academic staff<br />
Joost Knegjens MD Academic staff<br />
Han Krewinkel MSc Academic staff<br />
Joos Lebesque MD PhD Academic staff<br />
Corrie Marijnen MD PhD Academic staff<br />
Harry Masselink MD Academic staff<br />
Ben Mijnheer PhD Academic staff<br />
Luc Moonen MD PhD Academic staff<br />
Floris Pos MD PhD Academic staff<br />
Coen Rasch MD PhD Academic staff<br />
Babs Reichgelt MD Academic staff<br />
Peter Remeijer PhD Academic staff<br />
Nicola Russell MD PhD Academic staff<br />
Govert Salverda MD Academic staff<br />
Christoph Schneider PhD Academic staff<br />
Joep Stroom PhD Academic staff<br />
Bart Van Bunningen MD Academic staff<br />
Marcel Van Herk PhD Academic staff<br />
Baukelien Van Triest MD PhD Academic staff<br />
Karijn Verschueren MD Academic staff<br />
Corine Van Vliet-Vroegindeweij PhD<br />
Academic staff<br />
Marieke Van Zwienen MD PhD Academic staff<br />
Marcel Verheij MD PhD Academic staff<br />
Frits Wittkämper PhD Academic staff<br />
Monique Bloemers MD Temporary staff<br />
Gerben Borst MD Temporary staff<br />
Desiree Van Den Bongard MD PhD<br />
Temporary staff<br />
Birgit Hollmann MD Temporary staff<br />
Bas Kreike MD Temporary staff<br />
Oene Van Meer MD Temporary staff<br />
Philip Meijnen MD PhD Temporary staff<br />
Dimitry Nuyten MD Temporary staff<br />
Gabriele Speijer Temporary staff<br />
Roel Steenbakkers MD PhD Temporary staff<br />
Ben Vanneste MD Temporary staff<br />
Francine Voncken MD Temporary staff<br />
Martijn Eenink PhD Post-doc<br />
Marnix Maas PhD Post-doc<br />
DIVISION OF RADIOTHERAPY<br />
In <strong>2008</strong> our department continued to invest in its major research themes: improving<br />
treatment accuracy, efficacy and safety. Intensity-modulated radiotherapy leads to<br />
superior dose distributions and is applied in an increasing number of indications.<br />
Optimal geometrical precision before and during treatment is achieved by cone-beam<br />
CT-based image guidance, now routinely in use on 5 of our 9 linear accelerators.<br />
Both technologies have been pivotal for allowing dose escalation and the clinical<br />
implementation of extreme hypofractionated radiation schedules, e.g. in early stage<br />
lung cancer and solitary brain tumors/metastases. To further enhance radiation<br />
response new combined modality approaches are explored using both classical<br />
chemotherapeutic agents and novel biological response modifiers. These new<br />
generation biologicals have become available through our own research as well as<br />
through collaborations with pharmaceutical companies, and are making their way<br />
from the lab to the clinic as potential radiosensitizers. Given the increasing<br />
complexity and potential toxicity of these treatments, we have given high priority to<br />
dose verification by in vivo dosimetry. All curative treatments are currently verified by<br />
means of EPID dosimetry, minimizing the risk of over- and underdosage.<br />
Functional imaging becomes increasingly important in tumor staging, target<br />
delineation and response prediction. In collaboration with the departments of<br />
Radiology and Nuclear Medicine we therefore evaluate the value of several imaging<br />
modalities in these processes, including MRI spectroscopy, 18F-FAZA-PET, 99mTc-<br />
MIBI-SPECT and 99mTc-Annexin V scintigraphy. The integration of both anatomical<br />
and functional information in our treatment protocols creates the possibility to adapt<br />
therapy at an early stage and on an individual basis.<br />
In September <strong>2008</strong>, the European Comprehensive Cancer Centres Alliance (ECCCA)<br />
was founded during an international symposium at the <strong>NKI</strong>-AVL. This collaboration<br />
between three European cancer centers (Institut Gustave Roussy, Karolinska<br />
Institutet and <strong>NKI</strong>-AVL), brings together powerful technological platforms of<br />
genomics/proteomics based selection assays and preclinical evaluation tools to<br />
identify promising agents for combined application in early clinical trials. There are<br />
currently three clinical trial recruiting patients on a jointly basis.<br />
The Radiotherapy department is also an active partner in a new initiative in proton<br />
therapy. The Holland Particle Therapy Center (HollandPTC) project is a joint<br />
initiative of Erasmus MC, <strong>NKI</strong>-AVL, Leiden University MC and TU Delft, and aims at<br />
establishing a particle therapy center in Delft for both patient care, research and<br />
technological development.<br />
IMAGE ACQUISITION AND PROCESSING<br />
A Al-Mamgani1 , Anja Betgen, Jose Belderbos, Suzanne Van Beek, Roman Bohoslavsky, Jan De Boer,<br />
Josien De Bois, X Chai2 , Eugene Damen, H Dees-Ribbers, Joop Duppen, Rick Haas,<br />
Wilma Heemsbergen, David Jaffray3 , Edwin Jansen, Rianne De Jong, Simon Van Kranen,<br />
Corrie Marijnen, Vanessa Mexner, Danny Minkema, Jasper Nijkamp, Erik-Jan Rijkhorst, Floris Pos,<br />
Simon Rit, Christoph Schneider, Monique Smitsmans, Corine Van Vliet-Vroegindeweij, Marnix Witte,<br />
Jochem Wolthaus, Lambert Zijp, Coen Rasch, Joos Lebesque, Peter Remeijer, Jan-Jakob Sonke,<br />
Marcel Van Herk<br />
Cone beam CT reconstruction Respiratory motion induces artifacts in conebeam<br />
(CB) CT images of the thorax which can prevent an accurate localization of<br />
lung tumors at treatment time for patient positioning. Respiration-correlated<br />
reconstruction has been used to correct for this motion but requires a long<br />
acquisition time. We therefore developed an online motion-compensated CBCT<br />
reconstruction technique based on a prior model of the respiratory motion obtained<br />
from the 4D planning CT with deformable registration. The motion was compensated<br />
1 EMC Rotterdam / 2 Academic Medical Center<br />
3 University of Toronto/Princess Margaret Hospital, Ontario, Canada
during filtered-backprojection of scans made on the treatment machine. The method<br />
was evaluated on 26 CBCT scans of 3 patients acquired on an Elekta Synergy system.<br />
Visual observation of reconstructed MC-CBCT images showed that most of the<br />
respiratory motion artifacts were removed obtaining image quality comparable to<br />
static CBCT, even with a 1 min acquisition time protocol where 4D CBCT is hardly<br />
usable (see figure 1). Using the off-line computed motion model, the computational<br />
time 67 s for the 1 min acquisition protocol (concurrent with CB acquisition),<br />
providing a high quality image within a few seconds after the end of the acquisition.<br />
Figure 1: Effect of motion compensation reconstruction for a typical lung cone beam CT scan used for<br />
image guided radiotherapy. a) 3D reconstruction without motion compensation for a 4 minute acquisition<br />
time (intended for 4D acquisition). b) Motion compensated reconstruction (4 min. acquisition time).<br />
Note the improved sharpness of tumor and diaphragm. c) Motion compensated reconstruction (1 min.<br />
acquisition time). The image quality is more than acceptable for image guidance of the tumor and<br />
normal structures.<br />
Image guidance Recently, promising results have been reported for stereotactic<br />
body radiotherapy (SBRT) of primary and metastatic liver cancer. For conventional<br />
RT of liver cancer, the bony anatomy is often used as a surrogate for tumor location,<br />
which is too imprecise for SBRT. We implanted markers close to the lesion as a<br />
surrogate for tumor location and applied four dimensional (4D) cone beam CT<br />
guidance for registration and correction of the marker location. Four patients with<br />
metastasic liver cancer were included in this study so far (2 – 4 markers per patient).<br />
Patients were treated with 15 fractions of 3 Gy with an online or offline bony anatomy<br />
setup correction protocol using CBCT guidance. Retrospectively, markers were<br />
localized using locally rigid registration on 4D CBCT. Baseline variations were<br />
quantified by the difference in the (time weighted average) position of the markers<br />
and the bony anatomy. The results were analyzed in terms of group mean (M),<br />
standard deviation. The average peak-to-peak tumor amplitude assessed by 4D CBCT<br />
was 1.2 cm [range 0.7 cm - 1.6 cm]. Interfraction baseline shifts were about 1-2 mm<br />
in each direction. Baseline variations of 1-2 mm (1 SD) have been observed over the<br />
course of RT of liver cancer patients.<br />
Correction strategies Current image guided systems provide efficient on-line<br />
correction of target positioning. Significant motion, however, can occur between<br />
target and organs-at-risk (OARs), for instance for lung tumors (baseline shifts).<br />
Then, proper target positioning may lead to overdosage of OARs. We devises a<br />
practical image guidance system that can deal with such differential motion. During<br />
planning for lung SBRT patients, the cord was expanded by 1 cm, but sometimes less<br />
expansion was required to reach an acceptable plan. The guidance system registration<br />
regions, placed on spinal column and target. Image registration is performed for<br />
both regions providing setup error data of cord and target. The software checks the<br />
proposed correction against predefined limits for both regions. When the correction<br />
would move the high dose region outside limits towards the OAR, a warning is given.<br />
In a separate review procedure, the operator ‘fades’ between correct target and OAR<br />
positioning to reach a compromise with both regions within limits. The system was<br />
applied in 86 patients. In 10 patients, the proximity of the tumor and OAR required<br />
reduced OAR expansions. In 8 patients, a compromise had to be made because the<br />
baseline shift exceeded the OAR margin during one or more fractions. In all cases,<br />
it was possible to find a correction that satisfied both target and OAR constraints.<br />
This study showed that especially in hypofractionated radiotherapy of lung tumors,<br />
differential motion of tumor and OAR may lead to serious overdosages when OARs<br />
are not considered in the guidance procedure.<br />
127<br />
RADIOTHERAPY<br />
Anton Mans PhD Post-doc<br />
Leah McDermott PhD Post-doc<br />
Vanessa Mexner PhD Post-doc<br />
Anke Van Mourik PhD Post-doc<br />
Juup Nijholt PhD Post-doc<br />
Erik-Jan Rijkhorst PhD Post-doc<br />
Simon Rit PhD Post-doc<br />
Jan-Jakob Sonke PhD Post-doc<br />
Mine Temurhan PhD Post-doc<br />
Rajko Topolnjak PhD Post-doc<br />
Markus Wendling PhD Post-doc<br />
Marnix Witte PhD Post-doc<br />
Johan De Boer MSc PhD student<br />
Simon Van Kranen MSc PhD student<br />
Monique Smitsmans MSc PhD student<br />
Jochem Wolthaus MSc PhD student<br />
Jonathan Yang MSc PhD student<br />
Robin Kalisvaart Physician Assistant<br />
Margriet Kwint Physician assistant<br />
Gül Yüksel Physician assistant<br />
Anja Betgen Technical staff<br />
Josine De Bois Technical staff<br />
Joop Duppen Technical staff<br />
Angelo Mencarelli Technical staff<br />
Tom Minderhoud Technical staff<br />
Jasper Nijkamp MSc Technical staff<br />
Agnieszka Olszewska MSc Technical staff<br />
Carmen Panneman MSc Technical staff<br />
Carolien Peters Technical staff<br />
Kenneth Pengel Technical staff<br />
Lennert Ploeger Technical staff<br />
Edwin Roosjen Technical staff<br />
Maddalena Rossi Technical staff<br />
René Tielenburg Technical staff<br />
Lambert Zijp MSc Technical staff
128<br />
RADIOTHERAPY<br />
Publications<br />
Image acquisition and processing<br />
Fitton I, Steenbakkers RJ, Gilhuijs K,<br />
Duppen JC, Nowak PJ, van Herk M,<br />
Rasch CR. Impact of anatomical location<br />
on value of CT-PET co-registration for<br />
delineation of lung tumors. Int J Radiat<br />
Oncol Biol Phys <strong>2008</strong>;70:1403-1407<br />
Rit S, Wolthaus J, van Herk M, Sonke JJ.<br />
On-the-fly motion-compensated cone-beam<br />
CT using an a priori motion model. Med<br />
Image Comput Comput Assist Interv Int<br />
Conf Med Image Comput Comput Assist<br />
Interv <strong>2008</strong>;11:729-736<br />
Smitsmans MH, Pos FJ, de Bois J,<br />
Heemsbergen WD, Sonke JJ,<br />
Lebesque JV, van Herk M. The influence of<br />
a dietary protocol on cone beam CT-guided<br />
radio therapy for prostate cancer patients.<br />
Int J Radiat Oncol Biol Phys <strong>2008</strong>;71:<br />
1279-1286<br />
Sonke JJ, Lebesque J, van Herk M.<br />
Variability of four-dimensional computed<br />
tomography patient models. Int J Radiat<br />
Oncol Biol Phys <strong>2008</strong>;70:590-598<br />
Sonke JJ, Rossi M, Wolthaus J, van Herk M,<br />
Damen E, Belderbos J. Frameless Stereotactic<br />
Body Radiotherapy for Lung Cancer Using<br />
Four-Dimensional Cone Beam CT<br />
Guidance. Int J Radiat Oncol Biol Phys.<br />
<strong>2008</strong> Nov 27. (epub ahead of print)<br />
Van Herk M. Will IGRT live up to its<br />
promise? Acta Oncol <strong>2008</strong>;47:1186-1187<br />
Construction of a midposition CT scan for planning of lung cancer patients using<br />
deformable registration. Lower lobe lung tumors move with amplitudes of up to 2 cm<br />
due to respiration. To reduce respiration imaging artifacts in planning CT scans, 4D<br />
imaging techniques are used. Currently, we use a single (midventilation) frame of the<br />
4D data set for clinical delineation of structures and radiotherapy planning. A single<br />
frame, however, often contains artifacts due to breathing irregularities. The aim of<br />
this work is to develop a framework for the acquisition of a good quality scan<br />
representing all scanned anatomy in the mean position by averaging transformed<br />
(deformed) CT frames, i.e. canceling out motion. 4D and inspiration breath-hold<br />
(BH) CT scans were acquired for 13 patients. An iterative multiscale motion estimation<br />
technique was applied to the 4D CT scan, similar to optical flow but using image<br />
phase (gray-value transitions from bright to dark and vice versa) instead. From the<br />
(4D) deformation vector field (DVF) derived, the local mean position in the<br />
respiratory cycle was computed to deform all structures of the original 4D CT scan to<br />
this mean position. A 3D midposition (MidP) CT scan was then obtained by averaging<br />
of the deformed 4D CT scan. The shape of the tumor, with respect to that of the BH<br />
CT scan, was better represented by the MidP reconstructions than any of the 4D CT<br />
frames (including MidV; reduction of ‘shape differences’ was 66%). The MidP scans<br />
contained about one-third the noise of individual 4D CT scan frames. Tumor shape<br />
and position in the MidP CT scan represents that of the BH CT scan better than<br />
MidV CT scan and, therefore, was found to be appropriate for treatment planning.<br />
Geometrical Uncertainties in Soft Tissue Sarcomas of Extremities Standard of<br />
care for almost all extremity soft tissue sarcomas (ESTS) patients is surgery, followed<br />
by radiotherapy (RT). Several studies however, show benefit of preoperative RT,<br />
especially concerning late morbidity endpoints such as fibrosis and joint stiffness.<br />
The purpose of this study was to quantify volume and positional changes of the Gross<br />
Target Volume (GTV) during preoperative RT and investigate the possibility for more<br />
advanced correction strategies. Ten patients with lower limb ESTS were included in<br />
this retrospective study. Planning CT-scan and 2 CBCT-scans acquired at the start of<br />
the treatment and after five weeks were used for delineation of the GTV. Delineation<br />
was performed after a registration on bony anatomy, which was used as a surrogate<br />
for tumor position during treatment for setup correction. In four patients volumes<br />
did not change more than 3%. However, significant changes were discovered in six<br />
patients. In three patients an increase of the GTV volume was measured (10%, 19%<br />
and 26%) and in three patients a decrease was noted (12%, 17% and 23%). The COG<br />
of these delineations changed for the last CBCT with a mean vector length of 0.39<br />
cm (range 0.03 – 0.86 cm). The changes in the increasing volumes are always away<br />
from the bones, and vice versa in the decreasing volumes in the direction of the<br />
bones. Clinically applied margins were adequate to account for these uncertainties.<br />
To further reduce morbidity, reduction of geometrical uncertainties and margins are<br />
essential. This might require soft tissue guidance because the bony anatomy is not<br />
always a good surrogate for GTV position and adaptive re-planning to account for<br />
volume changes.<br />
Figure 2: Comparison of the so-called mid-ventilation image, and a novel concept: the mid-position image.<br />
a) The mid-ventilation image is used clinically as most representative image of a breathing patient. It is<br />
selected from a respiration correlated CT scan by choosing that (animation) frame that is closest to the<br />
mean position of the tumor. b) the mid-position image is constructed by deforming all pixels of all frames<br />
to their mean position and then averaging the results. This image gives an optimal representation of the<br />
patients’ anatomy and has a much better image quality.
Target shape variability in rectum cancer patients Changes in filling of rectum<br />
and bladder cause large geometrical uncertainties during radiotherapy of rectum<br />
cancer patients. The purpose of this study was to quantify the interfraction CTV<br />
shape changes of rectum cancer patients treated pre-operatively with 5x5 Gy. We<br />
assessed the interfraction CTV shape variability on Cone-Beam CT (CBCT) scans<br />
acquired just prior to irradiation for 27 patients (prone position). We focused on<br />
mesorectum first adnd delineated this structure on the planning CT and on each<br />
CBCT scan. In addition, rectum and bladder were delineated. It turns out that<br />
interfraction shape variability is substantial in certain parts of the mesorectum: The<br />
systematic (]) and random (]) errors varied substantially for different regions of the<br />
mesorectum: ] and ] of up to 7 mm were found on the anterior upper part of the<br />
mesorectum. At the lateral and dorsal borders of the mesorectum the errors were<br />
relatively small except for below the os coccyx (tail bone). The large systematic and<br />
random errors in the upper-middle part were mainly caused by differences in rectum<br />
filling and not so much by differences in bladder filling. Errors below the os coccyx<br />
can be explained by differences in muscle tension.<br />
Margins for deformable objects Image guided radiotherapy in combination with<br />
advanced registration techniques revealed substantial deformation for tumor sites<br />
such as head-and-neck, rectum and lung. Classical margin recipes (e.g. M=2.5S+<br />
0.7s), assume the target to be a single rigid object, which is not true in the presence<br />
of deformations. The purpose of this study was to derive margins for deforming<br />
objects. The framework of the classical margin recipe was extended by analyzing<br />
multiple points in space (differential motion) or on a spherical surface with<br />
deformations perpendicular to the surface. All points move with a Gaussian<br />
distribution with a constant standard deviation ] and a single correlation r between<br />
the distributions, which could be varied. By generating many possible instances the<br />
margin required to encompass all points for 90% of the instances was derived as a<br />
function of the number of points, r and ] (Monte Carlo simulations). In this analysis,<br />
we focused on the effect of the systematic error. The effect of random errors is<br />
unchanged compared to a single rigid object. Complete target coverage for 90% of<br />
the population depended on the number of points and the their correlation. If points<br />
move with r=1, the solution for classical 90% coverage probability was retrieved, i.e.<br />
a margin of 2.5 ] for multiple targets and 1.3] for deformations (single sided 1D<br />
probability distribution). For the fully uncorrelated situation, margins depended on<br />
the number of points. For deformations 20 points results in a margin of 2.5], 200<br />
points in 3.2]. For a situation with multiple rigid targets, the margins required<br />
increased from 2.8] (2 targets) to 3.1] (5 targets). These data show that applying<br />
classical margins for deformations is incorrect for two reasons. First, target coverage<br />
is unaffected for surface-points moving inwards. Second, with more than one point<br />
the probability of having all points within classical margins simultaneously<br />
decreases. We expect that larger tumors need more points to describe observed<br />
deformations and margins will increase accordingly.<br />
High precision objectives for IMRT optimization using Pinnacle Although<br />
triangulated surface meshes are available in the Pinnacle treatment planning system,<br />
the objective functions for IMRT optimization rely on volumes described as contours<br />
on the CT slices. A set of replacement objectives was developed to exploit the<br />
increased definition of mesh-based volumes, enabling high precision IMRT<br />
optimization. A customized set of objectives that perform Monte Carlo volume<br />
integration of triangulated regions of interest (ROIs) resulting in a higher accuracy of<br />
the cost value evaluation. For a head and neck cancer patient, we first optimized<br />
applying the original Pinnacle functions until a minimum of the total cost was<br />
achieved. Next, we applied our new functions and continued optimization. The<br />
higher accuracy of the cost evaluation did require more computation time during<br />
initialization (a few minutes), but the optimization process was not noticeably slowed<br />
down. PTV coverage was improved somewhat without violating organs at risk (OARs)<br />
dose constraints because the OARs are described with a higher resolution. However,<br />
the differences are small unless a very high spatial accuracy is required, such as for<br />
the optic nerve.<br />
129<br />
RADIOTHERAPY
130<br />
RADIOTHERAPY<br />
Advanced analysis of clinical trial results Trials with centrally collected digital<br />
planning data allow, in principle, relating dose distribution details with clinical<br />
outcome. However, this type of analysis is rare because of the difficulty of comparing<br />
dose distributions between patients. The purpose of this study is to relate the dose to<br />
the anatomy outside the prostate with observed and predicted failure free survival.<br />
CT, delineations and 3D dose cubes of the Dutch 68 vs. 78 Gy prostate trial were<br />
collected digitally for all patients. Failure at 4y was defined according to the Phoenix<br />
definition (clinical or biochemical). Maps of the dose surrounding prostate and<br />
rectum were created for each patient, giving the dose at different distances (up to<br />
4 cm) from the organ surface, in fixed directions from the center of the organ. In this<br />
way, map points have reasonable anatomical correspondence. Average dose maps<br />
were computed for patient subgroups with and without failure, and differences of<br />
these maps were computed. Both rectum and prostate based-maps show areas with<br />
significant dose differences at some distance from the prostate. A very significant<br />
(p
EPID DOSIMETRY<br />
Priscilla Camargo, Anton Mans, Leah McDermott, Jan-Jakob Sonke, Joep Stroom, Marcel Van Herk,<br />
Markus Wendling, Ben Mijnheer<br />
EPID dosimetry has been clinically used for IMRT verification in our department,<br />
both pre-treatment and in vivo, since 2005. In recent years, the method has gradually<br />
been applied to more treatment sites, and starting this year, the plans of all our IMRT<br />
patients (~2000/year) are verified with EPID dosimetry. Basically EPID dosimetry<br />
serves therefore two purposes: (1) it is our only form of patient-specific IMRT<br />
verification, used for all curative patient treatments; (2) with a few exceptions (too<br />
large fields with respect to the EPID dimensions, about 3%, and single high dose<br />
fractions, about 1%) it is applied in vivo thus guaranteeing the correct dose delivery to<br />
the patient. Three years of clinical experience have proven the system to be<br />
sufficiently accurate and highly efficient (25 min/plan, 50 plans/week, 1 radiographer).<br />
EPID dosimetry provides a safety net as the final check that all patients are correctly<br />
treated, given the fact that several types of errors have been detected that would have<br />
led to erroneous patient treatments if undetected.<br />
In our standard procedure, the dose in a plane parallel to the EPID intersecting the<br />
isocentre is reconstructed per beam from EPID images by means of a back-projection<br />
algorithm and compared to the planned dose distribution with the g-evaluation<br />
method (3%/3mm). No inhomogeneity correction is used and the EPID should be<br />
aligned with the central axis of the linac. Verification of the treatment of breast and<br />
lung cancer needed special attention, as our standard dose reconstruction based on<br />
EPID transmission measurements was not accurate enough for these two cancer sites.<br />
In our department, breast treatments (~700 patients/year) consist of two opposing,<br />
non-wedged, tangential half fields, each with an open and IMRT part. When lymph<br />
nodes have to be treated as well, quarter fields are used. To apply our method to<br />
breast cancer treatments, two modifications are made: (1) the dose specification point<br />
is used, since the isocentre is located on a field edge, and (2) an off-axis correction is<br />
applied, taking into account the modified pixel response due to spectral changes<br />
when the EPID is shifted. For quarter fields, a large EPID shift is necessary to image<br />
the field without irradiating the edge of the detector. When applying these corrections,<br />
in vivo EPID dosimetry for breast treatments shows now good results.<br />
Our current back-projection dose-reconstruction algorithm uses water-based scattercorrection<br />
kernels and does therefore not account for tissue inhomogeneities, which<br />
gives large deviations for lung cancer treatments. With in aqua vivo dosimetry, the<br />
dose is reconstructed in the patient as if the patient would have consisted entirely of<br />
water. By using in aqua vivo EPID dosimetry, dose verification for lung cancer<br />
patients during the actual treatment is possible, except for the tissue inhomogeneity<br />
correction of the TPS. The method was proven to be accurate within g criteria of 3%<br />
and 3 mm. This method has been implemented clinically and is applied for all our<br />
lung cancer treatments with curative intent.<br />
If the patient is in a different position, the radiation beams directions alter and in<br />
some cases the dose distribution will change from the planned dose. Whether or not<br />
EPID dosimetry picks up this deviation depends on the change in transmission for<br />
each field. We therefore studied the direction and magnitude of the shifts for which<br />
an alert would be raised for a single field and typical lung IMRT plans based on in<br />
aqua vivo EPID dosimetry. For this purpose an anthropomorphic thorax phantom was<br />
irradiated and shifted in ±3 orthogonal directions. From the absolute dose reconstructed<br />
inside the patient it could be concluded that EPID in aqua vivo dosimetry raises an<br />
alert for positioning differences of ~1-2 cm for typical lung cancer treatment plans.<br />
A start has been made with adapting our model for the use of EPID dosimetry in<br />
wedged beams. Because of the change in photon energy spectrum when placing a<br />
wedge in the beam, some of the parameters in our model, e.g. the beam hardening<br />
correction, have to be modified to get a better prediction of the reconstructed dose<br />
distribution in patients irradiated with wedged beams.<br />
Publication (continued)<br />
Epid dosimetry<br />
131<br />
RADIOTHERAPY<br />
McDermott LN, Wendling M, Nijkamp J,<br />
Mans A, Sonke JJ, Mijnheer BJ, van Herk M.<br />
3D in vivo dose verification of entire hypofractionated<br />
IMRT treatments using an<br />
EPID and cone-beam CT. Radiother Oncol<br />
<strong>2008</strong>;86:35-42<br />
Van Elmpt W, McDermott L, Nijsten S,<br />
Wendling M, Lambin P, Mijnheer B.<br />
A literature review of electronic portal<br />
imaging for radiotherapy dosimetry.<br />
Radiother Oncol <strong>2008</strong>;88:289-309
132<br />
RADIOTHERAPY<br />
Publication (continued)<br />
Treatment planning<br />
Wolthaus JW, Sonke JJ, van Herk M,<br />
Belderbos JS, Rossi MM, Lebesque JV,<br />
Damen EM. Comparison of different<br />
strategies to use four-dimensional computed<br />
tomography in treatment planning for lung<br />
cancer patients. Int J Radiat Oncol Biol<br />
Phys <strong>2008</strong>;70:1229-1238<br />
Wolthaus JW, Sonke JJ, van Herk M,<br />
Damen EM. Reconstruction of a timeaveraged<br />
midposition CT scan for<br />
radiotherapy planning of lung cancer<br />
patients using deformable registration. Med<br />
Phys <strong>2008</strong>;35:3998-4011<br />
TREATMENT PLANNING<br />
Harry Bartelink, Jose Belderbos, Gerben Borst, Joop Duppen, Paula Elkhuizen, Martijn Eenink,<br />
Barry Doodeman, Wilma Heemsbergen, Mariëlle Van Heumen, Suzanne Den Hollander,<br />
Rianne De Jong, Annemarie Lakeman, Emmy Lamers, Corrie Marijnen, Vanessa Mexner,<br />
Danny Minkema, Anke Van Mourik, Floris Pos, Coen Rasch, Jan-Jakob Sonke, Mine Temürhan,<br />
Corine Van Vliet-Vroegindeweij, Eugene Damen<br />
Lung<br />
Shift invariance of the dose distribution for online corrections of stereotactic<br />
body radiotherapy of lung cancer patients Substantial baseline variation (motion<br />
of the mean tumor position relative to the bony anatomy) has been observed in lung<br />
cancer patients treated with stereotactic body radiotherapy (SBRT). We therefore use<br />
online corrections based on the actual tumor position. This procedure assumes shift<br />
invariance of the dose distribution for small corrections. In a treatment planning<br />
study on five patients this assumption was tested. Clinical treatment plans were<br />
recalculated in Pinnacle after shifting the isocenter and the planning target volume<br />
(PTV), without re-optimizing. Shifts (0.5 cm, 1.0 cm and 1.5 cm) were applied in<br />
12 equiangular spaced directions of the sagittal plane. The main objective for the<br />
original plans was that at least 95% of the PTV volume received 54 Gy (3 x 18 Gy).<br />
As expected, increasing shifts led to increasing deterioration of the PTV coverage,<br />
but varied over the patient group. For half of the patients, D95 dropped up to 1 Gy for<br />
0.5 cm shifts, 1-2 Gy for 1.0 cm shifts and 1-3 Gy for 1.5 cm shifts. For the other half,<br />
D95 dropped up to 1-4 Gy, 2-5 Gy and 3-6 Gy, respectively. Tumor locations near the<br />
thoracic wall show the largest deterioration, due to the effect of a varying penumbra<br />
width when the PTV is moved towards or from the thoracic wall. From the results we<br />
concluded that in case of lung tumors located free from the thoracic wall, baseline<br />
shifts up to 1.0 cm are acceptable without re-optimization. In other cases, the<br />
robustness of the treatment plan to correct for baseline shifts must be validated.<br />
Gastroenterology<br />
An improved drinking protocol to decrease irradiated volume of small bowel<br />
during radiotherapy of rectal cancer The amount of irradiated small bowel can be<br />
reduced by treating patients with a full bladder. The effect of two drinking protocols<br />
on bladder volume was evaluated. Old protocol (OP, 28 patients): patients were<br />
instructed to empty their bladder one hour before start of treatment and subsequently<br />
drink 250 ml of water. New protocol (NP, 9 patients): patients were instructed to void<br />
one hour before start of treatment and drink 350 ml of water, in addition with the<br />
instruction to drink 2 liters of fluids per day during the course of treatment. The<br />
bladder was delineated on the planning CT and on cone-beam CT (CBCT) images,<br />
which were acquired for each fraction (fractionation schedule: 5 x 5 Gy). Average<br />
bladder volumes increased from 240 ml for the OP to 336 ml for the NP. In a<br />
multivariate analysis, this difference was significant (p = 0.02), while patient sex, age<br />
and position (prone or supine) were not. However, day-to-day variations in bladder<br />
volume increased with the average volume, and a significant negative time trend in<br />
bladder volume was seen during the course of the treatment. Moreover, bladder<br />
filling on the planning CT was on average larger than during the course of treatment<br />
(293 ml vs. 233 ml, p = 0.001). From these results it was concluded that NP increases<br />
bladder volume during treatment. NP results in an increase of inter-fraction<br />
variability in bladder volume, which should be taken into account when determining<br />
required PTV margins.
Breast<br />
Unraveling influences of patient error sources on breast IMRT plans by<br />
means of deformable registration Setup error and tissue deformation during<br />
treatment of breast cancer patients lead to variations in position and shape of the<br />
target volume and alter the originally planned dose distribution. We determined the<br />
sensitivity of 3 breast RT planning strategies for these error sources by evaluation of<br />
accumulated dose. For 5 patients, 3 plans were made with identical tangential,<br />
glancing beam setup: a non-IMRT technique with wedges and two IMRT techniques.<br />
For the SimpleIMRT technique 80% of the dose was delivered by the tangentials,<br />
while additional computer-optimized segments delivered the remaining 20%. In the<br />
FullIMRT technique, 100% of the dose was delivered by computer-optimized<br />
segments. The dose was accumulated over 5 fractions, taking the separate and<br />
combined errors into account. To that end, the dose was recalculated on the original<br />
CT after setup translations (T), a deformed CT (see below) (D), and a deformed CT<br />
after setup translations (T&D), and compared to the original plan (O). The original<br />
CT was deformed to the CBCT of the day to account for shape changes. Resulting<br />
deformation fields were used to generate a deformed CT for recalculating the dose<br />
distributions, and to deform these distributions back into the original planning CT<br />
for accumulation.<br />
Table 1 Coverage (V95 mean (sd)) of original dose (O) and dose including different types error sources<br />
(T, D, T&D) for different planning strategies (1-3). Results are preliminary in view of ongoing analyses.<br />
V95 O T D T&D<br />
Correction protocol Original plan Offline Online Offline<br />
Wedge 95(3) 91(4) 93(4) 90(3)<br />
SimpleIMRT 98(2) 95(2) 97(1) 94(1)<br />
FullIMRT 97(1) 95(2) 94(1) 93(1)<br />
The impact of error sources is reflected in reduction of the V95 compared to the<br />
original plan (Table 1). Translations represent the larger error source for the wedged<br />
and SimpleIMRT plans. For FullIMRT deformations seem to have a larger impact.<br />
Underdosage was mainly located near the skin. From these results we conclude that<br />
the impact of error sources on breast RT depends on planning technique. Full IMRT<br />
plans do not have glancing beams and are therefore more susceptible to deformation<br />
errors of the breast contour. The difference between D and T&D reveals the gain that<br />
can be achieved by clinically using an online instead of an offline correction<br />
Interobserver variability in target volume delineation of boost volume in<br />
breast irradiation across institutions Thirteen radiation oncologists from eleven<br />
different institutions delineated the volumes of surgical area, CTV boost and PTV<br />
boost of two left-sided and three right sided breast cancer patients. All observers used<br />
a written instruction and had access to all clinical available information. Three<br />
patients had no seroma (patient 1-3), one patient had some seroma (patient 4) and<br />
one patient had a clearly visible seroma (patient 5). The conformity index (CI) of two<br />
delineated structures (defined as the ratio of the overlapping volume and the<br />
encompassing total delineated volume) was used to quantify the difference in<br />
delineation between observers. For all patients a median delineation was defined by<br />
the volume consisting of all pixels that were delineated by at least 50% of the<br />
observers. To pinpoint specific areas of variation the median was subdivided in<br />
several anatomic regions: caudal, cranial, lateral, medial, dorsal, ventral and skin.<br />
A large variety in the delineated volumes was seen. In general, some observers<br />
consistently delineated larger volumes than the median volume for all patients, while<br />
others were consistently delineating smaller than the median volumes. The CI values<br />
showed a correlation with the presence of seroma. When seroma was hardly present,<br />
average CI-values below 0.5 were found, whereas in the case of a clearly visible<br />
seroma an average CI-value of 0.8 was found. Hardly any variations over the different<br />
anatomic regions were found. We concluded that significant interobserver variations<br />
could be found in the delineated volumes. The presence of seroma resulted in low<br />
interobserver variations.<br />
133<br />
RADIOTHERAPY
134<br />
RADIOTHERAPY<br />
Publication (continued)<br />
Clinical application of image guided<br />
radiotherapy<br />
Belderbos JS, Kepka L, Spring Kong FM,<br />
Martel MK, Videtic GM, Jeremic B. Report<br />
from the International Atomic Energy<br />
Agency (IAEA) consultants’ meeting on<br />
elective nodal irradiation in lung cancer:<br />
non-small-Cell lung cancer (NSCLC). Int J<br />
Radiat Oncol Biol Phys <strong>2008</strong>;72:335-342<br />
Kappers I, Belderbos JS, Burgers JA,<br />
van Zandwijk N, Groen HJ, Klomp HM.<br />
Non-small cell lung carcinoma of the<br />
superior sulcus: favourable outcomes of<br />
combined modality treatment in carefully<br />
selected patients. Lung Cancer <strong>2008</strong>;59:<br />
385-390<br />
Videtic GM, Belderbos JS, Spring Kong FM,<br />
Kepka L, Martel MK, Jeremic B. Report<br />
from the International Atomic Energy<br />
Agency (IAEA) consultants’ meeting on<br />
elective nodal irradiation in lung cancer:<br />
small-cell lung cancer (SCLC). Int J Radiat<br />
Oncol Biol Phys <strong>2008</strong>;72:327-334<br />
Effect of interobserver variation on dose distributions of simultaneous<br />
integrated boost (SIB) breast irradiation Delineating a boost target volume for<br />
breast cancer is difficult and interobserver variation is a fact. We investigated how<br />
much tumor coverage of a dose distribution planned on an initial boost planning<br />
target volume (PTV b) changes when evaluating on a PTV b of other physicians. CTscans<br />
of 5 patients after breast conserving surgery were used. Three physicians<br />
delineated the tumor bed and subsequently expanded to CTV and PTV. Two<br />
simultaneous integrated boost (SIB) IMRT planning techniques were used (PTV b<br />
31 x 2.38Gy): SIB 1: Simultaneous inverse optimization of 2 tangential glancing open<br />
fields combined with 2 tangential IMRT fields for the breast and 2 tangential open<br />
fields for PTV b; SIB 2: like SIB1 but with 1 orthogonal and 2 tangential IMRT fields<br />
for PTV b. Coverage of the PTV b used for planning was >98% in all cases. SIB 2<br />
resulted in a much tighter dose distribution. Coverage of PTVb in the presence of<br />
interobserver variation remains more than 90% in the majority of the cases for SIB 1.<br />
For SIB 2 a minimum of 80% coverage is observed for most plans. From these results<br />
we concluded that SIB 1 used at our institute is relatively robust against interobserver<br />
variation.<br />
CLINICAL APPLICATION OF IMAGE GUIDED RADIOTHERAPY<br />
Anja Betgen, Eugene Damen, Marcel Van Herk, Juup Nijholt, Peter Remeijer, Maddalena Rossi,<br />
Jan-Jakob Sonke, Jochem Wolthaus, Marieke Van Zwienen, Floris Pos, Coen Rasch, Jose Belderbos<br />
Stereotactic body radiation therapy (SBRT) for non-small cell lung cancer<br />
(NSCLC) patients or pulmonary metastases SBRT is characterized by the delivery<br />
of one or a few fractions (hypofraction) with a high dose per fraction and a high<br />
accuracy. With this treatment regime high local control rates can be reached in early<br />
stage lung cancer patients. At the <strong>NKI</strong>-AVL, SBRT has been introduced since mid-<br />
2005 to treat medically inoperable primary NSCLC or pulmonary metastases. We<br />
analyzed the response rate and toxicity of SBRT from July 2005 till November 2007<br />
in 70 patients treated for 74 tumors. All patients (median age 73 years) had<br />
peripheral tumors outside the proximal bronchial tree (diameter ≤ 5 cm) and were<br />
irradiated, frameless, with 3 fractions of 18 Gy (within 8-10 days). Dose prescription<br />
was according to the RTOG 0236 protocol. Stage was T1 in 85% and T2 in 15% of the<br />
patients. In 45% of the patients pathological proof of NSCLC was available. In 55%<br />
of patients a growing tumor on chest X-ray or CT scan was diagnosed with high<br />
FDG-PET uptake and a contra-indication for trans-thoracic puncture. Inoperability<br />
was due to poor pulmonary function tests in 44% of patients, heart disease in 14%,<br />
combination of both in 15%, previous thoracotomy in 7%, other malignancy in 8%,<br />
poor condition in 7% and thoracotomy refusal in 5%. Patients received a 4D midventilation<br />
CT approach in treatment planning. Prior to- and after the irradiation,<br />
4D- cone-beam CT (4D-CBCT) was used to verify and correct patient set-up errors<br />
and tumor baseline variation.<br />
Acute toxicity (with a median follow-up time of 9 months) was very mild. Late toxicity<br />
revealed radiation pneumonitis grade 2 in 8% and grade 3 in 1% of the patients.<br />
The local and loco-regional control in our SBRT group was excellent (90%).<br />
A B<br />
Figure 4: A: 5/<strong>2008</strong> central tumor right hilar side<br />
B: 8/<strong>2008</strong> 4 weeks after completion of chemo-radiation:complete metabolic response, however uptake in the<br />
esophagus due to radiation esophagitis
We investigated the accuracy of frameless SBRT using 4D-CBCT guidance by<br />
determining the intra-fraction stability in 58 patients. The time from the patient<br />
entering until leaving the treatment room was on average 50 min. The residual interfraction<br />
group mean, systematic and random errors for all directions were < 1mm.<br />
Even with considerable tumor motion the PTV-margins can safely be kept relatively<br />
small allowing patients with larger tumor volumes to benefit from the advantages of<br />
SBRT. For all patients, post treatment validation scans did not show unacceptable<br />
deviations confirming the stability of our frameless approach.<br />
We compared the overall survival in the SBRT treated patients with a very similar<br />
patient group treated with conventional fractionation from our dose-escalation (DE)<br />
trial [Belderbos et al. 2006]. The 28 patients from the DE trial were selected based on<br />
a small Gross Tumor Volume (GTV < 65cc). These patients were irradiated to a dose<br />
of 81-95 Gy (2.25 Gy per fraction; within 6 weeks). The 12 and 18 months overall<br />
survival in the patients treated with SBRT (n=67) compared to the DE patients was<br />
69% (SBRT) versus 68% (DE) and 60% (SBRT) versus 53% (DE). SBRT is a safe and<br />
effective alternative (compared to conventional fractionated radiotherapy) for<br />
peripheral early stage NSCLC or pulmonary metastases.<br />
STUDIES OF RADIATION RESPONSE IN TUMORS AND<br />
ORGANS AT RISK<br />
Abrahum Al-Mamgani1 , Jose Belderbos, Gerard Bengua2 , Gerben Borst, M Dielwart3 ,<br />
Michael Hauptmann4 , W Heemsbergen, Michel Van den Heuvel5 , Luca Incrocci1, M Ishikawa2 ,<br />
Peter Levendag1 , Jasper Nijkamp, Rikaya Onimaru2 , Stephanie Peeters, Wim Van Putten1 , H Shirato2 ,<br />
A Slot6 , Jan-Jakob Sonke, Joos Lebesque<br />
Prostate cancer<br />
The Dutch dose escalation randomized trial We recently updated the Dutch dose<br />
escalation randomized trial of 68 Gy versus 78 Gy with 669 prostate cancer patients.<br />
The primary endpoint was freedom from failure (FFF), defined as clinical or<br />
biochemical failure (BF). Two definitions of BF were used; the ASTRO-definition<br />
(3 consecutive rises of PSA level) and the Phoenix definition (the nadir plus 2). After<br />
a median follow-up of 70 months, FFF (ASTRO) was significantly better in the 78-Gy<br />
arm compared with the 68-Gy arm (7-year FFF rate is 54% vs. 47%; p= 0.04). FFF<br />
(Phoenix) was also significantly better in the 78-Gy arm with 7-year FFF rate of 56%<br />
vs. 45% (p = 0.03) (figure IX.5) However, no differences in freedom from clinical<br />
failure (FFCF) and overall survival were observed. The incidence of late genitourinary<br />
(GU) toxicity grade ≥ 2 was similar in both arms (40% vs. 41% at 7 years; p= 0.6),<br />
while the cumulative incidence of late gastrointestinal (GI) toxicity grade ≥ 2 was<br />
increased in the 78-Gy arm (35% vs 25% at 7 years; p= 0.04).<br />
We further performed a subgroup analysis, in which we divided the patients into two<br />
dose groups according to the actually given dose:
136<br />
RADIOTHERAPY<br />
dose radiotherapy. For patients, who received hormonal therapy, FFF was significantly<br />
improved for the high-dose group compared to the low-dose group (67% vs. 45%,<br />
p=0.05), indicating that high dose radiotherapy should also be given to this group of<br />
patients.<br />
Figure 5: Kaplan-Meier curve of 7-year rates of freedom from failure (FFF) by dose randomization defined<br />
according to the Phoenix definition (the nadir plus 2 µg/L after RT)<br />
The role of intensity modulated radiation therapy in reducing toxicity The<br />
results of a subgroup of 78 patients were studied. They were treated in the <strong>NKI</strong>-AVL<br />
to a total dose of 78 Gy with either a 3D-conformal radiotherapy technique with a<br />
sequential boost (SEQ) or a simultaneous integrated boost using IMRT (SIB-IMRT).<br />
The 5-year freedom from biochemical failure (Phoenix) was not worse in the SIB-<br />
IMRT patients (70% vs. 61% for the SEQ, p = 0.3). However, there was a significant<br />
lower incidence of acute grade ≥ 2 GI toxicity in patients treated with SIB-IMRT<br />
compared to SEQ (20% vs. 61%, p = 0.001). For acute GU toxicity and late GI and<br />
GU toxicity, the incidences were lower with SIB-IMRT, but these differences were not<br />
statistically significant.<br />
Lung<br />
We cooperated with the Department of Radiology at the Hokkaido University School<br />
of Medicine (Sapporo, Japan) where they have an extensive experience of treating<br />
pulmonary lesions using Stereotactic Body Radiotherapy (SBRT). The aim of the<br />
study was to find the relation between the incidence radiation pneumonitis (RP) and<br />
dose volume parameters.<br />
Linear quadratic (LQ) model and hypofractionated lung irradiation We first<br />
evaluated the validity of the LQ model for the high doses per fraction, given in SBRT.<br />
The lung radiation dose for 128 SBRT patients was calculated as the equivalent dose<br />
given in fractions of 2 Gy (i.e. Normalized Total Dose), according to the LQ model<br />
using a range of a/b values. The mean lung dose was calculated for a/b ratios of 1 Gy<br />
(MLD 1), 2 Gy (MLD 2), 3 Gy (MLD 3), 4 Gy (MLD 4), 5 Gy (MLD 5), 7.5 Gy (MLD 7.5) 10 Gy<br />
(MLD 10) and infinity (i.e. physical dose, MLD phys). In addition, other dose volume<br />
parameters like the volume lung receiving more than a threshold dose of x Gy (V x)<br />
and the MLD calculated with a modification of the LQ model (MLD LQL) were evaluated.<br />
Strong correlations were observed between all these parameters. Fitting the Normal<br />
Tissue Complication Probability (NTCP) model of Lyman to the observed incidence<br />
of RP, the MLD 3 had the highest log likelihood (best NTCP fit) compared to the MLD<br />
corrected with the other a/b ratios and the MLD LQL. However, these differences were<br />
not statistically significant. The MLD3 was significantly better predicting RP than the<br />
V 5, V 13 (p 0.025) and V 20 but was not significantly better the V 40.
Comparing the dose-effect relationship between the incidence of radiation<br />
pneumonitis (RP) after SBRT and conventional fractionated radiation therapy<br />
(CFRT) For both SBRT (n=128) and CFRT (n=142) patients, RP grade 2 or higher was<br />
scored. The incidence of RP was 10.9 % and 17.6 % for the SBRT and CFRT patients,<br />
respectively. The dose-response relationship for RP for SBRT was not significantly<br />
different from the dose-response relationship for CFRT (p=0.37). No difference in<br />
incidence in any dose range was observed between SBRT and CFRT (p>0.1).<br />
Breast cancer<br />
Ninja Antonini, Liesbeth Boersma1 , Eugene Damen, Paula Elkhuizen, Guus Hart, Heather Jones,<br />
Bas Kreike, Ben Mijnheer, Nicola Russell, Laura Van ’t Veer, Marc Van de Vijver, Conny Vrieling,<br />
Harry Bartelink<br />
The impact of an extra dose of 16 Gy was investigated in 251 patients with involved<br />
surgical margins in patients who underwent breast-conserving therapy and were part<br />
of the EORTC ‘boost versus no boost’ trial. Thirty-seven patients initially relapsed<br />
locally. At 10 years, the cumulative incidence of local recurrence was 17.5% (95% CI:<br />
10.4-24.6%) versus 10.8% (95% CI: 5.2-16.4%) for the low and high boost dose<br />
groups, respectively (HR=0.83, 95% CI: 0.43-1.57, p>0.1). Overall, 64 patients have<br />
died (25.5%), 47 of them of breast cancer, without a difference in duration of survival<br />
between the two groups (HR=0.97, 95% CI=0.59-1.5, p>0.1). In summary: we could<br />
not demonstrate a statistically significant difference in local control or survival<br />
between the high boost dose of 26 Gy and the low boost dose of 10 Gy in patients<br />
with microscopically incomplete excision of early breast cancer.<br />
In the same trial 5178 conservatively treated early breast cancer patients had a<br />
complete excision. Previously we showed that a 16 Gy boost dose significantly<br />
improved local control, but increased the risk of breast fibrosis. To investigate<br />
predictors for the long-term risk of fibrosis, Cox regression models of the time to<br />
moderate or severe fibrosis were developed on a random set of 1797 patients with<br />
and 1827 patients without a boost, and validated in the remaining set. The median<br />
follow-up was 10.7 years. The risk of fibrosis significantly increased (p
138<br />
RADIOTHERAPY<br />
Publication (continued)<br />
Meijnen P, Oldenburg HS, Peterse JL,<br />
Bartelink H, Rutgers EJ. Clinical outcome<br />
after selective treatment of patients<br />
diagnosed with ductal carcinoma in situ of<br />
the breast. Ann Surg Oncol <strong>2008</strong>;15:235-243<br />
Pengel KE, Loo CE, Teertstra HJ, Muller SH,<br />
Wesseling J, Peterse JL, Bartelink H,<br />
Rutgers EJ, Gilhuijs KG. The impact of<br />
preoperative MRI on breast-conserving<br />
surgery of invasive cancer: a comparative<br />
cohort study. Breast Cancer Res Treat. <strong>2008</strong><br />
Sep 21 (epub ahead of print)<br />
Poortmans PM, Collette L, Horiot JC,<br />
Van den Bogaert WF, Fourquet A, Kuten<br />
A, Noordijk EM, Hoogenraad W,<br />
Mirimanoff RO, Pierart M,<br />
Van Limbergen E, Bartelink H; On behalf<br />
of the EORTC Radiation Oncology and<br />
Breast Cancer Groups. Impact of the boost<br />
dose of 10Gy versus 26Gy in patients with<br />
early stage breast cancer after a<br />
microscopically incomplete lumpectomy:<br />
10-year results of the randomised EORTC<br />
boost trial. Radiother Oncol. <strong>2008</strong> Aug 15<br />
(epub ahead of print)<br />
Poortmans PM, Collette L, Bartelink H,<br />
Struikmans H, Van den Bogaert WF,<br />
Fourquet A, Jager JJ, Hoogenraad W,<br />
Müller RP, Dubois JB, Bolla M,<br />
Van Der Hulst M, Wárlám-Rodenhuis CC,<br />
Pierart M, Horiot JC; On behalf of the<br />
EORTC Radiation Oncology and Breast<br />
Cancer Groups.The addition of a boost dose<br />
on the primary tumour bed after lumpectomy<br />
in breast conserving treatment for breast<br />
cancer. A summary of the results of EORTC<br />
22881-10882 ‘boost versus no boost’ trial.<br />
Cancer Radiother <strong>2008</strong>;12:565-570<br />
Mechanisms, modulation and prediction of<br />
radiation-induced cell death<br />
Hoebers FJ, Kartachova M, de Bois J,<br />
van den Brekel MW, van Tinteren H,<br />
van Herk M, Rasch CR, Valdés Olmos<br />
RA, Verheij M. 99mTc Hynic-rh-Annexin<br />
V scintigraphy for in vivo imaging of<br />
apoptosis in patients with head and neck<br />
cancer treated with chemoradiotherapy. Eur<br />
J Nucl Med Mol Imaging <strong>2008</strong>;35:509-518<br />
with lipid metabolism, inhibit proliferative and survival signaling, affect cell cycle<br />
distribution and stimulate apoptosis induction in a variety of tumor cell systems. In<br />
combination with radiation, APLs cause a synergistic apoptotic effect and reduce<br />
clonogenic cell survival. Besides these radiosensitizing properties, APLs show potent<br />
anti-angiogenic effects in vitro. In an ongoing separate project (in collaboration with<br />
W Van Blitterswijk, Division Cell Biology I) we study mechanisms of cellular APL<br />
uptake and transport, and address the functional implications of an altered<br />
membrane lipid composition on cellular drug uptake, signal transduction and<br />
radiosensitivity. We found that the uptake of APLs via lipid rafts is essential for their<br />
cytotoxic effect, because disruption of these membrane structures by pharmacological<br />
(SM degradation/cholesterol depletion) or genetic (SM synthase knock down)<br />
approaches, results in resistance to APL. Interestingly, these cells that lack SM and<br />
thus have defective rafts, show cross-resistance to a variety of other, unrelated<br />
apoptotic stimuli, including death receptor ligands and DNA-damaging regimens.<br />
This ‘multi-stress resistance’ is subject of ongoing research and is associated with<br />
downregulation of the enzymes SMS1 and SHIP1.<br />
Following these in vitro experiments, a proof-of-concept study was performed in a<br />
mouse xenograft KB carcinoma model showing complete and sustained tumor<br />
regression after Perifosine and radiation. These in vitro and in vivo observations<br />
prompted us to conduct a clinical phase I study defining the MTD, toxicity profile<br />
and optimal scheduling of the combined treatment of oral administration of<br />
Perifosine and radiotherapy in patients with locally advanced inoperable solid<br />
tumors. Next, we initiated an international multicenter placebo-controlled phase II<br />
study in locally advanced NSCLC, randomizing 168 patients between radiation +<br />
placebo and radiation + daily Perifosine (completed ultimo 2007). In the first quarter<br />
of 2009 disclosure of the results is scheduled. Other promising APL derivatives with<br />
potentially better bioavailability and radiosensitizing properties, like the i.v.<br />
compound ErPC, have become available and are studied in vitro and in vivo.<br />
Other radiosensitizers in preclinical development In collaboration with different<br />
groups we evaluate other radio-enhancing agents. In a separate project with J Borst<br />
(Division Immunology IV), we study the interaction between death receptor ligands<br />
(TRAIL) and DNA damaging regimens (radiation and etoposide). TRAIL induces<br />
apoptosis in a wide variety of tumor tissues, but lacks normal tissue toxicity in<br />
preclinical animal models. TRAIL is of particular interest for combination therapies,<br />
since TRAIL and radiation or chemotherapy activate partially distinct apoptosis<br />
signaling pathways, while a molecular basis for synergy lies in the sensitization of<br />
cells by radiation to TRAIL-induced apoptosis. In several leukemic and solid tumor<br />
cell lines, we demonstrated combined (additive to synergistic) effects of TRAIL +<br />
radiation/etoposide. The effectiveness and acceptable toxicity of the combined<br />
treatment of TRAIL and radiation was subsequently demonstrated in vivo in<br />
collaboration with Dr. Henning Walczak, DKFZ, Germany. These experiments have<br />
been expanded with another death receptor ligand (MegaFasL/APO010) using a Bcl-<br />
2-overexpressing T-leukemic cell line (Jurkat), a colon carcinoma cell line (HCT116)<br />
and a mesothelioma cell line. While APO010 and radiation had a clear combined<br />
cytotoxic effect on tumor cells in vitro, a combined therapeutic effect on the same<br />
cells subcutaneously grafted in mice was not achieved at APO010 doses<br />
approximating the maximally tolerable level.<br />
In collaboration with the University of Florida and the division of Radiobiology at the<br />
Free University of Amsterdam, we have initiated preclinical efficacy testing of (-)<br />
Gossypol, a potent small-molecule inhibitor of Bcl-XL, Bcl-2 and Mcl-1. Gossypol<br />
binds to the BH3 domains of these, and possibly other Bcl-2 family members, and<br />
induces apoptosis. Because overexpression of these anti-apoptotic molecules confers<br />
radioresistance and is often associated with poor prognosis, inhibition of Bcl-2/<br />
Bcl-XL/Mcl-1 represents a promising strategy to increase tumor response to radiation.<br />
In a panel of human leukemic and head and neck cancer cell lines Gossypol was<br />
found to increase radiation-induced cell death. Currently, this combination approach<br />
is evaluated in vivo and in a recently initiated clinical phase I/II study in locally<br />
advanced head and neck cancer.
Another strategy that aims at enhancing the tumor response to radiation focuses on<br />
inhibition of poly(ADP-ribose)polymerase (PARP), an important DNA repair enzyme.<br />
APO866 is a highly specific non-competitive inhibitor of nicotinamide phosphoribosyltransferase<br />
(NAPRT), a key enzyme in the regulation of NAD+ biosynthesis from the<br />
natural precursor nicotiname. Inhibition of NAPRT progressively depletes NAD+.<br />
Cells that have a high turnover of NAD+, e.g. through its cleavage by PARP, rely on<br />
the readily available nicotinamide pathway for NAD+ synthesis and undergo apoptosis<br />
after inhibition of NAPRT. Indeed, inhibition of NAD+ by APO866 has been reported<br />
to induce apoptosis in human leukemic, hepatocarcinoma cells in vitro and various<br />
types of tumor xenografts in vivo. We have studied the effect of APO866 in the human<br />
leukemic cell lines U937 and Jurkat T and found a dose- and time-dependent induction<br />
of cell death as measured by the MTT assay. Reduction of cellular NAD+ levels was<br />
essential for these effects. In addition, we demonstrated the radiosensitizing effect of<br />
nanomolar concentrations of APO866 in a clonogenic survival assay. These results<br />
indicate that this NAD+-depleting and PARP-inhibiting compound is effective in<br />
compromising DNA repair when used in combination with radiation.<br />
Prediction of tumor response To evaluate whether early treatment-induced cell<br />
death can be visualized in vivo and whether it predicts clinical outcome, we evaluated<br />
a novel in vivo apoptosis imaging technique in collaboration with the department of<br />
nuclear medicine (Valdés Olmos, Division XIII). This 99mTc-Annexin V scintigraphy<br />
(TAVS) is based on the exposure of phosphatidylserine (PS) at the outer leaflet of the<br />
plasma membrane lipid bilayer during the early phase of apoptosis. The human<br />
endogenous protein annexin V has a high affinity for PS, providing a detection<br />
method for apoptotic cells in vitro and in vivo. We have gained experience with this<br />
imaging technique in a variety of tumor types, including NHL, NSCLC, SCLC,<br />
H&NSCC and sarcomas treated by radiotherapy and/or chemotherapy. A statistically<br />
significant correlation was found between the increase in TAV tumor uptake on the<br />
post-treatment scan and tumor response in 65 patients. These results indicate that<br />
TAVS might be useful as a non-invasive predictive test for treatment outcome,<br />
ultimately allowing an early switch to more effective regimens. Recently, we<br />
examined a group of 11 patients (20 lesions) with advanced NSCLC who received<br />
99m Tc-MIBI single photon emission computed tomography (SPECT-CT) before their<br />
first course of chemotherapy and analyzed the relationship between uptake and<br />
response to treatment. A statistically highly significant relationship was found<br />
between 99m Tc-MIBI uptake and change in tumor size (r 2 =0.53, p
140<br />
RADIOTHERAPY<br />
Publication (continued)<br />
Combination of radiotherapy and<br />
chemotherapy<br />
Ackerstaff AH, Balm AJ, Rasch CR,<br />
de Boer JP, Wiggenraad R, Rietveld DH,<br />
Gregor RT, Kröger R, Hilgers FJ. First-year<br />
quality of life assessment of an intra-arterial<br />
(RADPLAT) versus intravenous chemoradiation<br />
phase III trial. Head Neck. <strong>2008</strong><br />
Oct 28 (epub ahead of print)<br />
Hoebers FJ, Pameijer FA, de Bois J,<br />
Heemsbergen W, Balm AJ, Schornagel JH,<br />
Rasch CR. Prognostic value of primary<br />
tumor volume after concurrent<br />
chemoradiation with daily low-dose<br />
cisplatin for advanced-stage head and neck<br />
carcinoma. Head Neck <strong>2008</strong>;30:1216-23<br />
Jansen EPM, Verheij M. Radiotherapy of<br />
gastric cancer. In: Gastrointestinal<br />
Oncology: A Critical Multidisciplinary<br />
Approach. Editors: J Jankowski,<br />
R Sampliner, D Kerr, Y Fong, Blackwell<br />
Publishing <strong>2008</strong>;164-166<br />
Topolnjak R, van Vliet-Vroegindeweij C,<br />
Sonke JJ, Minkema D, Remeijer P,<br />
Nijkamp J, Elkhuizen P, Rasch C. Breast-<br />
Conserving Therapy: Radiotherapy Margins<br />
for Breast Tumor Bed Boost. Int J Radiat<br />
Oncol Biol Phys. <strong>2008</strong>;72:941-948<br />
Verheij M. The role of radiotherapy in the<br />
treatment of gastric cancer. Asian Hospital<br />
& Healthcare Management <strong>2008</strong>;17:44-46<br />
Verheij M, Bartelink H.<br />
Chemoradiotherapy. In: Encyclopedia of<br />
Cancer <strong>2008</strong>. Editor M Schwab<br />
Zuur CL, Simis YJ, Verkaik RS,<br />
Schornagel JH, Balm AJ, Dreschler WA,<br />
Rasch CR. Hearing loss due to concurrent<br />
daily low-dose cisplatin chemoradiation for<br />
locally advanced head and neck cancer.<br />
Radiother Oncol <strong>2008</strong>;89:38-43<br />
COMBINATION OF RADIOTHERAPY AND CHEMOTHERAPY<br />
Berthe Aleman, Harry Bartelink, Jose Belderbos, Henk Boot, Annemieke Cats, Otilia Dalesio,<br />
Luc Dewit, Michel Van Den Heuvel, Edwin Jansen, Corrie Marijnen, Maurits Swellengrebel,<br />
Renato Valdes Olmos, Coen Rasch, Marcel Verheij<br />
Gastroenterology – gastric cancer Combined radiotherapy and chemotherapy<br />
(CRT) has improved treatment outcome and organ preservation in a growing number<br />
of solid tumors (uterine cervix, lung, esophagus, head and neck, anorectal). More<br />
recently, a significant survival benefit in postoperative CRT was reported in gastric<br />
cancer. Based on these results we finished three adjuvant chemoradiotherapy phase<br />
I-II trials in gastric cancer in collaboration with our department of Gastroenterology<br />
and the Christie Hospital in Manchester, UK. In the first trial, postoperative<br />
radiotherapy was combined with escalating doses of daily cisplatin and capecitabine.<br />
The maximal tolerated dose (MTD) was established as 5 mg/m2 for cisplatin and 650<br />
mg/m2 bid for capecitabine. Very recently we analyzed cisplatin in a weekly schedule<br />
in this combination. The maximum tolerated dose (MTD) was cisplatin 20 mg/m 2<br />
and capecitabine 575 mg/m 2 bid. In the third trial radiotherapy was combined with<br />
escalating doses of capecitabine only. In this study, with a dose of 1000 mg/m 2 the<br />
MTD was not reached. Until now, we have treated over 170 gastric cancer patients<br />
with CRT. In the first 91 patients that had an adequate follow up in only 16% in-field<br />
recurrences were found, which compares favorably with historical surgical series.<br />
Furthermore, with long-term follow up using serial renography to monitor kidney<br />
function, a 27% and 52% decrease in left renal function is observed at 18 and 24<br />
months, respectively. With ongoing follow up this functional deterioration further<br />
progresses. This has prompted us to apply more sophisticated radiotherapy strategies<br />
(IMRT; cone beam CT scan on the linear accelerator) in the postoperative treatment<br />
of gastric cancer to minimize normal tissue toxicity. We have started accrual in a large<br />
international phase III study (CRITICS) which has been granted by the Dutch Cancer<br />
Society (CKTO), in which patients with operable gastric cancer will receive preoperative<br />
chemotherapy, surgery and then are being randomized between continuation of<br />
chemotherapy or postoperative CRT in a schedule based on the above-mentioned<br />
phase I-II studies. At this moment over 100 patients have been randomized.<br />
In addition Sweden has joined the trial. Furthermore, we have designed a phase I-II<br />
study together with the AMC and VUmc, in which neoadjuvant chemoradiation with<br />
weekly paclitaxel and capecitabine is applied in patients with inoperable gastric<br />
cancer (NARCIS).<br />
Gastroenterology – rectal cancer We retrospectively evaluated acute and perioperative<br />
toxicity and efficacy of preoperative chemoradiotherapy using the oral 5-FU<br />
prodrug capecitabine in locally advanced rectal cancer. Between June 2004 and<br />
February <strong>2008</strong>, 147 patients with T3-4/N2 rectal cancer were treated with radiation<br />
(25x2 Gy) in combination with capecitabine (825 mg/m 2 , day 1-33). Surgery was<br />
performed approximately 6 weeks later in one of 11 regional hospitals. The population<br />
consisted of 86 males and 61 females, with a median age of 63 years. Dosis reduction<br />
of chemotherapy occurred in 12% and 4% did not complete all radiotherapy fractions.<br />
Grade 3-5 toxicity was seen in 22.4%; 1 patient died due to pancytopenia and mucositis.<br />
Fifteen patients (10%) developed grade 3 diarrhoea and 17 patients (12%) grade 3-4<br />
radiation dermatitis. Of the 147 patients, 138 underwent a laparotomy, while resection<br />
was performed in 131 patients. 62 patients underwent an abdominal perineal<br />
resection (APR), 47 a low anterior resection (LAR) and 22 patients a Hartmann’s<br />
procedure. Extra visceral resection was necessary in 15 of these patients (11%).<br />
Anastomotic leakage was seen in 12 of 47 patients (26%) who underwent a LAR,<br />
while perineal wound complications developed in 20 of 62 perineal resection<br />
patients (33%). In 21% of all operated patients re-operation was necessary while<br />
1patient died due to postoperative complications (1%). A pathological complete<br />
response was achieved in 15 patients (11%). In 6 patients (5%, 2 LAR and 4 APR) a<br />
positive circumferential resection margin was found. All exenterations were microscopically<br />
radical. These results demonstrate that preoperative chemo radio therapy<br />
with oral capecitabine is an effective treatment in locally advanced rectal cancer,<br />
associated with an acceptable acute but significant peri-operative morbidity.
NSCLC Combining concurrent chemotherapy with radiotherapy has been a step<br />
forward in the treatment of locally advanced NSCLC. Recently a meta-analysis based<br />
on individual patient data was performed to compare sequential and concurrent<br />
chemo-radiation (CRT) in inoperable NSCLC patients. The patients included in<br />
EORTC study 08972 and M03IVC were included in this analysis. In 1301 randomized<br />
patients the 3 year survival was 24.8% for patients treated with concurrent chemoradiotherapy<br />
versus 18.2% in sequentially treated patients (p = 0.0026).The locoregional<br />
control was significantly better in the concurrent arm (HR 0,76, 95% CI<br />
[0,62-0,94], p = 0,011) while the chance to develop distant metastases was not<br />
different in both arms.<br />
Despite this improvement after concurrent chemo-radiotherapy, prognosis remains<br />
poor emphasizing the need for more effective strategies. The epidermal growth factor<br />
receptor monoclonal antibody (Cetuximab) has been shown to be an active agent in<br />
metastasized NSCLC. We therefore tested the feasibility of combining Cetuximab<br />
with concurrent CRT. In a phase I trial In patients presenting with inoperable locally<br />
advanced NSCLC Cetuximab (loading dose 400 mg/m 2 on day 1, followed by a weekly<br />
dose of 250 mg/m 2 ) was added to our chemo-radiotherapy regimen (66 Gy in<br />
24 fractions and daily dose Cisplatin 6 mg/m 2 ) . Between April and July <strong>2008</strong>,<br />
12 patients were treated within this trial. The addition of Cetuximab to our concurrent<br />
chemo-radiotherapy with daily dose Cisplatin was well tolerated with dermatitis and<br />
radiation esophagitis being the most common side effects (maximum score 3 according<br />
to CTC Version 3.0). No unexpected toxicity occurred. Early response monitoring,<br />
using a repeat PET-CT scan, 4 weeks after the last dose of radiotherapy was done in<br />
10 patients and showed a metabolic response in 5 patents (3 complete and 2 partial<br />
metabolic responses) and one patient with progressive disease. We will continue in<br />
the near future with a multi-centre phase II trial randomizing patients to concurrent<br />
chemo-radiation with or without Cetuximab.<br />
Publication (continued)<br />
Other<br />
141<br />
RADIOTHERAPY<br />
Bartelink H, Begg AC. Prognostic and<br />
predictive markers in oncology.<br />
Introduction. Semin Radiat Oncol<br />
<strong>2008</strong>;18:73-74<br />
Bossema ER, Marijnen CA,<br />
Baas-Thijssen MC, van de Velde CJ,<br />
Stiggelbout AM. Evaluation of the<br />
Treatment Tradeoff Method in Rectal<br />
Cancer Patients: Is Surgery Preference<br />
Related to Outcome Utilities? Med Decis<br />
Making <strong>2008</strong>;28:888-898<br />
Broeks A, Braaf LM, Huseinovic A,<br />
Schmidt MK, Russell NS, van Leeuwen FE,<br />
Hogervorst FB, Van ‘t Veer LJ. The<br />
spectrum of ATM missense variants and<br />
their contribution to contralateral breast<br />
cancer. Breast Cancer Res Treat<br />
<strong>2008</strong>;107:243-248<br />
de Boer JP, Hiddink RF, Raderer M,<br />
Antonini N, Aleman BM, Boot H,<br />
de Jong D. Dissemination patterns in nongastric<br />
MALT lymphoma. Haematologica<br />
<strong>2008</strong>;93:201-206<br />
de Bruin EC, van de Velde CJ,<br />
van Krieken JH, Marijnen CA, Medema JP.<br />
Epithelial human leukocyte antigen-DR<br />
expression predicts reduced recurrence rates<br />
and prolonged survival in rectal cancer<br />
patients. Clin Cancer Res <strong>2008</strong>;14:1073-1079<br />
De Bruin ML, Huisbrink J, Hauptmann M,<br />
Kuenen MA, Ouwens GM, van ‘t Veer MB,<br />
Aleman BM, van Leeuwen FE. Treatment-<br />
related risk factors for premature menopause<br />
following Hodgkin lymphoma. Blood<br />
<strong>2008</strong>;111:101-108<br />
de Vreeze RS, de Jong D, Tielen IH,<br />
Ruijter HJ, Nederlof PM, Haas RL,<br />
van Coevorden F. Primary retroperitoneal<br />
myxoid/round cell liposarcoma is a<br />
nonexisting disease: an immunohistochemical<br />
and molecular biological<br />
analysis. Mod Pathol. <strong>2008</strong> Sep 26.<br />
(epub ahead of print)<br />
de Vreeze RS, Koops W, Haas RL,<br />
van Coevorden F. An unusual case of<br />
hemosiderotic fibrohistiocytic lipomatous<br />
lesion: correlation of MRI and pathologic<br />
findings. Sarcoma <strong>2008</strong>;<strong>2008</strong>:893918
142<br />
RADIOTHERAPY<br />
Publication (continued)<br />
de Vreeze RS, de Jong D, Haas RL,<br />
Stewart F, van Coevorden F. Effectiveness<br />
of Radiotherapy in Myxoid Sarcomas is<br />
Associated with a Dense Vascular Pattern.<br />
Int J Radiat Oncol Biol Phys.<br />
<strong>2008</strong>;72:1480-1487<br />
den Dulk M, Krijnen P, Marijnen CA,<br />
Rutten HJ, van de Poll-Franse LV, Putter H,<br />
Meershoek-Klein Kranenbarg E,<br />
Jansen-Landheer ML, Coebergh JW,<br />
van de Velde CJ. Improved overall survival<br />
for patients with rectal cancer since 1990:<br />
the effects of TME surgery and pre-operative<br />
radiotherapy. Eur J Cancer <strong>2008</strong>;44:<br />
1710-1716<br />
Heideman WH, Russell NS, Gundy C,<br />
Rookus MA, Voskuil DW. The frequency,<br />
magnitude and timing of post-diagnosis<br />
body weight gain in Dutch breast cancer<br />
survivors. Eur J Cancer. <strong>2008</strong> Oct 16 (epub<br />
ahead of print)<br />
Hooning MJ, Aleman BM, Hauptmann M,<br />
Baaijens MH, Klijn JG, Noyon R,<br />
Stovall M, van Leeuwen FE. Roles of<br />
radiotherapy and chemotherapy in the<br />
development of contralateral breast cancer.<br />
J Clin Oncol <strong>2008</strong>;26:5561-5568<br />
Hoving S, Heeneman S, Gijbels MJ,<br />
te Poele JA, Russell NS, Daemen MJ,<br />
Stewart FA. Single-dose and fractionated<br />
irradiation promote initiation and<br />
progression of atherosclerosis and induce<br />
an inflammatory plaque phenotype in<br />
ApoE(-/-) mice. Int J Radiat Oncol Biol<br />
Phys <strong>2008</strong>;71:848-857<br />
Kappers I, Klomp HM, Burgers JA,<br />
van Zandwijk N, Haas RL, van Pel R.<br />
Neoadjuvant (induction) erlotinib response<br />
in stage IIIA non-small-cell lung cancer.<br />
J Clin Oncol <strong>2008</strong>;26:4205-4207<br />
Kunkler IH, Canney P, van Tienhoven G,<br />
Russell NS; MRC/EORTC (BIG 2-04)<br />
SUPREMO Trial Management Group.<br />
Elucidating the role of chest wall irradiation<br />
in ‘intermediate-risk’ breast cancer: the<br />
MRC/EORTC SUPREMO trial. Clin<br />
Oncol (R Coll Radiol) <strong>2008</strong>;20:31-34<br />
Lange MM, Maas CP, Marijnen CA, Wiggers T,<br />
Rutten HJ, Kranenbarg EK, van de Velde CJ;<br />
Cooperative Clinical Investigators of the Dutch<br />
Total Mesorectal Excision Trial. Urinary<br />
dysfunction after rectal cancer treatment is<br />
mainly caused by surgery. Br J Surg<br />
<strong>2008</strong>;95:1020-1028<br />
Messick TE, Russell NS, Iwata AJ, Sarachan KL,<br />
Shiekhattar R, Shanks JR, Reyes-Turcu FE,<br />
Wilkinson KD, Marmorstein R. Structural basis<br />
for ubiquitin recognition by the Otu1 ovarian<br />
tumor domain protein. J Biol Chem<br />
<strong>2008</strong>;283:11038-11049<br />
Nijkamp J, Pos FJ, Nuver TT, de Jong R,<br />
Remeijer P, Sonke JJ, Lebesque JV. Adaptive<br />
radiotherapy for prostate cancer using kilovoltage<br />
cone-beam computed tomography: first clinical<br />
results. Int J Radiat Oncol Biol Phys<br />
<strong>2008</strong>;70:75-82<br />
Pieterse AH, Baas-Thijssen MC, Marijnen CA,<br />
Stiggelbout AM. Clinician and cancer patient<br />
views on patient participation in treatment<br />
decision-making: a quantitative and qualitative<br />
exploration. Br J Cancer <strong>2008</strong>;99:875-882<br />
Rutten HJ, den Dulk M, Lemmens VE,<br />
van de Velde CJ, Marijnen CA. Controversies of<br />
total mesorectal excision for rectal cancer in<br />
elderly patients. Lancet Oncol <strong>2008</strong>;9:494-501<br />
Steggerda MJ, van der Poel HG, Moonen LM.<br />
An analysis of the relation between physical<br />
characteristics of prostate I-125 seed implants<br />
and lower urinary tract symptoms: bladder<br />
hotspot dose and prostate size are significant<br />
predictors. Radiother Oncol <strong>2008</strong>;88:108-114<br />
Straver ME, van Adrichem JC, Rutgers EJ,<br />
Rodenhuis S, Linn SC, Loo CE, Gilhuijs KG,<br />
Oldenburg HS, Wesseling J, Russell NS,<br />
Antonini N, Vrancken Peeters MT. Primary<br />
systemic therapy in patients with operable<br />
primary breast cancer: more benefits than breastconserving<br />
therapy. Ned Tijdschr Geneeskd.<br />
<strong>2008</strong>;152:2519-25<br />
Thomas G, Ali S, Hoebers FJ, Darcy KM,<br />
Rodgers WH, Patel M, Abulafia O, Lucci JA<br />
3rd, Begg AC. Phase III trial to evaluate the<br />
efficacy of maintaining hemoglobin levels above<br />
12.0 g/dL with erythropoietin vs above 10.0 g/dL<br />
without erythropoietin in anemic patients<br />
receiving concurrent radiation and cisplatin for<br />
cervical cancer. Gynecol Oncol <strong>2008</strong>;108:317-325<br />
van Sandick JW, Kappers I, Baas P, Haas RL,<br />
Klomp HM. Surgical treatment in the<br />
management of malignant pleural<br />
mesothelioma: a single institution’s experience.<br />
Ann Surg Oncol <strong>2008</strong>;15:1757-1764<br />
Voskuil DW, Vrieling A, Korse CM, Beijnen JH,<br />
Bonfrer JM, van Doorn J, Kaas R,<br />
Oldenburg HS, Russell NS, Rutgers EJ,<br />
Verhoef S, van Leeuwen FE, van ‘t Veer LJ,<br />
Rookus MA. Effects of lycopene on the insulinlike<br />
growth factor (IGF) system in<br />
premenopausal breast cancer survivors and<br />
women at high familial breast cancer risk. Nutr<br />
Cancer <strong>2008</strong>;60:342-353<br />
Vrieling C, de Jong D, Boot H, de Boer JP,<br />
Wegman F, Aleman BM. Long-term results of<br />
stomach-conserving therapy in gastric MALT<br />
lymphoma. Radiother Oncol <strong>2008</strong>;87:405-411<br />
Weichselbaum RR, Ishwaran H, Yoon T,<br />
Nuyten DS, Baker SW, Khodarev N et al.<br />
An interferon-related gene signature for DNA<br />
damage resistance is a predictive marker for<br />
chemotherapy and radiation for breast cancer.<br />
Proc Natl Acad Sci USA <strong>2008</strong>;105:18490-18495<br />
Weigelt B, Kreike B, Reis-Filho JS. Metaplastic<br />
breast carcinomas are basal-like breast cancers:<br />
a genomic profiling analysis. Breast Cancer Res<br />
Treat <strong>2008</strong>,September 25 (epub ahead of print)<br />
Wildiers H, Kunkler I, Biganzoli L, Fracheboud J,<br />
Vlastos G, Bernard-Marty C, Hurria A,<br />
Extermann M, Girre V, Brain E, Audisio RA,<br />
Bartelink H, Barton M, Giordano SH, Muss H,<br />
Aapro M; International Society of Geriatric<br />
Oncology. Management of breast cancer in<br />
elderly individuals: recommendations of the<br />
International Society of Geriatric Oncology.<br />
Lancet Oncol. 2007;8:1101-115 (review)<br />
Thesis<br />
Heemsbergen WD. Radiation treatment in<br />
prostate cancer: Balancing between tumor<br />
control and toxicity. University of Amsterdam,<br />
Amsterdam <strong>2008</strong>
DIVISION OF SURGICAL ONCOLOGY<br />
GYNAECOLOGY<br />
Marc van Beurden, Willemien van Driel, Lotti Lubsen-Brandsma, Jan Lange, Manon van Seters<br />
The department focuses on treatment and screening for ovarian cancer, on<br />
immunology and treatment for (premalignant) vulvar neoplasms and on the<br />
development of new treatment for premature menopausal symptoms.<br />
Ovary A randomized phase III clinical trial for stage III ovarian carcinoma has started<br />
investigating the effect of secondary debulking surgery with or without hyperthermic<br />
intraperitoneal cisplatinum. This multicenter study started inclusion in march 2007<br />
and currently 5 centers in the Netherlands are participating. Within short time it is<br />
expected that this will expand with 4 additional centers in Belgium and France.<br />
During interval debulking patients are randomized between interval debulking alone<br />
or adding infusion of cisplatinum under hyperthermic condition at the end of surgery.<br />
Primary endpoint of this study is progression free survival (KWF CKTO2006-16).<br />
Participation in a randomised, multicenter, phase III study of erlotinib versus<br />
observation in patients with no evidence of disease progression after first line,<br />
platinum-based chemotherapy for high-risk stage I and stage II-IV ovarian epithelial,<br />
primary peritoneal, or fallopian tube cancer is finished in <strong>2008</strong> and results are<br />
expected in 2010. Primary endpoint will be progression free survival; secondary<br />
endpoint will be overall survival, safety and quality of life (EORTC 55041).<br />
In a multicenter trial the effect of different hormonal replacement regimen on bone<br />
density, breast density and quality of life after prophylactic bilateral salpingo<br />
oophorectomy are examined in a randomized control trial (M05HIR Hirise).<br />
The effect of hormonal replacement therapy on menopausal complaints related to<br />
biochemical changes in surgically and naturally postmenopausal women is<br />
investigated in a prospective observational comparative study (M06HRT Novaria).<br />
A multicenter randomized trial to investigate the effect of cognitive behavioral<br />
therapy (CBT) and physical exercise for climacteric symptoms in breast cancer<br />
patients experiencing treatment-induced menopause is ongoing in 10 hospitals.<br />
285 of the 325 patients needed are randomized (KWF <strong>NKI</strong> 2006-3470).<br />
Vulva Participation in the GROINSS-V II study, an international multicenter<br />
observational study on patients with vulvar cancer during which patients with<br />
positive lymph nodes are treated with radiation instead of inguinofemoral<br />
lymphadenectomy is ongoing. Endpoints of this study are recurrence free survival<br />
and quality of life. For patients with locally advanced carcinoma of the vulva an<br />
efficacy study is ongoing during which patients are treated with induction<br />
chemoradiation and if necessary followed by surgery in order to reduce postoperative<br />
morbidity (AMC locally advanced vulvar cancer efficacy study).<br />
The benefit of wearing therapeutic elastic stockings in order to prevent lymphedema<br />
for patients treated with inguinal lymph node dissection is conducted in a nonblinded,<br />
randomized controlled trial (M06PRO Kousen study). This study is finished<br />
in September <strong>2008</strong> and results will be available in 2009.<br />
The efficacy of a immune respons modifier, imiquimod 5% cream, has been<br />
inevstigated in a randomized placebo controlled trial in patients with VIN. Thirty-five<br />
percent obtained a clinical and histological complete remission. There was a strong<br />
association between viral clearance and histologisc regression (P
144<br />
SURGICAL ONCOLOGY<br />
Adriaan Timmers DDS Academic staff<br />
Corina Van As-Brooks PhD Academic staff<br />
Michiel Van den Brekel MD PhD<br />
Academic staff<br />
Joost van Tongeren MD Resident<br />
C Verhagen MD Research physician<br />
Karel Zuur MD Reseach physician<br />
Urologic Oncology<br />
Simon Horenblas MD PhD FEBU Head<br />
Axel Bex MD PhD Academic staff<br />
Wim Meinhardt MD PhD Academic staff<br />
Henk Van de Poel MD PhD Academic staff<br />
Joris Leijte Research physician<br />
Niel Joshi MD Fellow<br />
Niels Graafland Research physician<br />
Gynaecology<br />
Marc Van Beurden MD PhD Academic staff<br />
Willemien Van Driel MD PhD Academic staff<br />
Jan Lange MD Academic staff<br />
Lotti Lubsen–Brandsma MD Academic staff<br />
Manon van Seters MD Research fellow<br />
Plastic and Reconstructive Surgery<br />
Joris Hage MD PhD Academic staff (head)<br />
Brigit Drost MD Academic staff<br />
Leonie Woerdeman MD PhD Academic staff<br />
Marieke Van der Berg MD Academic staff<br />
Martine Van Huizum MD Academic staff<br />
Kalam Ahmed MD Research fellow<br />
Esther Moerman MD Fellow<br />
Jan Kortmann MD Research fellow<br />
Mona Mazgani MD Fellow<br />
Tomas Bulut Research fellow<br />
Wouter Van der Pot MD Fellow<br />
Anesthesiology<br />
Peter Schutte MD Academic staff (head)<br />
Dirk Buitelaar MD Academic staff<br />
Katina Efthymiou MD Academic Staff<br />
Christiaan Keijzer MD PhD Academic staff<br />
May Ronday MD Academic staff<br />
Michael Srámek MD PhD Academic staff<br />
Tino Stoppa MD Academic staff<br />
Julia Ten Cate MD Academic staff<br />
Ingeborg Vergouwe MD Academic staff<br />
Dermatology<br />
Wietze Van der Veen MD PhD Academic<br />
staff (head)<br />
Inka Nieuweboer-Krobotova MD<br />
Academic staff<br />
Sanne Vos MD Academic staff<br />
Biljana Zupan-Kajcovski MD Academic staff<br />
GASTRO-INETSTINAL CANCER<br />
Theo Ruers, Frits van Coevorden, Vic Verwaal, Johanna van Sandick, Marie-Jeanne Vrancken Peeters,<br />
Ewout Courrech Staal, Sjoerd Bruin<br />
Oesophageal cancer In January <strong>2008</strong>, Ewout Courrech Staal started as a full-time<br />
research physician on a thesis-project ‘Multimodality treatment of oesophageal<br />
cancer’. A retrospective database was constructed including 1499 patients who<br />
presented at the <strong>NKI</strong>-AVL between 1995-2007 with primary, recurrent or metastatic<br />
oesophageal cancer. Of the 87 patients who were operated, 56 patients received neoadjuvant<br />
treatment. A microscopic radical resection was achieved in 96%. Pathologic<br />
response was complete in 25%. Postoperative mortality was 1%, and 5-year overall<br />
survival rate 42%. Thus, results of low-volume oesophageal cancer surgery in our<br />
institute meet high-quality standards. In an other study, different regimens of<br />
concurrent chemoradiotherapy were studied. Overall, tolerance was acceptable<br />
(77 of 94 patients [82%] completed the planned regimen). In September <strong>2008</strong>,<br />
a prospective study was initiated to investigate whether gene expression and/or<br />
serum protein profiling can improve individual treatment planning in patients with<br />
non-metastatic oesophageal cancer.<br />
Gastric cancer Participation in the so-called CRITICS study (A multicenter<br />
randomized phase III trial of neo-adjuvant chemotherapy followed by surgery and<br />
chemotherapy or by surgery and chemoradiotherapy in resectable gastric cancer) has<br />
led to an increasing number of patients referred for gastric surgery.<br />
Colorectal cancer The CGH analysis of colorectal tumors with metastasis to liver<br />
peritoneum and colorectal tumor without metastasis showed a loss in chromosome<br />
20q in those patients who have liver metastasis while there was no loss in chromosome<br />
20q in patients without liver metastasis. The genes associated with preferred metastasis<br />
to the liver will further be investigated.<br />
Furthermore new grading of psuedomyxoma peritonei was established by connecting<br />
the results of pathologic characteristics like mitotic activity, atypia and amount of<br />
mucous to survival. The results showed that histology with > 90 % mucous, limited<br />
mitotic activity and no mitotic activity were related to superior outcome, all other<br />
patients should be regarded as metastasized colorectal cancer. These results sharpen<br />
the indication of cytoreduction and HIPEC.<br />
The randomized trial comparing cytoreduction followed by HIPEC to systemic<br />
chemotherapy in patients with peritoneal metastasis of colorectal origin was updated,<br />
showing a 45% 5-year survival in those selected patients who underwent a complete<br />
cytoreduction.<br />
Interim results of a randomized multicentre European study investigating the effect<br />
of radiofrequency ablation (RFA) for unresectable colorectal liver metastases<br />
(EORTC 40004) were presented at ASCO this year. This study, under guidance of<br />
dr T. Ruers, showed a superior disease free survival after combined treatment of RFA<br />
plus chemotherapy versus chemotherapy alone (median DFS 17 mo versus 10 mo).<br />
A number of new studies are started. These studies are: in vivo scanning with radioactive<br />
labeled bevacuzimab, accelerated growth of synchronous colorectal liver metastasis,<br />
tumor destruction and dendritic cell activation in situ leading towards in vivo loaded<br />
DC vaccines.<br />
BREAST CANCER<br />
Emiel Rutgers, Hester Oldenburg, Marie-Jeanne Vrancken Peeters, Omgo Nieweg, Marieke Straver,<br />
Jelle Wesseling, Hans Peterse, Claudette Loo, Leonie Woerdeman, Reinie Kaas, Philip Meijnen<br />
Neomamma data base Over the past 7 years we have treated 329 patients with<br />
breast cancer according to different regimes of primary systemic therapy (PST).<br />
We have focused on the axillary response. An axillary pCR was seen in 20%, thus<br />
residual axillary disease was seen in 80% of the patients that underwent ALND.<br />
Axillary pCR was higher in patients with a positive sentinel node, namely 32%.<br />
Multivariate analysis identified two predictors of axillary pCR: subtype according to<br />
receptor-status and response of the primary breast tumor.
Future plans are to optimize response monitoring by using PET-CT scan in addition<br />
to MRI.<br />
New localization method for breast tumors This year we have introduced the<br />
radioactive jodium seed to localize both areas of DCIS and carcinoma in the breast.<br />
In a pilot study of 10 patients we have shown that this is a safe procedure; average<br />
migration of the seed was 0.62 mm (SD 0.43mm) with a minimum of 0.18 mm and<br />
a maximum migration of 1.3 mm.<br />
Future plans are to use radioactive seed localization in both the breast and axilla for<br />
surgery in the context of PST.<br />
Prophylactic mastectomy in BRCA 1-2 mutation carriers 260 BRCA1/2 carriers<br />
had uni- (n = 115) or bilateral (n = 145) prophylactic mastectomy. In 12 carriers DCIS<br />
was diagnosed and in one a small invasive breast cancer. Afterwards in one carrier a<br />
breast cancer was detected. During follow up (average 5 years), only in one patient<br />
after contralateral prophylactic mastectomy, a cancer was foud in the axilla.<br />
Prophylactic mastectomy is highly effective in preventing breast cancer in women at<br />
very high risk, so continued surveillance is not warranted.<br />
Effectiveness of cognitive behavioural therapy and physical exercise on<br />
climacteric symptoms in women with breast cancer experiencing treamentinduced<br />
menopause (EVA project) Patient accrual and data collection for this<br />
randomized multicenter study started, as planned, in January <strong>2008</strong>. To date,<br />
268 women have been randomly allocated to the CBT group, (n=69), the physical<br />
exercise group (n=65), the combined intervention group (n=67), or to the control<br />
group (n=67). Patient recruitment, intervention, and data collection (including<br />
follow-up) will continue until approximately medio 2010.<br />
LYMPHATIC MAPPING<br />
Omgo Nieweg, Iris Van der Ploeg, Maartje Van Rijk, Niels Graafland, Joost Leijte, Pieter Tanis,<br />
Renato Valdés Olmos, Emiel Rutgers, Simon Horenblas, Michiel Van den Brekel, Cornelis Hoefnagel,<br />
Bin Kroon<br />
The aims of our research program are to outline indications and contra-indications,<br />
to refine the technique of lymphoscintigraphy, of surgery and pathology, and to<br />
determine implications in terms of more individualized treatment, survival and<br />
regional tumor control. Reduction of the extent of the operations was a theme of<br />
research in <strong>2008</strong>.<br />
Sentinel node biopsy became standard of care in breast cancer patients before<br />
consensus on the technique was reached and without randomized studies having<br />
shown a similar or decreased axillary recurrence rate. A systematic review and metaanalysis<br />
of the literature was performed of 48 studies concerning 14,959 patients<br />
with a tumour-negative sentinel node biopsy and no subsequent axillary node<br />
dissection. With a median follow-up of 34 months, the axillary recurrence rate was<br />
found to be 0.3%, which is well within the desired range. This percentage was 0.25 in<br />
our own patient population.<br />
The extent of a groin dissection in melanoma patients in whom sentinel node biopsy<br />
reveals metastasis is controversial. The high quality lymphoscintigrams at the<br />
Netherlands Cancer Institute enable the extent of a completion groin lymph node<br />
dissection to be determined by the location of the higher-tier nodes. Eighteen patients<br />
had femoro-inguinal second-echelon nodes on their lymphoscintigram and<br />
underwent a superficial lymph node dissection only. The median follow-up duration<br />
was 61 months. One of the eighteen patients developed a deep (obturator) lymph<br />
node recurrence after twelve months. Revision of the lymphoscintigram showed that<br />
the images had been interpreted incorrectly and that the second-echelon node was<br />
located in the obturator area after all. This result suggests that the strategy to<br />
determine the extent of the groin dissection based on the location of the second-tier<br />
nodes is valid.<br />
Publications<br />
145<br />
SURGICAL ONCOLOGY<br />
Balm AJM, Smeele LE, Lohuis PJ.<br />
Optimizing exposure of the posterolateral<br />
maxillary and pterygoid region: the lower<br />
cheek flap. Eur J Surg Oncol. <strong>2008</strong>,34:<br />
699-703<br />
Bex A, De Vries R, Pos F, Kerst M,<br />
Horenblas S. Long-term survival after<br />
sequential chemoradiation for limited<br />
disease small cell carcinoma of the bladder.<br />
World J Urol. <strong>2008</strong> Jul 8.<br />
Bex A, Haanen JB, Vyth-Dreese FA,<br />
Horenblas S, De Gast GC. Cytokine<br />
therapy response as a selection criterion for<br />
cytoreductive nephrectomy in metastatic<br />
renal clear-cell carcinoma of intermediate<br />
prognosis. Results and conclusions from a<br />
combined analysis. Urol Int. <strong>2008</strong>;80:367-71<br />
Bex A. Review: treatment of small cell<br />
carcinoma of the urinary bladder: can we<br />
learn from small cell lung cancer? Clin Adv<br />
Hematol Oncol. <strong>2008</strong> May;6:385-6. Review.<br />
No abstract available<br />
Bleeker MC, Heideman DA, Snijders PJ,<br />
Horenblas S, Dillner J, Meijer CJ. Penile<br />
cancer: epidemiology, pathogenesis and<br />
prevention. World J Urol. <strong>2008</strong> Jul 8<br />
Borgemeester MC, van den Brekel MWM,<br />
van Tinteren H, Smeele LE, Pameijer FA,<br />
van Velthuysen ML, Balm AJM.<br />
Ultrasound-guided aspiration cytology for<br />
the assessment of the clinically N0 neck:<br />
factors influencing its accuracy. Head Neck<br />
<strong>2008</strong>;30:1505-1513<br />
Bueno-de-Mesquita JM, Linn SC, Keijzer R,<br />
Wesseling J, Nuyten DS, van Krimpen C,<br />
et al. Validation of 70-gene prognosis<br />
signature in node-negative breast cancer.<br />
Breast Cancer Res Treat <strong>2008</strong> Sep 26<br />
Buitelaar D, Huitink J, Oldenburg H,<br />
Rutgers E, Schutte P, van Tinteren H. Field<br />
Block: an additional technique of potential<br />
value for breast surgery under general<br />
anaesthesia. Eur J Anaesthesiology<br />
<strong>2008</strong>;25:253-255<br />
Buitelaar D, Huitink J, Oldenburg H,<br />
Rutgers E, Schutte P, van Tinteren H.<br />
Field block: an additional technique of<br />
potential value for breast surgery under<br />
general anaesthesia. Eur J Anaesthesiol<br />
<strong>2008</strong>;25:253-5
146<br />
SURGICAL ONCOLOGY<br />
Publications (continued)<br />
De Vries RR, Nieuwenhuijzen JA,<br />
Meinhardt W, Bais EM, Horenblas S.<br />
Long-term survival after combined<br />
modality treatment in metastatic bladder<br />
cancer patients presenting with supraregional<br />
tumor positive lymph nodes<br />
only. Eur J Surg Oncol. <strong>2008</strong> Aug 20.<br />
Dirven R, Hilgers FJM, Plooij JM, Maal TJJ,<br />
Bergé SJ, Verkerke GJ, Marres HAM. 3D<br />
Stereophotogrammetry for the assessment of<br />
tracheostoma anatomy. Acta Otolaryngol.<br />
<strong>2008</strong>;128:1248-1254<br />
Geurts TW, Balm AJ, van Velthuysen ML,<br />
van Tinteren H, Burgers JA,<br />
van Zandwijk N, Klomp HM. Survival<br />
after surgical resection of pulmonary<br />
metastases and second primary squamous<br />
cell lung carcinomas in head and neck<br />
cancer. Head Neck. <strong>2008</strong> Oct 28. [Epub<br />
ahead of print]<br />
Hage JJ, Woerdeman LAE. The design of<br />
the lateral thoracodorsal flap. Ann Plast<br />
Surg <strong>2008</strong>;61:302-305<br />
Hage JJ. Heraklas on knots - Sixteen<br />
surgical nooses and knots from the first<br />
century A.D. World J Surg <strong>2008</strong>;32:<br />
648-655<br />
Hennink S, Wouters MW, Klomp HM,<br />
Baas P. Necrotizing pneumonitis caused by<br />
postoperative pulmonary torsion. Interact<br />
Cardiovasc Thorac Surg. <strong>2008</strong>;7:144-5<br />
Hilgers FJM, Soolsma J, van den Brekel MWM,<br />
Ackerstaff AH, Balm AJM, Tan IB.<br />
A thin tracheal silicone washer solving<br />
periprosthetic leakage in laryngectomees;<br />
direct results and long-term clinical effects.<br />
Laryngoscope <strong>2008</strong>;118:640-645<br />
Hilgers FJM, van den Brekel MWM,<br />
van Tinteren H. Views on Acton el al.<br />
‘Investigation of tracheoesophageal voice<br />
prosthesis leakage patterns: Patient’s selfreport<br />
versus clinician’s confirmation’, Head<br />
Neck <strong>2008</strong>;30:618-621. Letter to the editor.<br />
Head Neck <strong>2008</strong>;30:161660-1661<br />
Ho VK, van der Heiden-van der Loo,<br />
Rutgers EJ, van Diest PJ, Hobbelink MG,<br />
Tjan-Heijnen VC, et al. Implementation of<br />
sentinel node biopsy in breast cancer<br />
patients in the Netherlands. Eur J Cancer<br />
<strong>2008</strong>;44:683-91<br />
THORACIC CANCER SURGERY<br />
Houke Klomp, Ingrid Kappers, Rachel Numan, Lotte Nieuwenhuis, Jeffrey Schaap, Michel Wouters,<br />
Johanna van Sandick<br />
Clinical research was focused on neoadjuvant treatment with EGFR tyrosinekinase<br />
inhibitors in operable non-small cell lung cancer (NSCLC), optimalization of<br />
treatment strategies in locally advanced NSCLC and in thoracic sarcomas,<br />
multimodality treatment for pleural mesothelioma, local treatment for oligometastatic<br />
disease and introduction and evaluation of a tailored care path in thoracic surgery.<br />
Preoperative EGFR tyrosinekinase inhibitor treatment in selected patients with<br />
NSCLC (females, non-smokers, adenocarcinoma) proved to be feasible. In a pilot<br />
study, (near) complete responses were observed within 3 weeks. This study will<br />
continue in unselected patients and further tests will be performed to validate<br />
pathologic tumour response.<br />
In locally advanced stage IIIA-N2 NSCLC, the role of surgery is a hot topic.<br />
Evaluation of induction chemotherapy followed by either surgical resection or<br />
radiotherapy, showed that outcome was favourable after lobectomy, but not after<br />
pneumonectomy (5-yr survival 43% versus 16%, 5-yr survival after radiotherapy 16%).<br />
In malignant pleural mesothelioma, introduction of neoadjuvant chemotherapy<br />
(followed by extra-pleural pneumonectomy and hemithoracic radiotherapy) showed<br />
disappointing results as to (local) disease control.<br />
Evaluation of all resections for chest wall sarcomas in the <strong>NKI</strong> and DDHK from<br />
1980-2006 showed that full-thickness surgical resection is a safe and effective<br />
procedure, even for recurrent chest wall sarcoma. Favourable overall survival<br />
supports repeated attempts to obtain local control.<br />
In patients with limited pulmonary metastatic disease, increased use of minimally<br />
invasive treatment proved to be safe in terms of procedure-related events and did not<br />
significantly affect disease control rates.<br />
Base line data from the prospective evaluation of clinical recovery after thoracic surgery<br />
showed a strong association of experienced pain during the first postoperative days<br />
and length of hospital stay as well as long-term quality of life. This is one of many<br />
items to be re-evaluated after adjustment of the thoracic surgery care path.<br />
SARCOMA<br />
Frits van Coevorden, Ronald de Vreeze, Houke Klomp, Rick Haas, Luc Dewit, Martijn Kerst,<br />
Daphne de Jong, Hester van Boven, Petra Nederlof, Joris Hage, Gerard Schaap (AMC),<br />
Frank de Nies (OLVG)<br />
Clinical aspects and development of mixed-type liposarcoma: A radiological,<br />
histological, immunohistochemical and molecular biological analysis The WHO<br />
divides liposarcoma in the morphological subtypes well-differentiated, dedifferentiated,<br />
myxoid, round cell, and pleomorphic. Recent cytogenetic studies<br />
found two alterations. Where well-differentiated and de-differentiated liposarcoma is<br />
characterized by amplification, myxoid and round cell liposarcoma is characterized<br />
by a translocation. In this study we analyzed mixed-type liposarcomas which show a<br />
radiological and histomorphological combined pattern of these two subgroups.<br />
Preoperative radiotherapy, shown in the Canadian randomized trial, to have a<br />
comparable local control to postoperative radiotherapy, can be considered in<br />
radiosensitive tumors as myxoid/roundcell liposarcoma, whereas in well<br />
differentiated liposarcoma radiotherapy will only be considered in recurrent tumors..<br />
Differentiation between subtypes is important to indicate prognosis and choose the<br />
appropriate treatment. In this study we found that when MRI images suggest a<br />
mixed-type liposarcoma, biopsies for immunohistochemical and molecular biological<br />
analyses are needed to characterize these tumors in order to choose the appropriate<br />
treatment. The heterogeneity in these mixed liposarcomas is probably explained by<br />
different maturation levels within a single developmental pathway from an<br />
undifferentiated stem cell.
Multifocal myxoid liposarcoma; metastasis or second primary? A molecular biological<br />
analysis. For details of this study see contribution of the DOD.<br />
HEAD AND NECK ONCOLOGY AND SURGERY<br />
Fons Balm, Michiel van den Brekel, Marcel Copper, Frans Hilgers, Martin Klop, Peter Lohuis,<br />
Ludi Smeele, Bing Tan, Annemieke Ackerstaff, Adrian Begg, Guido van den Broek, Jan Paul de Boer,<br />
O Hamming-Vrieze, Frank Hoebers, Saar Muller, Coen Rasch, Tom Geurts, Boudewijn Plaat,<br />
Renske Scheenstra, Margot Tesselaar, Louise van Velthuysen<br />
Pulmonary rehabilitation after total laryngectomy The short-term effect of three<br />
different HME devices on endotracheal temperature and humidity was tested in a<br />
randomized cross-over study in 14 laryngectomized individuals. The endotracheal<br />
temperature and humidity were measured without HME, with the ‘Normal, the<br />
‘HiFlow’ and the ‘Micron HME’ (with integrated bacterial filter).The absolute<br />
humidity during maximal inspiration without HME was 17.4 mg/l (mean inhalation<br />
breath length 1.3 sec). Breathing with Normal HME, HiFlow HME and Micron HME<br />
increased the humidity values with 5.5 mg/l (P
148<br />
SURGICAL ONCOLOGY<br />
Publications (continued)<br />
Kaas R, Muller SH, Hart AA, Rutgers EJ.<br />
Stage of breast cancers found during the<br />
surveillance of women with a familial or<br />
hereditary risk. Eur J Surg Oncol<br />
<strong>2008</strong>;34:501-7<br />
Kaas R, Rutgers EJ. [Systematic breast selfexamination<br />
is not a useful screening<br />
procedure, except in hereditary or familial<br />
increased risk of breast cancer]. Ned<br />
Tijdschr Geneeskd <strong>2008</strong>25;152:2317-8<br />
Kappers I, Belderbos JS, Burgers JA,<br />
van Zandwijk N, Groen HJ, Klomp HM.<br />
Non-small cell lung carcinoma of the<br />
superior sulcus: favourable outcomes of<br />
combined modality treatment in carefully<br />
selected patients. Lung Cancer. <strong>2008</strong><br />
Mar;59:385-90.<br />
Kappers I, Klomp HM, Burgers JA,<br />
Van Zandwijk N, Haas RL, van Pel R.<br />
Neoadjuvant (induction) erlotinib response<br />
in stage IIIA non-small-cell lung cancer.<br />
J Clin Oncol. <strong>2008</strong>;26:4205-7<br />
Karim RB, de Lint CA, van Galen SR,<br />
van Rozelaar L, Nieuwkerk PT,<br />
Askarizadeh E, Hage JJ. Long-term effect of<br />
polyalkylimide gel injections on severity of<br />
facial lipoatrophy and quality of life of<br />
HIV-positive patients. Aesth Plast Surg<br />
<strong>2008</strong>;32:873-878<br />
Keijzer C, Perez RS, de Lange JJ.<br />
Inspiratory Carbon Monoxide and<br />
Compound A Concentrations During<br />
Desflurane and Sevoflurane Anesthesia in<br />
Humans: An Observational Study. The<br />
Open Anesthesiology Journal <strong>2008</strong>;2:26-29<br />
Kerst JM, Pos FJ, Visser O, Horenblas S.<br />
[New developments in the treatment of<br />
muscle-invasive bladder cancer] Ned<br />
Tijdschr Geneeskd. <strong>2008</strong> Jan 26;152:187-92.<br />
Review. Dutch<br />
Kroon BBR, Hoefnagel CA,<br />
Valdés Olmos RA, Nieweg OE. Regionale<br />
lymfekliergebieden op afstand. Ned Tijdschr<br />
Geneeskd <strong>2008</strong>;152:1997-2000<br />
Kroon BBR, Noorda EM, Vrouenraets BC,<br />
Van Slooten GW, Nieweg OE. Isolated<br />
limb perfusion for melanoma. Surg Oncol<br />
Clin N Am. <strong>2008</strong>;17:785-794.<br />
6,2 years), who underwent a selective or (modified) radical SalvND (only) for<br />
suspected regional residual or recurrent disease after Cisplatin containing concurrent<br />
chemoradiation (70 Gy). Cisplatin regimen: intra-arterially 4x150mg/m 2 (n=21) or<br />
3x100mg/ m2 i.v. (n=15) and 6mg/m 2 i.v. (n=14). Postoperative complications<br />
occurred in 25 patients and were mostly wound related (38%): infection in 28%,<br />
delayed wound healing in 10%. Swallowing deteriorated in 12% after SalvND. In<br />
patients with complications, surgery was more extensive (in duration and number of<br />
dissected levels) and peroperative blood loss was more pronounced (p
UROLOGIC ONCOLOGY<br />
Simon Horenblas, Wim Meinhardt, Axel Bex, Henk Van der Poel<br />
Research in urologic oncology has been centered around the following themes:<br />
Improved staging of urologic tumors, organ and function sparing, fundamental<br />
research in prostate, kidney and penis cancer, improved quality control of treatment<br />
results.<br />
Improved staging of urologic tumors Research has been focussed on the role of<br />
sentinel node biopsy in prostate, testicular and penile cancer. In all above mentioned<br />
tumors, sentinel node biopsy has proven to be extremely useful in minimizing the<br />
morbidity of surgery of the lymph nodes and at the same time maximizing the<br />
detection rate of occult lymph node metastases. The experience with penile cancer,<br />
where the false negative rate dropped from 20% to an acceptable 5.1%, led to a prize<br />
winning publication. New aspects of research in sentinel nodes has been the use of<br />
SPECT /CT scans and intra-operative imaging with a mobile gamma camera<br />
(Sentinella®). These adjuncts have been able to increase accuracy of detection of<br />
sentinel nodes. In prostate cancer no false negative findings were observed. The<br />
SPECT/CT scan together with the Sentinella was effective in localizing sentinel nodes<br />
in a variety of anatomical locations, otherwise not removed during standard lymph<br />
node dissection. In testicular cancer also no false negative findings have been<br />
observed. Accrual in this clinical study is very slow unfortunately, mainly because of<br />
the fact that stage I germ cell tumors are mostly been treated outside of <strong>NKI</strong>-AVL. For<br />
renal cancer a sentinel node study is ongoing since 2007 (N06SNR). The removed<br />
sentinel node is also used for advanced studies on immunological changes. This part<br />
of research is being conducted together with the department of immunology.<br />
Research is also focussed on better delineation of the primary tumor, especially<br />
prostate cancer during robotic surgery. This is being investigated by dr Van der Poel<br />
and H Wijkstra (AMC) using transrectal ultrasound and microbubble enhanced<br />
vascular imaging. In animal experiments the use indocyanine green was shown to<br />
improve sentinel node visualization intraoperatively.<br />
Organ and function sparing While nephrectomy has been standard treatment for<br />
renal cancer, this is increasingly being replaced by partial nephrectomy and<br />
percutaneous Radio Frequency Ablation (RFA). These modalities play an important<br />
role in the integrative approach of metastatic renal cancer. Together with the dept. of<br />
urology of the Academic Medical Center in Amsterdam the value of contrast<br />
enhanced ultrasound is being assessed as staging modality and during follow up<br />
instead of the currently used CT-scan.<br />
The role of cytoreductive surgery is being investigated in a prospective phase II study<br />
with neo-adjuvant Sutent (tyrosine kinase inhibitor), in collaboration with the<br />
department of immunology (N06SUN). An EORTC-study, under guidance of Axel<br />
Bex and John Haanen, investigating the sequence of nephrectomy in metastatic renal<br />
cancer will be launched this year. In bladder cancer we continue to expand our<br />
experiences with the so called Sexuality Preserving Cystectomy and Neobladder<br />
(SPCN). In a recent analysis excellent continence and potency rates were seen<br />
without any danger of increased recurrences. Despite international scepsis we<br />
continue to advocate this procedure in well selected patients. In line with the wish to<br />
decrease the morbidity of the surgery robotic assisted cystectomy has been<br />
introduced. A randomized study comparing standard and robotic cystectomy is<br />
anticipated if funding can be found.<br />
In prostate cancer the introduction of the Da Vinci robot led to an increase of prostate<br />
cancer patients and operations. First results show good tumor margin free rates,<br />
continence and potency rates. All patients are carefully monitored using EORTC-<br />
QLQ questionnaires.<br />
Translational research<br />
Prostate cancer<br />
Cell signalling in hormone refractory prostate cancer is studied in a variety of in vitro<br />
and in vivo mouse modelling systems. Pim1 expression in advanced prostate cancer<br />
was shown to increase proliferation but suppress cell invasiveness explaining the<br />
149<br />
SURGICAL ONCOLOGY<br />
Publications (continued)<br />
Kroon BK, Leijte JA, Van Boven H,<br />
Wessels LF, Velds A, Horenblas S,<br />
Van ‘t Veer LJ. Microarray gene-expression<br />
profiling to predict lymph node metastasis<br />
in penile carcinoma. BJU Int. <strong>2008</strong><br />
Aug;102:510-5<br />
Langenhoff BS, Krabbe PF, Ruers TJ.<br />
Efficacy of follow-up after surgical treatment<br />
of colorectal liver metastases. Eur J Surg<br />
Oncol <strong>2008</strong> Aug 6<br />
Leijte JA, Gallee M, Antonini N,<br />
Horenblas S. Evaluation of current TNM<br />
classification of penile carcinoma. J Urol.<br />
<strong>2008</strong>;180:933-8;discussion 938<br />
Leijte JA, Horenblas S. Shortcomings of the<br />
current TNM classification for penile<br />
carcinoma: time for a change? World J<br />
Urol. <strong>2008</strong> Aug 9<br />
Leijte JA, Kirrander P, Antonini N,<br />
Windahl T, Horenblas S. Recurrence<br />
patterns of squamous cell carcinoma of the<br />
penis: recommendations for follow-up based<br />
on a two-centre analysis of 700 patients.<br />
Eur Urol. <strong>2008</strong> Jul;54:161-8<br />
Leijte JA, Valdés Olmos RA, Nieweg OE,<br />
Horenblas S. Anatomical mapping of<br />
lymphatic drainage in penile carcinoma<br />
with SPECT-CT: implications for the extent<br />
of inguinal lymph node dissection. Eur<br />
Urol. <strong>2008</strong>;54:885-90<br />
Leijte JAP, Valdés Olmos RA, Nieweg OE,<br />
Horenblas S. Anatomical mapping of<br />
lymphatic drainage in penile carcinoma<br />
with SPECT/CT: implications for the extent<br />
of inguinal lymph node dissection.<br />
Eur Urol <strong>2008</strong>;54:885-892<br />
Li WW, Visser O, Ubbink DT, Klomp HM,<br />
Kloek JJ, de Mol BA. The influence of<br />
provider characteristics on resection rates<br />
and survival in patients with localized nonsmall<br />
cell lung cancer. Lung Cancer.<br />
<strong>2008</strong>60:441-51<br />
Linthorst Homan MW, de Korte J,<br />
Grootenhuis MA, Bos JD, Sprangers MA,<br />
van der Veen JP. Impact of childhood<br />
vitiligo on adult life. Br J Dermatol<br />
<strong>2008</strong>;159:915-20
150<br />
SURGICAL ONCOLOGY<br />
Publications (continued)<br />
Linthorst Homan MW, Sprangers MA, de<br />
Korte J, Bos JD, van der Veen JP.<br />
Characteristics of patients with universal<br />
vitiligo and health-related quality of life.<br />
Arch Dermatol <strong>2008</strong> Aug;144:1062-4<br />
Loonen MPJ, Hage JJ, Kon M. Plastic<br />
surgery classics – Characteristics of 50 topcited<br />
articles in four plastic surgery journals<br />
since 1946. Plast Reconstr Surg<br />
<strong>2008</strong>;121:320e-327e<br />
Lordick F, Ruers T, Aust DE, Collette L,<br />
Downey RJ, El Hajjam M, et al. European<br />
Organisation of Research and Treatment of<br />
Cancer (EORTC) Gastrointestinal Group:<br />
Workshop on the role of metabolic imaging<br />
in the neoadjuvant treatment of<br />
gastrointestinal cancer. Eur J Cancer <strong>2008</strong><br />
Sep;44:1807-19<br />
Meijnen P, Oldenburg HS, Peterse JL,<br />
Bartelink H, Rutgers EJ. Clinical outcome<br />
after selective treatment of patients<br />
diagnosed with ductal carcinoma in situ of<br />
the breast. Ann Surg Oncol <strong>2008</strong><br />
Jan;15:235-43<br />
Meijnen P, Peterse JL, Antonini N,<br />
Rutgers EJ, van de Vijver MJ.<br />
Immunohistochemical categorisation of<br />
ductal carcinoma in situ of the breast.<br />
Br J Cancer <strong>2008</strong> Jan 15;98:137-42<br />
Meijnen P, Rutgers EJ. Meta-analysis of<br />
sentinel node biopsy in ductal carcinoma in<br />
situ of the breast (Br J Surg <strong>2008</strong>;95:547-<br />
554). Br J Surg <strong>2008</strong> Oct;95:1305<br />
Meinhardt W, Valdés Olmos RA,<br />
Van der Poel HG, Bex A, Horenblas S.<br />
Laparoscopic sentinel node dissection for<br />
prostate carcinoma: technical and<br />
anatomical observations. BJU Int. <strong>2008</strong><br />
Sep;102:714-7<br />
Mol AJ, Geldof AA, Meijer GA,<br />
Van der Poel HG, Van Moorselaar RJ. New<br />
experimental markers for early detection of<br />
high-risk prostate cancer: role of cell-cell<br />
adhesion and cell migration. J Cancer Res<br />
Clin Oncol. 2007 Oct;133:687-95<br />
Mulier S, Ni Y, Jamart J, Michel L,<br />
Marchal G, Ruers T. Radiofrequency<br />
ablation versus resection for resectable<br />
colorectal liver metastases: time for a<br />
randomized trial? Ann Surg Oncol <strong>2008</strong><br />
Jan;15:144-57<br />
variety of Pim1 expression levels found in human prostate cancer tissue. In preclinical<br />
analysis a synergistic antitumor effect of the combination of cyclophosphamide and<br />
mTOR inhibition was observed. This is the basis for a phase I clinical study in men<br />
with hormone refractory prostate cancer.<br />
Penis cancer<br />
The role of HPV in penis cancer was further investigated. Using molecular and<br />
serological analyses for a wide range of HPV types and comparing serological<br />
findings with age-matched male controls. Primarily HPV 16 infection is directly<br />
involved in penile carcinogenesis. In a prospective study, serum and cervical<br />
scrapings are being collected and investigated to look into the role of HPV<br />
transmission between patients with penile cancer and partners.<br />
Renal cancer<br />
Renal cancer tissue from the N06SUN trial exposed to the active metabolite of the<br />
tyrosine kinase inhibitor sunitinib is investigated in the Department of Immunology<br />
and in cooperation with the Angiogenesis Laboratory of the Department of Pathology<br />
at the University of Maastricht to establish an angiogenic profile of the post-treatment<br />
tissue in correlation to activated and suppressed immune cells infiltrating tumor<br />
tissue. These investigations are further substantiated by material from phase II trials<br />
of presurgical sunitinib and bevacizumab at the MD Anderson Cancer Center,<br />
Houston, TX and the St. Batholomews Hospital, London.<br />
RECIST criteria of anatomical response may not be adequate to document response<br />
of tumors treated with tyrosine kinase inhibitors. Dynamic contrast enhanced MRI of<br />
the primary tumors pre- and posttreatment are correlated to microvessel density to<br />
investigate if changes on imaging reflect a biological response.<br />
Improved quality control of treatment results Using prospective data collection,<br />
almost all uro-surgical treatments at the <strong>NKI</strong>-AVL can be analysed now. Together with<br />
the Comprehensive Cancer Center Amsterdam a comparative study has been finished<br />
looking at recurrences in bladder cancer as a quality control tool. Treatment related<br />
parameters were analysed in low volume and high volume hospitals and compared<br />
with the results in the <strong>NKI</strong>-AVL. Although the recurrence rate did not differ between<br />
the <strong>NKI</strong>-AVL (high volume hospital) and the other hospitals, the early post–operative<br />
death was less than in other hospitals. Despite difference, this was not statistically<br />
significant due to relatively small numbers. The same analysis has been performed<br />
nation wide. This time statistically significant differences between mortality rates of<br />
low and high volume hospitals were found.<br />
ANAESTESIOLOGY AND INTENSIVE CARE<br />
Peter Schutte, Dirk Buitelaar, Katina Efthymiou, Christiaan Keijzer, May Ronday, Michael Srámek,<br />
Tino Stoppa, Julia ten Cate, Ingeborg Vergouwe<br />
Inspiratory Carbon Monoxide and Compound A Concentrations During Desflurane<br />
and Sevoflurane Anesthesia in Humans: An Observational Study<br />
All modern vapor anesthetics are capable of carbon monoxide (CO) production as a<br />
result of interaction with desiccated strong base containing carbon dioxide absorbents.<br />
In desiccated absorbents, desflurane produces the highest concentrations of CO.<br />
Sevoflurane is known to produce the nephrotoxic compound A (CA) independently<br />
from water content of the carbon dioxide absorbent. The purpose of this study was to<br />
register the average CO concentrations in forty patients receiving anesthesia with<br />
desflurane or sevoflurane after implementation of a safety protocol adapted from<br />
Woehlck et al.. This protocol was developed to prevent desiccation of the strong base<br />
containing absorbent Drägersorb 800 Plus®. Methods: In 40 patients a low-flow<br />
anesthesia was maintained using an oxygen/air mixture with either sevoflurane or<br />
desflurane in combination with the CO2 absorbent Drägersorb 800 plus®. CO and<br />
CA production was measured in the inspiratory limb of the anesthesia machine<br />
using a portable gas chromatograph. Results: No carbon monoxide was measured in<br />
any of the desflurane or sevoflurane anesthesia’s. The mean concentration of CA for<br />
anesthesia with sevoflurane was 17.1 ± 5.5 parts per million. Conclusion: With the
introduction of a safety protocol no carbon monoxide was measured in anesthesia<br />
performed with desflurane or sevoflurane. Compound A is almost continuously<br />
detected in anesthetic procedures with the use of sevoflurane in very low<br />
concentrations. Implementation of a simple safety protocol possibly prevents<br />
desiccation of the absorbent and could subsequently reduce the risk of carbon<br />
monoxide intoxication.<br />
Comparison of postoperative throat and neck complaints after use of the i-gel® and<br />
the La Premiere® disposable laryngeal mask. A double blinded randomized<br />
controlled trial.<br />
The i-gel® supraglottic airway device has been designed to match the peripharyngeal<br />
anatomy without the use of an inflatable cuff. We therefore hypothesized that the<br />
i-gel® should have less postoperative throat and neck complaints compared to a<br />
standard disposable laryngeal mask (La Premiere®).<br />
Methods: 244 patients were randomized to receive either the i-gel® (i-gel) or La<br />
Premiere ® standard laryngeal mask (LM) for airway management. Ease of<br />
insertion, anatomical positioning and leak pressure of the devices were registered.<br />
Patients were interviewed postoperatively by trained recovery room personnel who<br />
were blinded for the used device, for throat or neck complaints after 1, 24 and 48<br />
hours. Results: Sore throat occurred in 6, 6 and 5% of the patients in the i-gel group<br />
after 1, 24 and 48 hours. For the laryngeal mask this was 32*,47* and 24*%<br />
(*=p
152<br />
SURGICAL ONCOLOGY<br />
Publications (continued)<br />
Peeters CF, de Waal RM, Wobbes T,<br />
Ruers TJ. Metastatic dormancy imposed by<br />
the primary tumor: does it exist in humans?<br />
Ann Surg Oncol <strong>2008</strong> Nov;15:3308-15<br />
Pengel KE, Loo CE, Teertstra HJ, Muller SH,<br />
Wesseling J, Peterse JL, et al. The impact<br />
of preoperative MRI on breast-conserving<br />
surgery of invasive cancer: a comparative<br />
cohort study. Breast Cancer Res Treat <strong>2008</strong><br />
Sep 21<br />
Ploeg AJ, Lardenoye JW, Vrancken Peeters MP,<br />
Hamming JF, Breslau PJ. Wound<br />
complications at the groin after peripheral<br />
arterial surgery sparing the lymphatic<br />
tissue: a double-blind randomized clinical<br />
trial. Am J Surg <strong>2008</strong> Oct 15<br />
Prevoo W, Van den Bosch MA, Horenblas S.<br />
[Guideline ‘Renal cell carcinoma’] Ned<br />
Tijdschr Geneeskd. <strong>2008</strong> Jun<br />
7;152:1350;author reply 1350. Dutch<br />
Rutgers EJ, Pusztai L, Bernards R. Are<br />
short-term or long-term recurrence rates<br />
more important in breast cancer screening?<br />
Ann Intern Med <strong>2008</strong> Sep 2;149:357-8<br />
Rutgers EJ. Sentinel node biopsy:<br />
interpretation and management of patients<br />
with immunohistochemistry-positive<br />
sentinel nodes and those with micrometastases.<br />
J Clin Oncol <strong>2008</strong> Feb<br />
10;26:698-702<br />
Santegoets LA, van Seters M,<br />
Heijmans-Antonissen C, Kleinjan A,<br />
van Beurden M, Ewing PC, et al. Reduced<br />
local immunity in HPV-related VIN:<br />
Expression of chemokines and involvement<br />
of immunocompetent cells. Int J Cancer<br />
<strong>2008</strong>;123:616-22<br />
Schagen SB, Boogerd W, Muller MJ,<br />
Huinink WT, Moonen L, Meinhardt W,<br />
Van Dam FS. Cognitive complaints and<br />
cognitive impairment following BEP<br />
chemotherapy in patients with testicular<br />
cancer. Acta Oncol. <strong>2008</strong>;47:63-70<br />
Scheer MG, Sloots CE, van der Wilt GJ,<br />
Ruers TJ. Management of patients with<br />
asymptomatic colorectal cancer and<br />
synchronous irresectable metastases. Ann<br />
Oncol <strong>2008</strong> Nov;19:1829-35<br />
Both patients and panelists expressed an overall preference for the adhesive side as of<br />
1 week after surgery. Patients’ visual analogue scale scores for scar comfort and scar<br />
appearance and panelists’ visual analogue scale scores for aesthetic outcome were<br />
significantly better for the adhesive side after 6 weeks and 6 months (p < 0.05), as<br />
was the total Hollander Wound Evaluation Scale score of the panelists after 6 weeks<br />
(p < 0.02). The total Patient and Observer Scar Assessment Scale score after<br />
6 months was significantly better for the adhesive side according to the patients<br />
(p < 0.01), but not according to the panelists (p = 0.11).<br />
We concluded that 2-octyl-cyanoacrylate is a sound alternative for wound closure.<br />
Skinning and resurfacing of the clitoris for vulvar intraepithelial neoplasia<br />
Partial or total clitoris amputation for vulvar intraepithelial neoplasia (VIN) affects<br />
quality of life and sexual function and is likely to constitute over-treatment as<br />
superficial excision only of the involved epithelium would suffice. Thus, we applied<br />
skinning clitorectomy and replacement of clitoral skin as an organ-sparing surgical<br />
therapy for clitoral VIN.<br />
Seven consecutive patients were treated from July 2003 through February <strong>2008</strong>.<br />
Only the skin of the glans clitoridis was resected and the spongiosum was<br />
subsequently covered with either a thin skin flap from the inner aspect of the<br />
remaining preputium or minor labium, or a split thickness skin graft.<br />
In all patients, the pre-operative diagnosis of VIN was confirmed histopathologically.<br />
Additionally, micro-invasive carcinoma was found in two. Preoperative sexual<br />
function was largely preserved and, after a mean follow-up of 21 months, no recurrence<br />
or invasion of the original lesion was observed in any of the patients.<br />
We concluded that skinning clitorectomy and skin replacement should be advocated<br />
as a sound organ-sparing alternative for clitoral amputation in the treatment of<br />
clitoral VIN.<br />
DERMATOLOGY-MELANOMA<br />
Esther Tjin, Henk Mallo, Wietze Van der Veen, Omgo Nieweg, Bin Kroon, John Haanen,<br />
Florry Vyth-Dreese, Rosalie Luiten<br />
Integrated analyses of melanoma – T cell interactions in situ: relevance for<br />
immunotherapy<br />
Clinical studies have shown that immunotherapy of melanoma by vaccination or<br />
adoptive transfer of effector T-cells can mediate tumor regression. However, a<br />
significant number of melanoma patients either respond poorly or show relapse.<br />
This might be either caused by tumor resistance to T-cell-mediated lysis or impaired<br />
effector T-cell function. To discriminate between these processes, we have developed<br />
an ex vivo tumor explant model in a patient-specific setting. This model is currently<br />
tested and offers the opportunity to predict the potential clinical outcome of<br />
immunotherapy for melanoma patients.
Publications (continued)<br />
Scheer MG, Stollman TH, Boerman OC,<br />
Verrijp K, Sweep FC, Leenders WP, et al.<br />
Imaging liver metastases of colorectal cancer<br />
patients with radiolabelled bevacizumab: Lack of<br />
correlation with VEGF-A expression. Eur J<br />
Cancer <strong>2008</strong> Sep;44:1835-40<br />
Scheer MG, Stollman TH, Vogel WV,<br />
Boerman OC, Oyen WJ, Ruers TJ. Increased<br />
metabolic activity of indolent liver metastases<br />
after resection of a primary colorectal tumor.<br />
J Nucl Med <strong>2008</strong> Jun;49:887-91<br />
Schouten van der Velden AP, Punt CJ,<br />
van Krieken JH, Derleyn VA, Ruers TJ. Hepatic<br />
veno-occlusive disease after neoadjuvant<br />
treatment of colorectal liver metastases with<br />
oxaliplatin: a lesson of the month. Eur J Surg<br />
Oncol <strong>2008</strong> Mar;34:353-5<br />
Seters M van, Beckmann I,<br />
Heijmans-Antonissen C, Beurden M van,<br />
Ewing PC, Zijlstra FJ, et al. Disturbed patterns<br />
of immunocompetent cells in usual-type vulvar<br />
intraepithelial neoplasia. Cancer Res<br />
<strong>2008</strong>;68:6617-22<br />
Seters M van, Beurden M van, ten Kate FWJ,<br />
Beckmann I, Ewing PC, Eijkemans MJC,<br />
Kagie MJ, et al. Treatment of vulvar<br />
intraepithelial neoplasia with topical<br />
imiquimod. New Eng J Med <strong>2008</strong>;358:1465-73<br />
Smeenk RM, Bruin SC, van Velthuysen ML,<br />
Verwaal VJ. In brief. Curr Probl Surg <strong>2008</strong><br />
Aug;45:522-5<br />
Smeenk RM, Bruin SC, van Velthuysen ML,<br />
Verwaal VJ. Pseudomyxoma peritonei.<br />
Curr Probl Surg <strong>2008</strong> Aug;45:527-75<br />
Smeenk RM, van Velthuysen ML, Verwaal VJ,<br />
Zoetmulder FA. Appendiceal neoplasms and<br />
pseudomyxoma peritonei: a population based<br />
study. Eur J Surg Oncol <strong>2008</strong> Feb;34:196-201<br />
Smit LHM, Nieweg OE, Mooij WJ,<br />
Bonfrer JMG, Haanen JBAG, Kroon BBR,<br />
De Gast GC. Value of serum S100-B for<br />
prediction of distant relapse and survival in<br />
stage III B/C melanoma. Anticancer Res,<br />
<strong>2008</strong>;28:2297-2302<br />
Soolsma J, van den Brekel MWM,<br />
Ackerstaff AH, Balm AJM, Tan IB, Hilgers FJM.<br />
Long-term results of Provox ActiValve, solving<br />
the problem of frequent Candida- and<br />
‘underpressure’-related voice prosthesis<br />
replacements. Laryngoscope <strong>2008</strong>;118:252-257<br />
Steggerda MJ, Van der Poel HG, Moonen LM.<br />
An analysis of the relation between physical<br />
characteristics of prostate I-125 seed implants<br />
and lower urinary tract symptoms: bladder<br />
hotspot dose and prostate size are significant<br />
predictors. Radiother Oncol. <strong>2008</strong> Jul;88:108-14.<br />
Stollman TH, Scheer MG, Leenders WP,<br />
Verrijp KC, Soede AC, Oyen WJ, et al. Specific<br />
imaging of VEGF-A expression with radiolabeled<br />
anti-VEGF monoclonal antibody. Int J Cancer<br />
<strong>2008</strong> May 15;122:2310-4<br />
Studer UE, Collette L, Whelan P, Albrecht W,<br />
Casselman J, De Reijke T, Knönagel H, Loidl W,<br />
Isorna S, Sundaram SK, Debois M; EORTC<br />
Genitourinary Group. Using PSA to guide<br />
timing of androgen deprivation in patients with<br />
T0-4 N0-2 M0 prostate cancer not suitable for<br />
local curative treatment (EORTC 30891).<br />
Eur Urol. <strong>2008</strong> May;53:941-9<br />
Tanis PJ, Nieweg OE, van den Brekel MWM,<br />
Balm AJM. Dilemma of clinically node-negative<br />
head and neck melanoma: outcome of ‘watch<br />
and wait’ policy, elective lymph node dissection,<br />
and sentinel node biopsy – a systemic review.<br />
Head Neck <strong>2008</strong>;30:380-389<br />
Tournoy KG, Burgers SA, Annema JT,<br />
Vermassen F, Praet M, Smits M, Klomp HM,<br />
van Meerbeeck JP, Baas P. Transesophageal<br />
endoscopic ultrasound with fine needle<br />
aspiration in the preoperative staging of<br />
malignant pleural mesothelioma. Clin Cancer<br />
Res. <strong>2008</strong> Oct 1;14:6259-63<br />
Valdés Olmos RA, Vidal-Sicart S,Nieweg OE.<br />
SPECT-CT and real-time intraoperative<br />
imaging: new tools for sentinel node localization<br />
and radioguided surgery? Eur J Nucl Med Mol<br />
Imaging, (e-pub ahead of print)<br />
Van den Berg JH, Nuijen B, Beijnen JH,<br />
Vincent A, van Tinteren H, Kluge J,<br />
Woerdeman LA, Hennink WE, Storm G,<br />
Schumacher T, Haanen JB. Optimization of<br />
intradermal vaccination by DNA tattooing in<br />
human skin. Hum Gene Ther <strong>2008</strong>;Nov 25<br />
[Epub ahead of print]<br />
Van der Bogt KE, Vrancken Peeters MP,<br />
van Baalen JM, Hamming JF. Resection of<br />
carotid body tumors: results of an evolving<br />
surgical technique. Ann Surg <strong>2008</strong><br />
May;247:877-84<br />
153<br />
SURGICAL ONCOLOGY<br />
Van der Ploeg IM, Kroon BB, Antonini N,<br />
Valdes Olmos RA, Rutgers EJ, Nieweg OE.<br />
Axillary and extra-axillary lymph node<br />
recurrences after a tumor-negative sentinel<br />
node biopsy for breast cancer using<br />
intralesional tracer administration. Ann<br />
Surg Oncol <strong>2008</strong> Apr;15:1025-31<br />
Van der Ploeg IM, Tanis PJ, Valdes Olmos RA,<br />
Kroon BB, Rutgers EJ, Nieweg OE.<br />
Breast cancer patients with extra-axillary<br />
sentinel nodes only may be spared axillary<br />
lymph node dissection. Ann Surg Oncol<br />
<strong>2008</strong> Nov;15:3239-43<br />
Van der Ploeg IM, Valdes Olmos RA,<br />
Kroon BB, Rutgers EJ, Nieweg OE. The<br />
hidden sentinel node and SPECT/CT in<br />
breast cancer patients. Eur J Nucl Med Mol<br />
Imaging <strong>2008</strong> Aug 20<br />
Van der Ploeg IMC, Nieweg OE, Antonini N,<br />
Valdés Olmos RA, Rutgers EJTh, Kroon BBR.<br />
Axillary and extra-axillary lymph node<br />
recurrences after a tumor-negative sentinel<br />
node biopsy for breast cancer using intralesional<br />
tracer administration Ann Surg<br />
Oncol <strong>2008</strong>;15:1025-1031<br />
Van der Ploeg IMC, Nieweg OE,<br />
Valdés Olmos RA, Kroon BBR. Tumorpositive<br />
sentinel node biopsy of the groin in<br />
clinically node-negative melanoma patients:<br />
superficial or superficial and deep lymph<br />
node dissection? Ann Surg Oncol<br />
<strong>2008</strong>,15:1485-1491<br />
Van der Ploeg IMC, Nieweg OE, Van Rijk MC,<br />
Valdés Olmos RA, Kroon BBR. Axillary<br />
recurrence after a tumour-negative sentinel<br />
node biopsy in breast cancer patients:<br />
A systematic review and meta-analysis of<br />
the literature. Eur J Surg Oncol<br />
<strong>2008</strong>;34:1277-1284<br />
Van der Ploeg IMC, Tanis PJ, Valdés Olmos RA,<br />
Kroon BBR, Rutgers EJTh, Nieweg OE.<br />
Breast cancer patients with extra-axillary<br />
sentinel nodes only may be spared axillary<br />
lymph node dissection. Ann Surg Oncol<br />
<strong>2008</strong>,15:3239-3243<br />
Van der Ploeg IMC, Valdés Olmos RA,<br />
Kroon BBR, Nieweg OE. The hybrid<br />
SPECT/CT as an additional lymphatic<br />
mapping tool in patients with breast cancer.<br />
World J Surg <strong>2008</strong>;32:1930-1934
154<br />
SURGICAL ONCOLOGY<br />
Publications (continued)<br />
Van der Ploeg IMC, Valdés Olmos RA,<br />
Nieweg OE, Rutgers EJTh, Kroon BBR,<br />
Hoefnagel CA. The hidden sentinel node<br />
and SPECT/CT in breast cancer patients.<br />
Eur J Nucl Med Mol Imaging (e-pub ahead<br />
of print)<br />
Van der Ploeg IMC, Van Rijk MC, Nieweg OE,<br />
Kroon BBR. Sentinel lymph node. In:<br />
Encyclopedia of cancer. Second edition.<br />
Schwab M, Ed. Springer, Heidelberg <strong>2008</strong>.<br />
ISBN 978-3-540-36847-2<br />
Van der Poel HG, De Blok W, Bex A,<br />
Meinhardt W, Horenblas S. Peroperative<br />
transrectal ultrasonography-guided bladder<br />
neck dissection eases the learning of robotassisted<br />
laparoscopic prostatectomy.<br />
BJU Int. <strong>2008</strong> Sep;102:849-52<br />
Van Der Poel HG, Moonen L, Horenblas S.<br />
Sequential treatment for recurrent localized<br />
prostate cancer. J Surg Oncol. <strong>2008</strong> Apr<br />
1;97:377-82<br />
Van der Poel HG. Editorial comment on:<br />
marked gene transcript level alterations<br />
occur early during radical prostatectomy.<br />
Eur Urol. <strong>2008</strong> Feb;53:344<br />
Van der Poel HG. Editorial comment on:<br />
Oncological and functional results of<br />
antegrade radical retropubic prostatectomy<br />
for the treatment of clinically localised<br />
prostate cancer. Eur Urol. <strong>2008</strong> Mar;53:561-<br />
2. No abstract available<br />
Van der Poel HG. Editorial Comment on:<br />
Predictive Value of the Differential<br />
Expression of the Urokinase Plasminogen<br />
Activation Axis in Radical Prostatectomy<br />
Patients. Eur Urol. <strong>2008</strong> Jun 23<br />
Van der Veldt AA, Meijerink MR, van den<br />
Eertwegh AJ, Bex A, De Gast G, Haanen<br />
JB, Boven E. Sunitinib for treatment of<br />
advanced renal cell cancer: primary<br />
tumor response. Clin Cancer Res. <strong>2008</strong><br />
Apr 15;14:2431-6<br />
Van der Zee J, Kroon BBR. Isolated limb<br />
perfusion for malignant melanoma;possibly<br />
better results with high dose hyperthermia.<br />
Int J Hyperthermia. <strong>2008</strong>;24:602-603<br />
Van Driel WJ, Dalesio OB, van der Donk E.<br />
OVHIPEC-studie in combinatie met<br />
het gebruik van het ALEA eCRFsysteem.<br />
NTvO <strong>2008</strong>;5:326-30<br />
Van Rossum MA, Jongmans P,<br />
Van As-Brooks CJ, Hilgers FJM. De ontwikkeling<br />
van een evidence-based therapieprogramma voor<br />
het verbeteren van spraakverstaanbaarheid bij<br />
tracheoesofageale sprekers. Stem-, Spraak-, en<br />
Taalpathologie <strong>2008</strong>;16:112-123<br />
Van Sandick JW, Kappers I, Baas P, Haas RL,<br />
Klomp HM. Surgical treatment in the<br />
management of malignant pleural<br />
mesothelioma: a single institution’s experience.<br />
Ann Surg Oncol <strong>2008</strong>;15:1757-64<br />
Van Sandick JW, Kappers I, Baas P, Haas RL,<br />
Klomp HM. Surgical treatment in the<br />
management of malignant pleural<br />
mesothelioma: a single institution’s experience.<br />
Ann Surg Oncol. <strong>2008</strong> Jun;15:1757-64<br />
Verwaal VJ, Bruin S, Boot H, van Slooten G,<br />
van Tinteren H. 8-year follow-up of randomized<br />
trial: cytoreduction and hyperthermic<br />
intraperitoneal chemotherapy versus systemic<br />
chemotherapy in patients with peritoneal<br />
carcinomatosis of colorectal cancer. Ann Surg<br />
Oncol <strong>2008</strong> Sep;15:2426-32<br />
Verwaal VJ, Kusamura S, Baratti D, Deraco M.<br />
The eligibility for local-regional treatment of<br />
peritoneal surface malignancy. J Surg Oncol<br />
<strong>2008</strong> Sep 15;98:220-3<br />
Voskuil DW, Vrieling A, Korse CM, Beijnen JH,<br />
Bonfrer JM, van Doorn J, et al. Effects of<br />
lycopene on the insulin-like growth factor (IGF)<br />
system in premenopausal breast cancer survivors<br />
and women at high familial breast cancer risk.<br />
Nutr Cancer <strong>2008</strong> May;60:342-53<br />
Weum S, de Weerd L, Klein S, Hage JJ<br />
Behandling av bløtdelsdefekter med<br />
perforatorlapper. Tidsskr Nor Lægeforen<br />
<strong>2008</strong>;128:313-315<br />
Wouters M. Outcome-based referral to improve<br />
quality of care in upper GI surgery. J Surg<br />
Oncol. <strong>2008</strong> Oct 1;98:307-8<br />
Wouters MW, van Geel AN,<br />
Menke-Pluijmers M, de Kanter AY,<br />
de Bruin HG, Verhoog L, Eggermont AM.<br />
Should internal mammary chain (IMC)<br />
sentinel node biopsy be performed? Breast. <strong>2008</strong><br />
Apr;17:152-8<br />
Wouters MW, van Geel AN, Nieuwenhuis L,<br />
van Tinteren H, Verhoef C, van Coevorden F,<br />
Klomp HM. Outcome after surgical resections of<br />
recurrent chest wall sarcomas. J. Clin Oncol<br />
<strong>2008</strong>;26:5113-8<br />
Wouters MW, Wijnhoven BP, Karim-Kos HE,<br />
Blaauwgeers HG, Stassen LP, Steup WH,<br />
Tilanus HW, Tollenaar RA. High-volume versus<br />
low-volume for esophageal resections for cancer:<br />
the essential role of case-mix adjustments based<br />
on clinical data. Ann Surg Oncol. <strong>2008</strong><br />
Jan;15:80-7<br />
Zuur CL, Simis YJ, Verkaik RS, Schornagel JH,<br />
Balm AJ, Dreschler WA, Rasch CR. Hearing<br />
loss due to concurrent low-dose cisplatin<br />
chemoradiation for locally advanced head and<br />
neck cancer Radiother Oncol <strong>2008</strong>;89:38-43<br />
Zuur JK, Muller SH, Vincent A, Sinaasappel M,<br />
de Jongh FHC, Hilgers FJM. Assessment of<br />
tracheal temperature and humidity in<br />
laryngectomized individuals and the influence of<br />
heat and moisture exchangers on tracheal<br />
climate. Head Neck <strong>2008</strong>;30:1072-1082<br />
In press<br />
Ackerstaff AH, Balm AJM, Rasch CRN,<br />
De Boer JP, Wiggenraad R, Rietveld DHF,<br />
Gregor RT, Kröger R, Hilgers FJM. First-year<br />
Quality of Life assessment of an intra-arterial<br />
(RADPLAT) versus intravenous chemoradiation<br />
Phase III trial. Head Neck<br />
Balch CM, Morton DL, Gershenwald J,<br />
McMasters K, Nieweg OE, Powell B, Ross M,<br />
Sondak VK, Thompson JF. Sentinel node bopsy<br />
and standard of care for melanoma. J Am Acad<br />
Dermatol<br />
De Vreeze RS, de Jong D, Tielen IH, Ruijter HJ,<br />
Nederlof PM, Haas RL, van Coevorden F.<br />
Primary retroperitoneal myxoid/round cell<br />
liposarcoma is a nonexisting disease: an<br />
immunohistochemical and molecular biological<br />
analysis. Mod Pathol <strong>2008</strong>, Sept <strong>2008</strong><br />
De Vreeze RS, Koops W, Haas, RL,<br />
van Coevorden F. An unusual case of<br />
hemosiderotic fibrohistiocytic lipomatous lesion:<br />
correlation of MRI and pathological findings.<br />
Sarcoma
Geurts TW, Balm AJM, Van Velthuysen MLF,<br />
Van Tinteren H, Burgers JA, Van Zandwijk N,<br />
Klomp HM. Survival after surgical resection of<br />
pulmonary metastases and second primary<br />
squamous cell lung carcinomas in head and neck<br />
cancer. Head Neck<br />
Geurts TW, Lohuis PJFM, Pameijer FA,<br />
Van den Brekel MWM. Lipoma of the<br />
nasopharynx. Arch Otolaryngol<br />
Kreeft A, Lohuis PJ, Smeele LE. Stent repair of<br />
an anastomotic pseudoaneurysm of the carotid<br />
artery after free fibula transplantation. Br J Oral<br />
Maxillofac Surg<br />
MA van Rossum, P Jongmans,<br />
CJ van As-Brooks, FJM Hilgers. De<br />
ontwikkeling van een evidence-based<br />
therapieprogramma voor het verbeteren van<br />
spraakverstaanbaarheid bij tracheoesofageale<br />
sprekers. Stem Spraak Taal Pathologie<br />
Nieweg OE, Van der Ploeg IMC, Kroon BBR.<br />
Naar sentinel-nodebiopsie bij melanoom:<br />
antwoorden en nieuwe vragen - interim-verslag<br />
van de Multicenter Selective Lymphadenectomy<br />
Trial. Oncollectie.<br />
Van der Molen L, Van Rossum MA,<br />
Burkhead LM, Smeele LE, Hilgers FJM.<br />
Functional outcomes and rehabilitation<br />
strategies in patients, treated with chemoradiation<br />
for advanced head and neck cancer:<br />
A systematic review. Eur Arch Otorhinolaryngol<br />
VB Wreesmann, LM Smeele, FJM Hilgers,<br />
PJFM Lohuis. Closure of tracheoesophageal<br />
fistula with prefabricated revascularized<br />
bilaminar radial forearm free flap. Head Neck<br />
Books<br />
Textbook of Surgical Oncology<br />
Graeme Poston, Daniel Beauchamp,<br />
Theo Ruers<br />
Informa Healthcare, London<br />
ISBN9781841845074<br />
Book chapters<br />
Van den Brekel MWM, Hilgers FJM.<br />
Preoperative workup of the neck in head and<br />
neck squamous cell carcinoma. In: Pearls and<br />
Pittfalls in Head and Neck Surgery;practical<br />
tips to minimize complications. Editor Cernea<br />
CR;associate editors: Dias FL, Fliss D, Lima<br />
RA, Myers EN, Wei WI. Karger Publishers,<br />
Basel, Freiburg, Paris, London, New York,<br />
Bangalore, Bangkok, Shanghai, Singapore,<br />
Tokio, Sydney;<strong>2008</strong>, pp.34-35. ISBN 978-3-<br />
8055-8425-8. (CATEGORY C)<br />
Hilgers FJM, Van den Brekel MWM. Practical<br />
tips for voice rehabilitation after<br />
pharyngolaryngectomy. In: Pearls and Pittfalls<br />
in Head and Neck Surgery;practical tips to<br />
minimize complications. Editor Cernea<br />
CR;associate editors: Dias FL, Fliss D, Lima<br />
RA, Myers EN, Wei WI. Karger Publishers,<br />
Basel, Freiburg, Paris, London, New York,<br />
Bangalore, Bangkok, Shanghai, Singapore,<br />
Tokio, Sydney;<strong>2008</strong>, pp 96-97. ISBN 978-3-<br />
8055-8425-8. (CATEGORY C)<br />
Giuliana Mariani, Armando E Giuliano, H.<br />
William Strauss. Radioguided Surgery. A<br />
comprehensive team approach. Contributors:<br />
S. Horenblas. Part II: 11: Dynamic sentinel<br />
lymph node biopsy in penile carcinoma.<br />
Simon Horenblas, Bin K. Kroon, Renato A.<br />
Valdés Olmos, Omgo E. Nieweg<br />
Nieweg OE. Een fellow en zijn PET. Groninger<br />
chirurgische oncologica. Hoekstra HJ,<br />
Wobbes Th, redacteuren. Chirurgische<br />
Oncologie, UMCG, Groningen <strong>2008</strong>. ISBN<br />
078-90-9023505-9. pg. 112-114<br />
Vinod H. Nargund, Derek Raghavan, Howard<br />
M. Sandler. Urological Oncology. Part III<br />
Systemic genitourinary oncology, 19: Cancer of<br />
the penis and scrotum. Simon Horenblas and<br />
Bin K. Kroon<br />
T. Ruers. Liver metastases. In Textbook of<br />
Surgical Oncology<br />
Editors: Graeme Poston, Daniel Beauchamp,<br />
Theo Ruers<br />
Informa Healthcare, London<br />
155<br />
SURGICAL ONCOLOGY
156<br />
BIOMETRICS<br />
Department head, group leader Otilia Dalesio<br />
Otilia Dalesio MSc Head<br />
Ninja Antonini MSc Academic staff<br />
Harm Van Tinteren PhD Academic staff<br />
Andrew Vincent PhD Academic staff<br />
Erik Van Werkhoven MSc Academic staff<br />
Danny Baars Technical staff<br />
Frauwkje Bessels Technical staff<br />
Monique Carreno Technical staff<br />
Daphne Daems Technical staff<br />
Gerda De Jong Technical staff<br />
Marjolijn De Waal MSc Technical staff<br />
Brigitte Dufournij Technical staff<br />
Ernesto Fernandez Salcedo MSc Technical staff<br />
Christiane Hagenaars Technical staff<br />
Song-Hieng Hau MSc Technical staff<br />
Annelies Hiemstra Technical staff<br />
Paula Hoekstra MSc Technical staff<br />
Joop Jonkman MSc Technical staff<br />
Josien Kant Technical staff<br />
Edwin Klerkx Technical staff<br />
Lies Kolmschate Technical staff<br />
Jenneke Koorn Technical staff<br />
Lissettee Lorist Technical staff<br />
Marianne Mahn MSc Technical staff<br />
Ingrid Mandjes MSc Technical staff<br />
Carla Modder Technical staff<br />
Pietje Muller Technical staff<br />
Lida Oostveen Technical staff<br />
Cecile Paulus van Pauwvliet Technical staff<br />
Loes Pronk MSc Technical staff<br />
Harriet Rehorst Technical staff<br />
Anneke Reinders Technical staff<br />
Jolanda Remmelzwaal Technical staff<br />
Daniel Roberts Technical staff<br />
Marielle Roskam Technical staff<br />
Dea Storm Technical staff<br />
Ria Tilgenkamp Technical staff<br />
Alex Torres Acosta Technical staff<br />
Heleen Vaessen MSc Technical staff<br />
Ludy Valkenet MD Technical staff<br />
Marjolijn Van den Haak MSc Technical staff<br />
Emile Van der Donk Technical staff<br />
Tony Van de Velde Technical staff<br />
Gabry Van Netten Technical staff<br />
Els Van Oosterwijk Technical staff<br />
Wil Van Waardenberg Technical staff<br />
Marcel Vlaskamp MSc Technical staff<br />
Anneke Wals Technical staff<br />
Lidwina Wever Technical staff<br />
Els Willemse Technical staff<br />
Jeroen Zandbergen MSc Technical staff<br />
Lonny Ziblat Technical staff<br />
BIOMETRICS DEPARTMENT<br />
ICT PROJECTS<br />
We have just reached the 2 year milestone of the 4 years TENALEA Initial Deployment<br />
(TENALEA ID) project. This is a very large project with a total investment of<br />
approximately 16 million euro, with a 3.7 million euro contribution from the eTEN<br />
program of the EC. As initiators of the project we stimulate different (European) Data<br />
Coordinating Centers to adopt our online tools to support the process of clinical trial<br />
data capture and data management. We also ensure the technical, financial and<br />
organizational coordination of the project. The consortium includes academic<br />
institutes and coordinating centers in Germany, Poland, France, UK and the<br />
Netherlands, including the Academic Medical Centre (AMC) in Amsterdam and the<br />
Clinical Trial Centre Maastricht (CTCM).<br />
Because of our involvement in the development of tools for clinical trials during the<br />
last 20 years, we have currently to our disposal the most modern tools to support<br />
clinical trial organization. The most important being ALEA, which is a system to<br />
register, randomize and automatically distribute notifications of the randomization;<br />
and an electronic CRF system, that allows the development of applications for<br />
studies, by which entry of data from the participating centers directly into the Data<br />
Center databases can be done (ALEA eCRFs). The eCRF system allows detection of<br />
inconsistencies at the source resulting in less data queries at later times. This should<br />
help local data managers in terms of immediate feed back on the consistency and<br />
completeness of the data and should improve quality standards and help working<br />
more efficiently. Most importantly, the electronic data is available much earlier and is<br />
of better quality, thereby speeding up the statistical analysis and reporting<br />
dramatically.<br />
ALEA eCRFs is based on generic packages, principally Microsoft Office Forms<br />
Services and InfoPath2007 from Office 2007 Enterprise (Microsoft). These tools<br />
provide a good basis for an electronic case record form system. Figure 1 shows that<br />
InfoPath provides an extensive feature set to define data entry checks in a userfriendly<br />
interface. The final product incorporates an audit trail in compliance with<br />
the requirements of the International Committee for Harmonization and produces<br />
standard based export facilities (CDISC, ODM and SDTM).<br />
The integration of PACS, allowing central review of images (eg for tumor responses<br />
evaluation) the in a clinical setting, is currently being developed in collaboration with<br />
the radiotherapy department within the CTMM project.<br />
During this year, we collaborated with UK partners to design a simulation system<br />
that can be used to evaluate the performance of the different randomization methods<br />
or parameterization of ALEA. Figure 2a and 2b present the results of 1000<br />
simulations of 1000 patients entered into a 3 armed study with 3 stratification factors<br />
of 2 levels each. Figure 2a shows the treatment allocation histogram depicting how<br />
often a treatment was allocated in each simulation. The expectation would be that the<br />
most regularly observed frequency occurs around the number of patients divided by<br />
the number of treatments. Similar to the treatment histogram, Figure 2b shows the<br />
treatment allocation graph that illustrates how often each patient received each<br />
treatment. Assuming the trial is entirely non-deterministic, we would expect to see a<br />
fairly even allocation of each treatment for each patient. If, however, the trial is<br />
deterministic, we would expect to see a more frequent allocation of a certain<br />
treatment to certain patients.<br />
These simulation tools are being included in the new version of the randomization<br />
program (ALEA3) for use of the different clinical trials coordination centers<br />
responsible for the central management, registration and randomization of patients<br />
in clinical trials.
Figure 1: Data Entry checks definition possibilities<br />
In <strong>2008</strong> the number of clinical trial coordination centers using our systems has<br />
increased to 27 and include centers in Europe, UK and also in New Zealand,<br />
Australia and Indonesia. The randomization methods possibilities in ALEA were<br />
extended and were described in the package manual. Our efforts also went to the full<br />
validation of our systems. The Standard Operating Procedures were reviewed and<br />
updated. The systems were audited by an independent auditor and a new<br />
validation cycle was initiated.<br />
Figure 2a: Treatment allocation histogram<br />
Figure 2b: Treatment allocation graph<br />
Publications<br />
157<br />
BIOMETRICS<br />
Zuur JK, Muller SH, Vincent A,<br />
Sinaasappel M, de Jongh FH, Hilgers FJ.<br />
Assessment of tracheal temperature and<br />
humidity in laryngectomized individuals<br />
and the influence of a heat and moisture<br />
exchanger on tracheal climate. Head Neck<br />
<strong>2008</strong>;30:1072-82<br />
Wouters MW, van Geel AN,<br />
Nieuwenhuis L, van Tinteren H,<br />
Verhoef C, van Coevorden F, Klomp HM.<br />
Outcome after surgical resections of<br />
recurrent chest wall sarcomas. J Clin Oncol<br />
<strong>2008</strong>;26:5113-8<br />
Verwaal VJ, Bruin S, Boot H,<br />
van Slooten G, van Tinteren H. 8-year<br />
follow-up of randomized trial: cytoreduction<br />
and hyperthermic intraperitoneal<br />
chemotherapy versus systemic chemotherapy<br />
in patients with peritoneal carcinomatosis of<br />
colorectal cancer. Ann Surg Oncol<br />
<strong>2008</strong>;15:2426-32<br />
van der Weide L, van Sornsen de Koste JR,<br />
Lagerwaard FJ, Vincent A, van Triest B,<br />
Slotman BJ, Senan S. Analysis of carina<br />
position as surrogate marker for delivering<br />
phase-gated radiotherapy. Int J Radiat<br />
Oncol Biol Phys <strong>2008</strong>;71:1111-7<br />
van der Ploeg IM, Kroon BB, Antonini N,<br />
Valdes Olmos RA, Rutgers EJ, Nieweg OE.<br />
Axillary and extra-axillary lymph node<br />
recurrences after a tumor-negative sentinel<br />
node biopsy for breast cancer using<br />
intralesional tracer administration. Ann<br />
Surg Oncol <strong>2008</strong>;15:1025-31<br />
van den Heuvel-Eibrink MM, Grundy P,<br />
Graf N, Pritchard-Jones K, Bergeron C,<br />
Patte C, van Tinteren H, Rey A,<br />
Langford C, Anderson JR, de Kraker J.<br />
Characteristics and survival of 750 children<br />
diagnosed with a renal tumor in the first<br />
seven months of life: A collaborative study<br />
by the SIOP/GPOH/SFOP, NWTSG, and<br />
UKCCSG Wilms tumor study groups.<br />
Pediatr Blood Cancer <strong>2008</strong>;50:1130-4<br />
van den Broek E, Richard W,<br />
van Tinteren H, de Vries N. UPPP<br />
combined with radiofrequency<br />
thermotherapy of the tongue base for the<br />
treatment of obstructive sleep apnea<br />
syndrome. Eur Arch Otorhinolaryngol<br />
<strong>2008</strong>;265:1361-5
158<br />
BIOMETRICS<br />
Publications (continued)<br />
van den Berg RM, Teertstra HJ,<br />
van Zandwijk N, van Tinteren H, Visser C,<br />
Pasic A, Sutedja TG, Baas P, Golding RP,<br />
Postmus PE, Smit EF. CT detected<br />
indeterminate pulmonary nodules in a<br />
chemoprevention trial of fluticasone. Lung<br />
Cancer <strong>2008</strong>;60:57-61<br />
van den Berg JH, Nuijen B, Beijnen JH,<br />
Vincent A, van Tinteren H, Kluge J,<br />
Woerdeman LA, Hennink WE, Storm G,<br />
Schumacher T, Haanen JB. Optimization<br />
of intradermal vaccination by DNA<br />
tattooing in human skin. Hum Gene Ther<br />
<strong>2008</strong>;<br />
Tol J, Cats A, Mol L, Koopman M, Bos MM,<br />
van der Hoeven JJ, Antonini NF,<br />
van Krieken JH, Punt CJ. Gastrointestinal<br />
ulceration as a possible side effect of<br />
bevacizumab which may herald perforation.<br />
Invest New Drugs <strong>2008</strong>;26:393-7<br />
Tol J, Koopman M, Rodenburg CJ, Cats A,<br />
Creemers GJ, Schrama JG, Erdkamp FL,<br />
Vos AH, Mol L, Antonini NF, Punt CJ.<br />
A randomised phase III study on<br />
capecitabine, oxaliplatin and bevacizumab<br />
with or without cetuximab in first-line<br />
advanced colorectal cancer, the CAIRO2<br />
study of the Dutch Colorectal Cancer<br />
Group (DCCG). An interim analysis of<br />
toxicity. Ann Oncol <strong>2008</strong>;19:734-8<br />
Stuiver MM, van Wilgen CP, de Boer EM,<br />
de Goede CJ, Koolstra M, van Opzeeland A,<br />
Venema P, Sterken MW, Vincent A,<br />
Dijkstra PU. Impact of shoulder complaints<br />
after neck dissection on shoulder disability<br />
and quality of life. Otolaryngol Head Neck<br />
Surg <strong>2008</strong>;139:32-9<br />
Simkens L, Tol J, Koopman M, Mol L,<br />
Antonini N, van Krieken H, Punt C.<br />
Current questions in the treatment of<br />
advanced colorectal cancer: the CAIRO<br />
studies of the Dutch Colorectal Cancer<br />
Group. Clin Colorectal Cancer <strong>2008</strong>;7:105-9<br />
Nieuwenhuijzen JA, de Vries RR, Bex A,<br />
van der Poel HG, Meinhardt W, Antonini N,<br />
Horenblas S. Urinary diversions after<br />
cystectomy: the association of clinical<br />
factors, complications and functional results<br />
of four different diversions. Eur Urol<br />
<strong>2008</strong>;53:834-42<br />
CLINICAL STUDIES AND OTHER COLLABORATIONS<br />
We have developed collaborations with several cooperative groups; academic groups,<br />
industry and clinical research organizations, and we function as partner for clinical<br />
studies and clinical trials, being involved from the generation of the idea, protocol<br />
setting, the planning, and providing randomization services, quality assurance, data<br />
handling and statistical expertise. In addition, the tools that we have developed within<br />
our ICT projects are attracting new associations. With the help of the training kits<br />
that we have developed, personnel of clinical trial coordinating centers can be<br />
incorporated along with our own form designers in the production of the applications<br />
for their own clinical trials. The systems so obtained are then hosted in our computer<br />
servers.<br />
We collaborate with the Dutch Chest Physician Association (NVALT) in planning<br />
designing and running of clinical trials in lung cancer and mesothelioma. More than<br />
75 hospital in the Netherlands have included in excess of 1600 patients in their 9<br />
randomized studies. We have obtained support from the Dutch Cancer Society for<br />
the management of data of 5 studies (the NVALT 3, 4, 5, 8 and 11).<br />
In <strong>2008</strong> the final reports of the 2 of the NVALT studies has been prepared. The role<br />
of platinum compounds as part of second line therapies in advanced NSCLC was<br />
studied in the NVALT7 study. This was a randomized trial comparing pemetrexed (P)<br />
and pemetrexed-carboplatin (PC) in patients relapsing after platinum-based<br />
chemotherapy. Main eligibility criteria: histological or cytological proof of advanced<br />
NSCLC, relapse > 3 months after (first-line) platinum-based chemotherapy, normal<br />
organ reserve, ECOG PS 0-2, age >18 years and signed informed consent. After<br />
stratification, patients were randomized to (A) P 500 mg/m2 i.v. q 3 wks or (B) C<br />
AUC 5 and P 500 mg/m2 both iv q 3 wks. Response was assessed 6 weekly according<br />
to RECIST and toxicity 3 weekly according to NCI-CTC criteria. The primary endpoint<br />
was time to progression (TTP). Secondary endpoints: objective response rate (ORR),<br />
overall survival (OS), toxicity. From October 2005 to May 2007 151 males and 89<br />
females were enrolled by 24 centers. The median (range) age was 59 (36-84), 33% of<br />
the patients had an ECOG PS score of 0, 58% 1 and 9% had a PS of 2. After a median<br />
follow up of 14.7 months, median TTP for arm A was 2.8 months and for arm B 4.2<br />
months; HR 0.67 (95% CI 0.51 – 0.89, p = 0.005). Median OS: 7.6 months versus<br />
8.0 months. The overall response rate was 4% in arm A and 9% in arm B. Subgroup<br />
analyses suggested that adenocarcinoma is associated with favorable outcome.<br />
Toxicities grade 3/4 was 29% in arm A and 44% in Arm B, mainly hematological<br />
without clinical sequel. It was concluded that PC as second line treatment for<br />
relapsed NSCLC resulted in a statistically significant 33% reduction of hazard of<br />
progression as compared to P alone. The paper has been recently accepted for<br />
publication in the JCO.<br />
The second final report concerned the NVALT3 study, which is randomized trial in<br />
elderly patients with non-small-cell lung cancer. This study incorporated a battery of<br />
geriatric and quality of life assessments and compared 2 chemotherapy combinations<br />
in terms of quality of life, toxicity and survival and attempt to identify possible<br />
interactions between the assessments and the results of treatment. Patients had to be<br />
70 years or older with cytological or histological confirmation of inoperable stage III<br />
or IV non small cell lung cancer. Using a minimization algorithm, patients were<br />
centrally assigned to receive a maximum of 4 three-week cycles of carboplatingemcitabine<br />
(CG) or carboplatin-paclitaxel (CP). In the CG arm carboplatin was<br />
administered at a dose targeting an area under the concentration-time curve (AUC) of 5,<br />
intravenously (i.v.) at day 1 followed by gemcitabine 1250 mg/m2 i.v. on days 1 and 8.<br />
Before chemotherapy, on day 1, patients received dexamethason i.v. and a serotonin<br />
receptor antagonist. Patients in the CP arm received paclitaxel 175 mg/m2 i.v.<br />
immediately followed by carboplatin AUC 5 mg/ml/min on day 1. Paclitaxel<br />
administration was preceded by routine medication including dexamethasone,<br />
clemastine, ranitidine, and a serotonin receptor antagonist. Between March 2003 and<br />
August 2006 a total of 182 patients were entered into the study by 14 hospitals. The<br />
average age was 75 years (range:70 to 87). The percentage of patients that completed
all 4 cycles was 64% and 65% in the CG and CP arms, respectively. Grade III/IV<br />
toxicities occurred in 75% of the patients in the CG arm and 60% in the CP arm.<br />
Treatment related SAE’s occurred in 19% of the patients in the CG arm and 15% in<br />
the CP arm. As expected, neurologic toxicity ≥ grade II occurred more frequently in<br />
the CP arm than in the CG arm (neuro-sensory 19% vs 5%, p = 0.002; neuro-motory:<br />
10% vs 2%, p = 0.03). The paper has been submitted to the JCO.<br />
Patient accrual to the NVALT4 trial, a randomized placebo-controlled study of docetaxel/<br />
carboplatin with celecoxib or placebo in patients with locally advanced or metastatic<br />
non-small cell lung cancer, has been completed in <strong>2008</strong>. Follow-up data is being collected<br />
for the 561 patients entered and final analysis is planned for the first half of 2009.<br />
The NVALT 5 is a phase III trial of the antiangiogenic agent Thalidomide in patients<br />
with malignant pleural mesothelioma after first line chemotherapy. Accrual has<br />
continued in <strong>2008</strong> and it is expected to be completed in 2010. Two studies in<br />
resectable NSCLC low and high risk patients as determined by preoperative PET<br />
SUVmax (NVALT8A and NVALT8B), have been open to patient entry for 1 year.<br />
However, the accrual has been disappointing probably due to the fact that the<br />
organization of the study is rather complex since, it is multidisciplinary involving<br />
surgeons, lung physicians, nuclear medicine experts and pathologists, and requiring<br />
central analysis of PET values and central collection of frozen tissue . For this study<br />
we have developed an e-CRFs application based on the ALEA eCRFS tool developed<br />
within our ICT projects. This should facilitate data capture on the different<br />
departments of the hospitals involved directly into our servers and should improve<br />
communication across professions, speed and quality.<br />
The NVALT 9, a phase II study to establish the efficacy of intravenous loading doses<br />
of Ibandronate 6 mg in patients with lung cancer and skeletal metastases<br />
experiencing moderate to severe bone pain is still slowly accruing patients.<br />
A new study, the NVALT11 was developed during <strong>2008</strong> and is currently accruing<br />
patients. In this study prophylactic cranial irradiation (PCI) versus observation is<br />
studied in radically treated patients with stage III non-small cell lung cancer. It is<br />
done as a cooperation of the NVALT, the Dutch Lung Cancer Research Group<br />
(DLCRG) and the National Platform for Radiotherapy in Lung Tumors (LPRL).<br />
We are also the coordinating and statistical center for another study of the DLCRG,<br />
the Rosel study, a randomized clinical trial of radiosurgery (stereotactic radiotherapy)<br />
or surgery in patients with stage A non-small cell lung cancer who are fit to undergo<br />
primary resection. This study is partially supported by the ZonW.<br />
Collaboration with the Dutch Colorectal Cancer Group (DCCG) has resulted in<br />
several large phase III randomized studies in patients with colorectal carcinoma for<br />
which we are the Statistical Center.<br />
After the successfully run CAIRO 1 study, which was published in the Lancet in<br />
2007, the CAIRO 2 study was carried out as rapidly as the first study and in <strong>2008</strong> the<br />
final analysis was completed. This is a randomized study in the same patient group,<br />
advanced colorectal cancer previously untreated patients, studying cetuximab added<br />
to capecitabine, oxaliplatin and bevacizumab. Between June 2005 and December<br />
2006 755 patients were randomized. The publication of the final results has been<br />
accepted in the New England Journal of Medicine.<br />
Multiple studies based on the patient data collected in the CAIRO studies are been<br />
performed and many have already resulted in publications as can be seen from the<br />
publication list.<br />
The CAIRO 3 is the third randomized phase III trial of the DCCG in patients with<br />
advanced colorectal carcinoma. The study compares maintenance treatment with<br />
capecitabine and bevacizumab versus observation after induction treatment with<br />
capecitabine, oxaliplatin, and bevacizumab as first-line treatment. For this study, we<br />
have developed an e-CRFs application. Accrual started in May 2007 and 232 patients<br />
have already been entered into the study.<br />
Publications (continued)<br />
159<br />
BIOMETRICS<br />
Meijnen P, Peterse JL, Antonini N,<br />
Rutgers EJ, van de Vijver MJ.<br />
Immunohistochemical categorisation of<br />
ductal carcinoma in situ of the breast.<br />
Br J Cancer <strong>2008</strong>;98:137-42<br />
Leijte JA, Kirrander P, Antonini N,<br />
Windahl T, Horenblas S. Recurrence<br />
patterns of squamous cell carcinoma of the<br />
penis: recommendations for follow-up based<br />
on a two-centre analysis of 700 patients.<br />
Eur Urol <strong>2008</strong>;54:161-8<br />
Leijte JA, Gallee M, Antonini N,<br />
Horenblas S. Evaluation of current TNM<br />
classification of penile carcinoma. J Urol<br />
<strong>2008</strong>;180:933-8<br />
Kweekel DM, Koopman M, Antonini NF,<br />
Van der Straaten T, Nortier JW,<br />
Gelderblom H, Punt CJ, Guchelaar HJ.<br />
GSTP1 Ile105Val polymorphism correlates<br />
with progression-free survival in MCRC<br />
patients treated with or without irinotecan:<br />
a study of the Dutch Colorectal Cancer<br />
Group. Br J Cancer <strong>2008</strong>;99:1316-21<br />
Kweekel DM, Gelderblom H,<br />
Van der Straaten T, Antonini NF, Punt CJ,<br />
Guchelaar HJ. UGT1A1*28 genotype and<br />
irinotecan dosage in patients with<br />
metastatic colorectal cancer: a Dutch<br />
Colorectal Cancer Group study. Br J Cancer<br />
<strong>2008</strong>;99:275-82<br />
Hoebers FJ, Kartachova M, de Bois J,<br />
van den Brekel MW, van Tinteren H,<br />
van Herk M, Rasch CR, Valdes Olmos RA,<br />
Verheij M. 99mTc Hynic-rh-Annexin V<br />
scintigraphy for in vivo imaging of apoptosis<br />
in patients with head and neck cancer<br />
treated with chemoradiotherapy. Eur J Nucl<br />
Med Mol Imaging <strong>2008</strong>;35:509-18<br />
Hilgers FJ, van den Brekel MW,<br />
van Tinteren H. Views on Acton et al’s<br />
“Investigation of tracheoesophageal voice<br />
prosthesis leakage patterns: patient’s selfreport<br />
versus clinician’s confirmation”.<br />
Head Neck <strong>2008</strong>;30:1660-1<br />
Geurts TW, Balm AJ, van Velthuysen ML,<br />
van Tinteren H, Burgers JA,<br />
van Zandwijk N, Klomp HM. Survival<br />
after surgical resection of pulmonary<br />
metastases and second primary squamous<br />
cell lung carcinomas in head and neck<br />
cancer. Head Neck <strong>2008</strong>;
160<br />
RADIOTHERAPY<br />
Publications (continued)<br />
de Witte MA, Bendle GM, van den Boom MD,<br />
Coccoris M, Schell TD, Tevethia SS,<br />
van Tinteren H, Mesman EM, Song JY,<br />
Schumacher TN. TCR gene therapy of<br />
spontaneous prostate carcinoma requires in<br />
vivo T cell activation. J Immunol<br />
<strong>2008</strong>;181:2563-71<br />
de Boer JP, Hiddink RF, Raderer M,<br />
Antonini N, Aleman BM, Boot H, de Jong D.<br />
Dissemination patterns in non-gastric<br />
MALT lymphoma. Haematologica<br />
<strong>2008</strong>;93:201-6<br />
Buitelaar D, Huitink J, Oldenburg H,<br />
Rutgers E, Schutte P, van Tinteren H.<br />
Field block: an additional technique of<br />
potential value for breast surgery under<br />
general anaesthesia. Eur J Anaesthesiol<br />
<strong>2008</strong>;25:253-5<br />
Buijs C, Rodenhuis S, Seynaeve CM,<br />
van Hoesel QG, van der Wall E, Smit WJ,<br />
Nooij MA, Voest E, Hupperets P,<br />
Ten Vergert EM, van Tinteren H,<br />
Willemse PH, Mourits MJ, Aaronson NK,<br />
Post WJ, de Vries EG. Prospective study of<br />
long-term impact of adjuvant high-dose and<br />
conventional-dose chemotherapy on healthrelated<br />
quality of life. J Clin Oncol<br />
2007;25:5403-9<br />
Bueno-de-Mesquita JM, van Harten WH,<br />
Retel VP, van ‘t Veer LJ, van Dam FS,<br />
Karsenberg K, Douma KF, van Tinteren<br />
H, Peterse JL, Wesseling J, Wu TS,<br />
Atsma D, Rutgers EJ, Brink G, Floore AN,<br />
Glas AM, Roumen RM, Bellot FE,<br />
van Krimpen C, Rodenhuis S,<br />
van de Vijver MJ, Linn SC. Use of 70-gene<br />
signature to predict prognosis of patients<br />
with node-negative breast cancer: a<br />
prospective community-based feasibility<br />
study (RASTER). Lancet Oncol<br />
2007;8:1079-87<br />
Borgemeester MC, van den Brekel MW,<br />
van Tinteren H, Smeele LE, Pameijer FA,<br />
van Velthuysen ML, Balm AJ. Ultrasoundguided<br />
aspiration cytology for the<br />
assessment of the clinically N0 neck: factors<br />
influencing its accuracy. Head Neck<br />
<strong>2008</strong>;30:1505-13<br />
Van Driel WJ, Dalesio OB, Van Der Donk E.<br />
OVHIPEC-studie in combinatie met het<br />
gebruik van het ALEA eCRF-systeem. Ned<br />
Tijdschr Oncol <strong>2008</strong>;5:326-30<br />
In addition, the design of a study after radical resection of liver metastases of<br />
colorectal cancer comparing bevacizumab in combination with capecitabine and<br />
eloxatin vs eloxatin alone as adjuvant treatment (the HEPATICA study) has recently<br />
been modified allowing patients receiving pre-operative chemotherapy, in an attempt<br />
to increase the accrual rate.<br />
We collaborate with medical departments in our Institute in carrying out and<br />
handling the data of large multicenter clinical trials they coordinate, like the<br />
OVHIPEC, which is a study of intraperitoneal chemotherapy and hyperthermia in<br />
ovarian cancer or the Matador study in breast cancer. With the radiotherapy<br />
department we are carrying out a large randomized study in young women with early<br />
breast cancer. More than 1200 patients have been entered by participating centers in<br />
the Netherlands, Belgium and Germany. Tumor material is being collected for<br />
translational research analyses. Cosmetics results are being evaluated with pictures<br />
taken in series.<br />
The statisticians have collaborated with other departments of the institute and other,<br />
academic and not academic, institutes in the region in a variety of studies many of<br />
which resulted in co-authorships.
CLINICAL TRIALS IN THE<br />
NETHERLANDS CANCER INSTITUTE<br />
161<br />
CLINICAL TRIALS<br />
type of nickname title study coordinator phase activated no pts.<br />
cancer study in <strong>NKI</strong>-AVL (closed) in <strong>NKI</strong>-<br />
AVL per<br />
18/12/<br />
ALL SITES<br />
<strong>2008</strong><br />
M03D24 Dose-finding and pharmacokinetic Trial of JHM Schellens I ‘02/04/2003 31<br />
Orally Administered D-24851 to patients with (11/01/<strong>2008</strong>)’<br />
Solid Tumors<br />
M05AZA A phase-I open-label, multicenter study to JHM Schellens I ‘23/05/2005 33<br />
assess the safety, tolerability and pharma- (22/02/<strong>2008</strong>)’<br />
cokinetics of AZD1152 given as as 2 hour<br />
intravenous infusion on 2 dose schedules in<br />
patients with advanced solid malignancies<br />
M05HOP An open label phase I dose escalation study JHM Schellens I ‘28/06/2005 41<br />
of E7080 (01/08/<strong>2008</strong>)’<br />
M05PAR A Phase 1, Pharmacokinetic and Biological JHM Schellens I ‘15/11/2005 29<br />
Evaluation of a Small Molecule Inhibitor of (01/03/<strong>2008</strong>)’<br />
Poly ADP-Ribose Polymerase-1 (PARP-1),<br />
KU-0059436, in patients with advanced tumors<br />
M05PFS A phase 1, open-label, dose escalation study to JHM Schellens I ‘27/09/2005 37<br />
evaluate safety, pharmacokinetics and pharma- (01/10/<strong>2008</strong>)’<br />
codynamics of 2 dosing schedules of<br />
PF 00299804 in patients with advanced<br />
malignant solid tumors<br />
M05RTB A phase III international randomized trial of M Verheij III 10-1-2006 4<br />
single versus multiple fractions for re-irradiation<br />
of painful bone metastases<br />
M06BBC Phase I study to determine the safety, maximum JHM Schellens I ‘19/05/2006 13<br />
tolerated dose and pharmacokinetics of (01/04/<strong>2008</strong>)’<br />
BAY57-9352 in combination with bevacizumab<br />
in subjects with advanced solid tumors<br />
M06BCI Phase I study to determine the safety, maximum JHM Schellens I ‘12/10/2006 12<br />
tolerated dose, pharmacokinetics and biomarker (18/04/<strong>2008</strong>)’<br />
status of BAY 57-9352 in combination with<br />
capecitabine and irinotecan in subjects with<br />
advanced solid tumors<br />
M06OGP Phase I dose escalation study of oral JHM Schellens I 29-8-2006 30<br />
gemcitabine prodrug (LY2334737) alone and in<br />
combination with erlotinib (Tarceva) in patients<br />
with advanced solid tumors<br />
M07CUP CUP-PRINT Molecular profiling as a strategy for the M Kerst other 27-3-<strong>2008</strong> 10<br />
identification of primary solid tumour in<br />
metastatic cancer patients
162<br />
CLINICAL TRIALS<br />
type of nickname title study coordinator phase activated no pts.<br />
cancer study in <strong>NKI</strong>-AVL (closed) in <strong>NKI</strong>-<br />
AVL per<br />
18/12/<br />
<strong>2008</strong><br />
M07ERI An open label parallel group study to explore JHM Schellens other 11-3-<strong>2008</strong> 8<br />
the pharmacokinetics of eribulin mesylate<br />
(E7389) in patients with advanced solid tumors<br />
and normal or reduced hepatic function<br />
according to the Child-Pugh system<br />
M07HHK Cost effectiveness of scalp cooling in the JW Baars other 20-5-<strong>2008</strong> *<br />
prevention of chemotherapy-induced hair loss<br />
M07KUC A phase I, open-label, study to assess the safety JHM Schellens I 25-6-2007 32<br />
and tolerability of KU-0059436 in combination<br />
with Carboplatin, KU-0059436 in combination<br />
with Paclitaxel/Carboplatin doublet and<br />
KU-0059436 in combination with Paclitaxel in<br />
the treatment of patients<br />
M07MKC A phase I dose escalating study evaluating JHM Schellens I 21-2-<strong>2008</strong> 20<br />
MK-1775 in both monotherapy and in<br />
combination with Gemcitabine, Cisplatin or<br />
Carboplatin in adult patients with advanced<br />
solid tumors<br />
M07OTS A phase I study of oral topotecan in subjects JHM Schellens I 18-2-<strong>2008</strong> 3<br />
with cancer and impaired renal function<br />
M07PFU Pharmacogenomic and pharmacokinetic JHM Schellens other 16-5-2007 7<br />
safety and cost-saving analysis in patients<br />
treated with fluoropyrimidines<br />
M07POB The relationship between cost-effectiveness WJ Nooijen other 10-1-2007 *<br />
of blood safety measures and the distribution<br />
of patients receiving blood<br />
M08AZD A Phase I, Open-Label Study to Assess the JHM Schellens I ‘01/07/<strong>2008</strong> 6<br />
Effect of Dosing AZD6244 Hyd Sulfate in the (01/09/<strong>2008</strong>)’<br />
Presence and Absence of Food in Patients<br />
With Advanced Solid Malignancies<br />
M08EVC A phase I/II, non-randomized, multicenter, JHM Schellens I/II ‘26/08/<strong>2008</strong> 1<br />
dose-escalating, two-stage efficacy and (suspended)’<br />
feasablity study of the combination of<br />
everolimus and capecitabine in patients with<br />
advanced malignancies<br />
M08HYT A phase I open-label study of the safety, JHM Schellens I 15-9-<strong>2008</strong> 5<br />
tolerability and pharmacokinetics of two<br />
schedules of oral topotecan in combination<br />
with pazopanib in subjects with advanced<br />
solid tumors<br />
N03JJP Relative bioavailability of Paclitaxel in JHM Schellens Pilot ‘26/01/2004 12<br />
combination with Cyclosporin A. (18/04/<strong>2008</strong>)’<br />
A pilot investigation.<br />
N05GEN Genotyping as tool for dose individualization A Huitema other ‘14/06/2005 9<br />
in high-dose chemotherapy with cyclo- (29/10/<strong>2008</strong>)’<br />
phosphamide, thiotepa and carboplatin<br />
* = not registered at trialbureau
163<br />
CLINICAL TRIALS<br />
type of nickname title study coordinator phase activated no pts.<br />
cancer study in <strong>NKI</strong>-AVL (closed) in <strong>NKI</strong>-<br />
AVL per<br />
18/12/<br />
<strong>2008</strong><br />
N06PFB Pleural fluid bank MM van den Heuvel other 27-9-2006 *<br />
N07DOW Weekly administration of oral Docetaxel in JHM Schellens I 14-11-2007 20<br />
combinaton with Ritonavir<br />
N07NEX Phase I study of gemcitabine and carboplatin JHM Schellens I 29-1-<strong>2008</strong> 10<br />
plus sorafenib in patients with advanced<br />
solid tumors<br />
N08BPM 24 hour ambulatory blood pressure monitoring JHM Schellens other ‘02/09/<strong>2008</strong> 0<br />
in patients with malignancies enrolled in (suspended)’<br />
phase I oncological studies<br />
N08CTC Validation of circulating tumor cells (CTC) JHM Schellens other 31-7-<strong>2008</strong> 8<br />
detection techniques in patients with advanced<br />
solid tumors<br />
N08NPM NVALT Palliative care Protocol on malignant<br />
pleural effusion MM van den Heuvel other 13-3-<strong>2008</strong> 38<br />
N08TKI An open-label, non-randomized, single center, JHM Schellens I ‘26/03/<strong>2008</strong> 13<br />
phase I study to determine the absorption, (03/11/<strong>2008</strong>)’<br />
distribution, metabolism and excretion (ADME)<br />
of TKI258 after a single oral administration of<br />
TKI258 in patients with advanced solid<br />
malignancies<br />
BRAIN / CNS<br />
N05THB Totale hersenbestraling met een eenmalige LGH Dewit other 7-4-2005 35<br />
boost bij patienten met solitaire hersenmetastase<br />
van een epitheliale tumor – een<br />
registratiestudie<br />
BREAST<br />
E10031 SOFT ‘A phase III trial evaluating the role of ovarian GS Sonke III 20-7-2005 4<br />
function suppression and the role of<br />
exemestane as adjuvant therapies for<br />
premenopausal women with endocrine<br />
responsive breast cancer; tamoxifen versus<br />
ovarian function suppression + tamoxifen<br />
versus ovaria’<br />
E10041 MINDACT Micro-array in node-negative disease may Avoid EJTh Rutgers III 2-1-2007 144<br />
Chemotherapy: a prospective randomized study<br />
comparing the 70-gene signature with the<br />
common clinical-pathological criteria in<br />
selecting patients for adjuvant chemotherapy<br />
in node-negative breast cancer<br />
E10981 AMAROS After Mapping of the Axilla: Radiotherapy EJTh Rutgers III 21-12-2000 419<br />
Or Surgery?<br />
* = not registered at trialbureau
164<br />
CLINICAL TRIALS<br />
type of nickname title study coordinator phase activated no pts.<br />
cancer study in <strong>NKI</strong>-AVL (closed) in <strong>NKI</strong>-<br />
AVL per<br />
18/12/<br />
<strong>2008</strong><br />
E22051 SUPREMO SUPREMO, an MRC phase III randomised trial NS Russell III 27-2-2007 11<br />
to assess the role of adjuvant chest wall<br />
irradiation in ‘intermediate risk’ operable breast<br />
cancer following mastectomy<br />
E90061 Multicenter parallel phase II trial of BI 2536 JHM Schellens II ‘16/01/<strong>2008</strong> 0<br />
administered as one hour IV infusion every (01/09/<strong>2008</strong>)’<br />
3 weeks in defined cohorts of patients with<br />
various solid tumors. A new drug screening<br />
program of the EORTC network of core<br />
institutions (NOCI)<br />
M03RBC YOUNG Radiation dose intensity study in breast cancer GMM Bartelink III 29-3-2004 115<br />
BOOST in young women: a randomized phase III trial<br />
of additional dose to the tumor bed<br />
(young boost)<br />
M04MAT MATADOR Microarray Analysis in breast cancer to Tailor SC Linn III 26-4-2004 49<br />
Adjuvant Drugs Or Regimens (MATADOR)<br />
M05BRI BRIGHT Long term risk of breast cancer following NS Russell other 5-1-2006 89<br />
treatment of Hodgkin’s disease<br />
M05HIR HIRISE Hormonal substitution after prophylactic M van Beurden other 31-5-2005 *<br />
adnectomy in women with an increased risk<br />
for breast- and ovarian cancer due to a genetic<br />
predisposition: HIRISE (High-Risk women<br />
and hormonal Substitution Exposure)<br />
M06DAT DATA A prospective randomised open multicentre SC Linn III 1-5-2007 34<br />
phase III study to assess different Durations<br />
of Anastrozol therapy after 2 to 3 years<br />
Tamoxifen as Adjuvant therapy in postmenopausal<br />
women with breast cancer<br />
M06ERB EGF 103659 An open-label expanded access SC Linn IV ‘11/01/2007 20<br />
study of lapatinib and capecitabine therapy in (30/06/<strong>2008</strong>)’<br />
subjects with ErbB2 overexpressing locally<br />
advanced or metastatic breast cancer<br />
M06HER CANDY ‘Prospective randomized pharmacological JHM Schellens III 18-6-2007 10<br />
intervention study; evaluating the effect of<br />
angiotensin II-receptor (AT1) blocker<br />
candesartan versus placebo in prevention of<br />
trastuzumab-associated cardiotoxicity in<br />
patients with primary breast cancer’<br />
M06TM2 TEAM II A randomised, multicenter, prospective, SC Linn III 26-9-2006 28<br />
phase III trial investigating (a) Neoadjuvant<br />
horminal therapy with exemestane for three<br />
versus six months and/or (b) The efficacy and<br />
safety of the addition of ibandronate to adjuvant<br />
hormonal therapy<br />
* = not registered at trialbureau
165<br />
CLINICAL TRIALS<br />
type of nickname title study coordinator phase activated no pts.<br />
cancer study in <strong>NKI</strong>-AVL (closed) in <strong>NKI</strong>-<br />
AVL per<br />
18/12/<br />
<strong>2008</strong><br />
M07ATX ATX Phase II randomized trial of combination SC Linn II 12-12-2007 6<br />
therapy of paclitaxel and bevacizumab versus<br />
paclitaxel, capecitabine and bevacizumab as<br />
first-line treatment for locally recurrent or<br />
metastatic breast cancer patients with<br />
HER2/neu negative tumor<br />
M07CBE Late effects of chemotherapy on brain SSB Schagen other 1-7-<strong>2008</strong> *<br />
functioning in the elderly<br />
M07FEP A randomized double-blind phase 2 trial of GS Sonke II 12-9-2007 7<br />
Fulvestrant plus Enzastaurin versus Fulvestrant<br />
plus placebo in aromatase inhibitor-resistant<br />
metastatic breast cancer<br />
M07LET IDEAL IDEAL: Investigation on the duration of EJTh Rutgers III 16-11-2007 19<br />
extended adjuvant letrozole treatment. An open<br />
lable, randomized phase III trial comparing<br />
2,5 year duration of letrozole (Femara)<br />
treatment with 5 year duration in patients<br />
previously treated for endocrine sensitiv<br />
M07LTB ALTTO ‘A randomised, multi-centre, open-label, GS Sonke III 27-5-<strong>2008</strong> 0<br />
phase III study of adjuvant lapatinib,<br />
trastuzumab, their sequence and their<br />
combination in patients with HER2/ErbB2<br />
positive primary breast cancer; ALTTO study’<br />
M08BCP Prospective and Retrospective register study SC Linn other 27-3-<strong>2008</strong> 27<br />
of the German Adjuvant Cancer Study Group<br />
(GABG) for diagnosis and treatment of breast<br />
cancer in pregnancy<br />
M08CPE A two-arm randomized open label phase 2 study SC Linn II 25-11-<strong>2008</strong> 0<br />
of CP-751,871 in combination with exemestane<br />
versus exemestane alone as first line treatment<br />
for postmenopausal patients with hormone<br />
receptor positive advanced breast cancer<br />
M08PTP A phase Ib open-label, two arm study of i.v. and JHM Schellens I 11-12-<strong>2008</strong> 0<br />
oral panobinostat(LBH589) in combination with<br />
i.v. trastuzumab (Herceptin) and i.v. paclitaxel<br />
as treatment for adult female patients with HER2<br />
overexpressing metastatic breast cancer (MBC)<br />
M08TRA TRAIN Trastuzumab in a neoadjuvant regimen for GS Sonke II 18-9-<strong>2008</strong> 6<br />
Her2+ breast cancer - the TRAIN study<br />
N04POM Tailored preoperative chemotherapy in stage II S Rodenhuis II 21-3-2005 84<br />
or III breast cancer with either a primary tumor<br />
over 3 cm in size or a clinically tumor-positive<br />
axilla<br />
N04RTB Effecten van bestraling op bloedvaten NS Russell Pilot 5-10-2004 70<br />
* = not registered at trialbureau
166<br />
CLINICAL TRIALS<br />
type of nickname title study coordinator phase activated no pts.<br />
cancer study in <strong>NKI</strong>-AVL (closed) in <strong>NKI</strong>-<br />
AVL per<br />
18/12/<br />
<strong>2008</strong><br />
N05ECP Prospectief, dubbelblind, gerandomiseerde JHM Schellens III 10-10-2005 70<br />
placebo gecontroleerde farmacologisch<br />
interventieonderzoek: evaluatie van de<br />
frequentie, duur en intensiteit van opvliegers<br />
na interventie met Venlafaxine (EfexorÆ) en<br />
Clonidine versus placebo<br />
N05MIB 99mTc-methoxyisobutylisonitrile (MIBI) for RA Valdes Olmos other 7-6-2005 *<br />
imaging of apoptosis and prediction of tumour<br />
respons to chemotherapy and/or radiotherapy<br />
in cancer patients<br />
N05RHN Feasibility study of intensive alkylating S Rodenhuis other ‘14/11/2005 13<br />
chemotherapy in residual HER2/neu-negative (18/04/<strong>2008</strong>)’<br />
breast cancer after pre-operative chemotherapy<br />
N05TOM a clinical trial to compare the efficacy of the HJ Teertstra other 7-2-2006 *<br />
lorad tomosynthesis mammography system to<br />
conventional mammography<br />
N06GLB Phase I study of gemcitabine plus lapatinib JHM Schellens I 16-5-2007 8<br />
(GW572016) in women with advanced breast<br />
cancer<br />
N06IAA randomized phase II/III study of intensified S Rodenhuis II/II 8-1-2007 9<br />
alkylating agent chemotherapy with peripheral<br />
blood progenitor cell support in the preoperative<br />
chemotherapy of breast tumors that are<br />
deficient for homologous recombination<br />
N06TRZ The effect of Trastuzumab treatment on B-cell JHM Schellens other 11-12-2006 20<br />
kinase activities assessed by microarray derived<br />
phosphorylation profiles<br />
N06VNI Meting van de functie van de arm en de bijdrage JJ Hage other 26-9-2006 *<br />
daaraan van de grote borstspieren voor en na de<br />
tweezijdige implantatie van een endoprothese<br />
ten behoeve van een borstreconstructie die<br />
direct uitgevoerd aansluitend aan de huidsparende<br />
borstoperatie<br />
N07BOS BOSOM Genetic determinants of survival and second EJTh Rutgers other 12-12-2007 *<br />
breast cancer development in premenopausal<br />
breast cancer patients<br />
N07MAN Randomized phase II/III study of second-line S Rodenhuis II/II 3-4-<strong>2008</strong> 0<br />
endrocrine treatment followed by capecitabine<br />
versus capecitabine followed by endrocrine<br />
treatment in patients with metastatic ER<br />
positive breast cancer<br />
N08AFT AFTER A randomized prospective trial of 2-6 weeks SC Linn II 4-8-<strong>2008</strong> 7<br />
pre-operative hormonal treatment for hormone<br />
receptor postive breast cancer: Anostrozole +/-<br />
fulvestrant or tamoxifen exposure – response in<br />
molecular profile (AFTER-study)<br />
* = not registered at trialbureau
167<br />
CLINICAL TRIALS<br />
type of nickname title study coordinator phase activated no pts.<br />
cancer study in <strong>NKI</strong>-AVL (closed) in <strong>NKI</strong>-<br />
AVL per<br />
18/12/<br />
<strong>2008</strong><br />
N08RMB Tumorresponse monitoring in patients with MTFD Vrancken other 23-9-<strong>2008</strong> 5<br />
breast cancer treated with primary systemic Peeters<br />
therapy: towards predicting response in both<br />
the primary tumor and in axillary lymph nodes<br />
GASTRO INTESTINAL<br />
M05RAX RAX Pre-operative chemoradiotherapy regimen with C Marijnen II 20-3-2006 5<br />
capecitabine and bevacuzimab in locally<br />
advanced rectal cancer. A feasability study (RAX)<br />
M06CRI CRITICS A multicenter randomized phase III trial of M Verheij III 11-1-2007 18<br />
neo-adjuvant chemotherapy followed by surgery<br />
and chemotherapy or by surgery and chemoradiotherapy<br />
in resectable gastric cancer<br />
(CRITICS-study)<br />
M06RCS M1 STUDY A phase II study evaluating short course VJ Verwaal II 22-2-2007 2<br />
radiotherapy, neoadjuvant bevacizumab,<br />
capecitabine and oxaliplatin and radical<br />
resection of primary tumor and metastases<br />
in primary stage IV rectal cancer<br />
M06SCR SCRIPT A multicenter phase III randomised trial A Cats III 9-5-2006 5<br />
comparing total mesorectal excision with<br />
pre-operative radiotherapy with or without<br />
post-operative oral capecitabine in the<br />
treatment of operable primary rectal cancer<br />
M06SUS SUSTENT ‘Surgical gastrojejunostomy or endoscopic A Cats III ‘06/09/2006 0<br />
duodenal stent placement for the palliation (10/05/<strong>2008</strong>)’<br />
of malignant gastric outlet obstruction;<br />
a randomized study. Surgery versus Stent for<br />
malignant gastro-duodenal obstruction;<br />
SUSTENT study’<br />
M07CBO CAIRO3 Maintenance treatment with capecitabine and A Cats III 4-9-2007 2<br />
bevacuzimab versus observation after induction<br />
treatment with capecitabine, oxaliplatin and<br />
bevacuzimab as first-line treatment in patients<br />
with advanced colorectal carcinoma,<br />
a randomised phase III study<br />
M07DDO D-DOCCS Development of a docetaxel, oxaliplatin, A Cats I 23-7-2007 9<br />
capecitabine combination schedule in patients<br />
with advanced cancer of the stomach or the<br />
gastro-esophageal junction, a phase I study<br />
M07HBT HerBerT Feasibility study of external beam radiation C Marijnen I/II 25-7-2007 9<br />
therapy followed by high-dose rate endorectal<br />
brachytherapy (HDBRT) in inoperable rectal<br />
cancer patients
168<br />
CLINICAL TRIALS<br />
type of nickname title study coordinator phase activated no pts.<br />
cancer study in <strong>NKI</strong>-AVL (closed) in <strong>NKI</strong>-<br />
AVL per<br />
18/12/<br />
<strong>2008</strong><br />
M07HEP HEPATICA A randomized two arm phase III study in patients A Cats III 28-4-<strong>2008</strong> 5<br />
post radical resection of liver metastasis of<br />
colorectal cancer to investigate Bevacizumab<br />
(q3w) in combination with capecitabine plus<br />
Eloxatin (XELOX) (q3w) as adjuvant<br />
chemotherapy versus XELOX alone (q3w)<br />
M07NAR NARCIS ‘Neo-Aduvant Radiotherapy-Chemotherapy E Jansen I/II 28-1-<strong>2008</strong> 0<br />
In Stomach Cancer. Induction therapy with<br />
carboplatin, paclitaxel and radiotherapy in<br />
patients with locally advanced gastric cancer.<br />
The ‘‘NARCIS’’ study’<br />
M07RBV ‘Clinical pilot study: radiolabeled bevacizumab TJM Ruers I 12-2-<strong>2008</strong> *<br />
as a tracer of VEGF expression in patients with<br />
colorectal liver metastases.<br />
Selecting patients who may benefit from<br />
anti-VEGF therapy and visualizing the response’<br />
M07RCM SILENT Simultaneous integrated boost radiation A Cats I 31-1-<strong>2008</strong> 6<br />
therapy with concomitant capecitabine and<br />
mitomycin-C chemotherapy for locally advanced<br />
anal carcinoma<br />
M08ACL Accelerated growth of synchronous colorectal TJM Ruers II 21-2-<strong>2008</strong> 4<br />
liver metastases: effects of neo-adjuvant therapy<br />
M08DCS Tumor destruction and DC activation in situ: TJM Ruers Pilot 18-9-<strong>2008</strong> *<br />
towards in vivo loaded DC vaccines<br />
M08GPO Genetic and protein profiling in patients with JW van Sandick other 1-9-<strong>2008</strong> 3<br />
oesophageal cancer<br />
N02ECC A single institution phase II study of ECC H Boot II 29-8-2002 133<br />
(Epirubicin, Cisplatin, Capecitabin) in locally<br />
advanced or metastatic gastric cancer and<br />
adenocarcinoma of the oesophago-gastric<br />
junction and distal oesophagus<br />
N02RCA Post operative chemo-radiotherapy after H Boot I/II ‘19/12/2002 70<br />
resection of gastric and esophageal cancer (11/09/<strong>2008</strong>)’<br />
N05GEA Saving the gastro-epiploic artery at VJ Verwaal III 21-3-2006 37<br />
omentectomy and the effect on post-surgical<br />
recovering gastric function<br />
N05MCT a phase II study of treatment with the ME Smits other 5-12-2005 3<br />
combination of unlabelled (cold) and<br />
radioactive (hot) meta-iodobenzylguanidin<br />
(MIBG) in metastatic carcinoid tumours<br />
N05STP Serum and tissue protein profiling and tumour ME Smits other 19-1-2006 133<br />
genetic analysis in patients with potential<br />
premalignant conditions or colorectal cancer<br />
* = not registered at trialbureau
169<br />
CLINICAL TRIALS<br />
type of nickname title study coordinator phase activated no pts.<br />
cancer study in <strong>NKI</strong>-AVL (closed) in <strong>NKI</strong>-<br />
AVL per<br />
18/12/<br />
<strong>2008</strong><br />
N06DCM Feasibility of the analysis of platinum-DNA JHM Schellens other 12-12-2006 4<br />
adducts in tumour tissue in cisplatinum treated<br />
with advanced gastric cancer<br />
GYNAECOLOGICAL<br />
E55041 A randomised, multicentre, phase III study of WJ van Driel III ‘25/09/2006 9<br />
Erlotinib vs observation in patients with no (20/02/<strong>2008</strong>)’<br />
evidence of disease progression after first line,<br />
platinum-based chemotherapy for high-risk<br />
stage I and stage II-IV ovarian epithelial,<br />
primary peritoneal, or fallo<br />
M05HIR HIRISE Hormonal substitution after prophylactic M van Beurden other 31-5-2005 *<br />
adnectomy in women with an increased risk for<br />
breast- and ovarian cancer due to a genetic<br />
predisposition: HIRISE (High-Risk women and<br />
hormonal Substitution Exposure)<br />
M05PPO Proteomic patterns in blood and tissue of WJ van Driel other 12-1-2006 *<br />
ovarian cancer patients<br />
M05SNV GROINSS-V II GROningen International study on sentinel WJ van Driel other 26-3-2007 9<br />
nodes in vulvar cancer (GROINSS-V) II<br />
M06HRT NOVARIA The effect of hormonal replacement therapy on CM Korse other 25-9-2006 48<br />
menopausal complaints related to biochemical<br />
changes in surgically and naturally postmenopausal<br />
women. A prospective observational<br />
comparative study<br />
M06OVH OVHIPEC Phase III randomised clinical trial for stage III WJ van Driel III 4-1-2006 15<br />
ovarian carcinoma randomising between<br />
secondary debulking surgery with or without<br />
hyperthermic intraperitoneal chemotherapy<br />
(OVHIPEC-1)<br />
M06RTE PORTEC-3 Randomised phase III trial comparing B van Triest III 28-3-2007 1<br />
concurrent chemoradiation and adjuvant<br />
chemotherapy with pelvic radiation alone in<br />
high risk and advanced stage endometrial<br />
carcinoma: PORTEC-3<br />
M07OVE OVERT-1 A phase II, double blind placebo controlled S Rodenhuis II 1-7-<strong>2008</strong> 5<br />
multicenter randomised study of AZD0530 in<br />
patients with ovarian cancer sensitive to<br />
platinum based chemotherapy (OVERT-1)<br />
M07RCV Phase II study of definitive radiochemotherapy WJ van Driel II 26-6-2007 0<br />
for locally advanced squamous cell cancer of<br />
the vulva: an efficacy study<br />
* = not registered at trialbureau
170<br />
CLINICAL TRIALS<br />
type of nickname title study coordinator phase activated no pts.<br />
cancer study in <strong>NKI</strong>-AVL (closed) in <strong>NKI</strong>-<br />
AVL per<br />
18/12/<br />
<strong>2008</strong><br />
N05CGO Randomized clinical pharmacological dose- JHM Schellens I 8-9-2005 18<br />
escalation study to explore both safety and<br />
preliminary efficacy and pharmaco-kinetics,<br />
-dynamics and -genomics of fixed dose rate<br />
gemcitabine and 30-minute standard<br />
gemcitabine infusion<br />
HEAD AND NECK<br />
E90061 Multicenter parallel phase II trial of BI 2536 JHM Schellens II ‘16/01/<strong>2008</strong><br />
administered as one hour IV infusion every (01/09/<strong>2008</strong>)’ 0<br />
3 weeks in defined cohorts of patients with<br />
various solid tumors. A new drug screening<br />
program of the EORTC network of core<br />
institutions (NOCI)<br />
M02STA The effect of a statin on the progression of the FJP Hoebers III 28-1-2003 79<br />
intima-media thickness induced by radiotherapy<br />
M05PET FDG-PET for avoidance of futile direct M van de Brekel other 11-5-2005 *<br />
laryngoscopies under general anaesthesia with<br />
taking of biopsies in patients with suspicion on<br />
recurrent laryngeal carcinoma after radiotherapy<br />
M07CMD Treatment of myogenic cranio mandibular FJM Hilgers III 29-8-2007 *<br />
dysfunction (CMD): a prospective randomised<br />
clinical trial, comparing a mechanical stretching<br />
device (Therabite) with standard physiotherapy<br />
M07LRC A randomized, double blind, placebo controlled, JP de Boer II 10-6-<strong>2008</strong> 5<br />
multicentre phase II study of oral lapatinib in<br />
combination with concurrent radiotherapy and<br />
cisplatin versus radiotherapy and cisplatin alone,<br />
in subjects with stage III and IVA,B squamous<br />
cell carcinoma<br />
M07MSH Molecular staging of surgically treated head and M van de Brekel other 12-2-<strong>2008</strong> *<br />
neck cancer patients: towards rationalized<br />
postoperative clinical management<br />
M08SNU SNUS Ultrasound guided fine needle aspiration M van de Brekel other 26-6-<strong>2008</strong> 1<br />
cytology and sentinel node biopsy in the<br />
detection of occult lymph node metastases of<br />
early oral and oropharyngeal cancer<br />
N02SNL Early detection of lymph node metastasis of PJFM Lohuis Pilot 3-4-2002 8<br />
squamous cell carcinoma of the larynx with<br />
lymphatic mapping and sentinel node biopsy<br />
N04LFV Longfunctie na toediening van inhalatie- FJM Hilgers III 7-2-2005 *<br />
medicatie (salbutamol-ipratropiumbromide<br />
dosisaerosol) met en zonder voorzetkamer bij<br />
gelaryngectomeerden<br />
N04RTB Effecten van bestraling op bloedvaten NS Russell Pilot 5-10-2004 70<br />
* = not registered at trialbureau
171<br />
CLINICAL TRIALS<br />
type of nickname title study coordinator phase activated no pts.<br />
cancer study in <strong>NKI</strong>-AVL (closed) in <strong>NKI</strong>-<br />
AVL per<br />
18/12/<br />
<strong>2008</strong><br />
N05HME De korte termijn invloed van een Heat and JK Zuur Pilot 1-9-2005 *<br />
Moisture Exchanger op de endotracheale<br />
temperatuur en luchtvochtigheid bij<br />
gelaryngectomeerden<br />
N05MIB 99mTc-methoxyisobutylisonitrile (MIBI) for RA Valdes Olmos other 7-6-2005 *<br />
imaging of apoptosis and prediction of tumour<br />
respons to chemotherapy and/or radiotherapy<br />
in cancer patients<br />
N05TSP Prevention of trismus, swallowing and speech FJM Hilgers other 14-11-2005 *<br />
problems in patients treated with chemoradiation<br />
for advanced head and neck cancer<br />
N07HME A prospective clinical feasibility study of a new FJM Hilgers other 16-5-2007 *<br />
heat and moisture exchanger with bacteria, virus<br />
and pollen filtration capabilities (HME-F)<br />
N07MCP Microcirculatory changes during photodynamic MP Copper Pilot 21-5-2007 *<br />
therapy (PDT) in squamous cell carcinoma of<br />
the oral cavity and oropharynx<br />
N07SHT Subglottic humidity and temperature in nasal RJ Scheenstra other 14-5-2007 *<br />
breathing and tracheotomy breathing with<br />
upper airway occlusion<br />
N07VEG Development of a next generation indwelling FJM Hilgers I/II ‘28/03/2007 *<br />
Provox voice rehabilitation system (Vega) (25/09/<strong>2008</strong>)’<br />
N07VIN Transoral resection of stage I-II carcinomas of M van de Brekel II 12-3-2007 5<br />
the oropharynx by robot-assisted surgery:<br />
a feasability study<br />
N08HHF Validation of hypoxia imaging of cancer in the WV Vogel II 22-10-<strong>2008</strong> *<br />
head and neck area with FAZA-PET<br />
LEUKAEMIA / MDS<br />
M05H65 HOVON 65 A randomized phase III study on the effect of JW Baars III ‘03/10/2005 4<br />
Bortezomib combined with Adriamycin, (16/05/<strong>2008</strong>)’<br />
Dexamethason (AD) for induction treatment,<br />
followed by High Dose Melphalan and<br />
Bortezomib alone during maintenance in<br />
patients with multiple myeloma<br />
M05H68 HOVON 68 A randomized phase III study in previously M Kerst III 21-3-2006 2<br />
untreated patients with biological high-risk CLL:<br />
fludarabine + cyclophophamide (FC) versus<br />
FC + low dose alemtuzumab<br />
LUNG<br />
E08062 Randomized phase II study of amrubicin as<br />
single agent or in combination with cisplatin<br />
versus etoposide-cisplatin as first-line treatment<br />
in patients with extensive stage SCLC (ES)<br />
P Baas II 28-2-<strong>2008</strong> 0<br />
* = not registered at trialbureau
172<br />
CLINICAL TRIALS<br />
type of nickname title study coordinator phase activated no pts.<br />
cancer study in <strong>NKI</strong>-AVL (closed) in <strong>NKI</strong>-<br />
AVL per<br />
18/12/<br />
<strong>2008</strong><br />
M03THA NVALT 5 Phase III trial of the antiangiogenic agent P Baas III 18-2-2004 86<br />
Thalidomide in patients with malignant pleural<br />
mesothelioma after first line chemotherapy<br />
(NVALT5)<br />
M05ATD Accurate target definition of non-small cell lung J Stroom other 5-1-2006 *<br />
tumors using pathology-validated PET and CT<br />
imaging for the optimization of radiation<br />
treatment<br />
M05GCP Pharmacogenomic and pharmacokinetic study JHM Schellens other 13-9-2005 124<br />
in patients with advanced non small-cell lung<br />
cancer (NSCLC) treated with first-line<br />
gemcitabine / platinum combination<br />
chemotherapy<br />
M06NEL Efficacy of neoadjuvant erlotinib in patients with HM Klomp I/II 8-11-2006 27<br />
clinical stage I/II non-small cell lung cancer<br />
(NSCLC)<br />
M07CCL RADITUX Open-label, randomised multi-center study MM van den Heuvel I/II 13-3-<strong>2008</strong> 15<br />
investigating Cetuximab, in combination with<br />
concurrent chemo-radiotherapy in locally<br />
advanced non-small cell lung carcinoma<br />
M07ESU Randomized double blind phase 2 study of P Baas II 29-5-2007 9<br />
erlotinib with or without SU11248 in the treatment<br />
of metastatic non small cell lung cancer<br />
M07OSM A phase III randomized, double blind, placebo P Baas II/II 9-10-2007 22<br />
controlled trial of oral suberoylanilide<br />
hydroxamic acid (L-001079038) in patients with<br />
advanced malignant pleural mesothelioma<br />
previously treated with systemic chemotherapy<br />
M07STN START A multi-center phase III randomized, double- MM van den Heuvel III 13-12-2007 3<br />
blind placebo-controlled study of the cancer<br />
vaccine Stimuvax (L-BLP25 or BLP25 liposome<br />
vaccine) in non-small cell lung cancer (NSCLC)<br />
subjects with unresectable stage III disease<br />
N00ALF Endoscopic detection op pre-neoplastic lesions P Baas Pilot 30-6-2000 41<br />
and carcinoma in the bronchial tree with deltaaminolevunic<br />
acid fluorescence<br />
N04LSN Feasibility of lymphoscintigraphy and sentinel HM Klomp Pilot 8-9-2004 5<br />
node biopsy in patients with non-small lung<br />
cancer<br />
N05MIB 99mTc-methoxyisobutylisonitrile (MIBI) for RA Valdes Olmos other 7-6-2005 *<br />
imaging of apoptosis and prediction of tumour<br />
respons to chemotherapy and/or radiotherapy<br />
in cancer patients<br />
* = not registered at trialbureau
173<br />
CLINICAL TRIALS<br />
type of nickname title study coordinator phase activated no pts.<br />
cancer study in <strong>NKI</strong>-AVL (closed) in <strong>NKI</strong>-<br />
AVL per<br />
18/12/<br />
<strong>2008</strong><br />
N07NRA A phase I/II study with NAMI-A, a Novel JHM Schellens I/II 31-7-<strong>2008</strong> 3<br />
Ruthenium Anticancer Agent, and gemcitabine<br />
combination second-line therapy in NSCLC<br />
patients<br />
LYMPHOMA - HODGKIN’S DISEASE<br />
E20012 BEACOPP (4 cycle’s escalated + 4 cycle’s JW Baars III 29-3-2004 1<br />
baseline) vs ABVD (8 cycle’s) in Stage III & IV<br />
Hodgkin’s Lymphoma.<br />
E20051 The H10 EORTC/GELA randomized Intergroup JW Baars III 23-11-2006 6<br />
trial on early PET-scan guided treatment<br />
adaptation versus standard combined modality<br />
treatment in patients with supradiaphragmatic<br />
stage I/II Hodgkin’s lymphoma<br />
LYMPHOMA - NON-HODGKIN’S<br />
E20971 A phase III study on low-dose totasl body RLM Haas III 2-2-2004 6<br />
irradiation and involved field radiotherapy in<br />
patients with localized, stages I and II, low<br />
grade non-hodgkin’s lymphoma<br />
M05H55 Efficacy of maintenance therapy with rituximab JP de Boer III 20-7-2005 1<br />
after induction therapy (R-CHOP versus R-FC)<br />
for elderly patients with mantle cell lymphoma<br />
not suitable for autologous stem cell<br />
transplantation<br />
M05H63 HOVON 63 Randomized phase III study of Rituximab with JW Baars III ‘21/03/2006 1<br />
intensified CHOP chemotherapy (R-iCHOP-14) (25/03/<strong>2008</strong>)’<br />
versus Rituximab with High-Dose Sequential<br />
Therapy and Autologous Stem Cell Transplantation<br />
(R-HDT+ASCT) in adult patients (18-65years)<br />
M06CMV Cytomegalovirus (CMV) – specific T cell M Kerst other 28-9-2006 *<br />
immunity in B cell malignancies<br />
M06H77 HOVON 77 Efficacy and safety of a single dose of JW Baars II 23-5-2006 0<br />
14.8 MBq/kg 90Y-ibritumomab tiuxetan (Zevalin)<br />
in elderly patient with diffuse large B-cell<br />
lymphoma and FDG-PET positive partial<br />
remission following first-line R-CHOP therapy.<br />
A phase II clinical trial<br />
M07H80 HOVON 80 Phase II study on the feasability and efficacy of JW Baars II 10-9-2007 1<br />
R-DHAP-MTX combined with i.t. rituximab and<br />
autologous SCT in patients with a recurrent<br />
aggressive B-cell NHL with CNS localisation<br />
M07H84 HOVON 84 Randomized phase III study on the effect of JW Baars III 22-1-<strong>2008</strong> 0<br />
early intensification of rituximab in combination<br />
with 2-weekly CHOP chemotherapy followed by<br />
rituximab maintenance in elderly patients with<br />
DLBCL<br />
* = not registered at trialbureau
174<br />
CLINICAL TRIALS<br />
type of nickname title study coordinator phase activated no pts.<br />
cancer study in <strong>NKI</strong>-AVL (closed) in <strong>NKI</strong>-<br />
AVL per<br />
18/12/<br />
<strong>2008</strong><br />
N03OFP ‘A phase II study of eradication therapy with JP de Boer II 6-10-2003 8<br />
additional oral fludarabine in t(11;18) positive<br />
gastric MALT lymphoma’<br />
N03RIM Radioimmunotherapy (131I-antiCD20 monoclonal JW Baars Pilot 25-6-2003 1<br />
antibody) as consolidation treatment after<br />
remission induction for patients with relapsed<br />
or refractory CD20+ B-cell NHL<br />
N05MIB 99mTc-methoxyisobutylisonitrile (MIBI) for RA Valdes Olmos other 7-6-2005 *<br />
imaging of apoptosis and prediction of tumour<br />
respons to chemotherapy and/or radiotherapy<br />
in cancer patients<br />
N07RIT Determination of the pharmacokinet ics of JW Baars 14-4-<strong>2008</strong> 22<br />
rituximab and Ab to rituximab<br />
N08IRP Iodine-124-rituximab positron emission WV Vogel Pilot 17-12-<strong>2008</strong> *<br />
tomography (IR-PET) for molecular imaging of<br />
CD20 expression<br />
MELANOMA / SKIN<br />
M05MSL MSLT-II A phase III multicenter randomized trial of OE Nieweg III 11-9-2006 3<br />
sentinel lymphadenectomy and complete<br />
lymph node dissection versus sentinel<br />
lymphadenectomy alone in cutaneous melanoma<br />
patients with molecular or histopathological<br />
evidence of metastases in the sentinel node<br />
M06MDX A randomized, double-blind, multicenter study JBAG Haanen III ‘13/09/2006 17<br />
comparing MDX-010 monotherapy, MDX-010 in (22/07/<strong>2008</strong>)’<br />
combinatio with a melanoma vaccine monotherapy<br />
in HLA-A*0201-positive patients with<br />
previously treated unresectable stage III or IV<br />
melanoma<br />
N03CYL Pilot study of topical application of salicylate in R Oosterkamp Pilot ‘26/06/2003 5<br />
familial cylindromas (30/01/<strong>2008</strong>)’<br />
N03LAM Longitudinal analysis of melanoma-specific JBAG Haanen II 22-8-2003 3<br />
immunity in stage III and IV melanoma patients<br />
N06TIS ‘Integrated analyses of melanoma-T cell JBAG Haanen other 29-8-2006 11<br />
interactions; relevance for immunotherapy’<br />
MISCELLANEOUS<br />
M06PRO PROTECT The efficacy of therapeutic compression hoses M Stuiver other ‘05/09/2006 59<br />
for prevention of lymphedema after inguinal (11/09/<strong>2008</strong>)’<br />
lymph node dissection in cancer patients<br />
N01RIT Identifications of molecular mechanisms NS Russell Pilot 7-5-2002 30<br />
involved in radiation-induced telangiectasia<br />
* = not registered at trialbureau
175<br />
CLINICAL TRIALS<br />
type of nickname title study coordinator phase activated no pts.<br />
cancer study in <strong>NKI</strong>-AVL (closed) in <strong>NKI</strong>-<br />
AVL per<br />
18/12/<br />
<strong>2008</strong><br />
N03THY Therapeutic management of thymoma and LGH Dewit Pilot 25-11-2003 15<br />
thymic carcinoma: – a prospective registration<br />
study based on preoperative risk assessment<br />
of local failure.<br />
N05CHO The efficacy of cordotomies in patients with A Lukas II 17-1-2006 *<br />
chest pain due to primariy localized malignancies<br />
of the chest<br />
SOFT TISSUE / OSTEOSARCOMA<br />
E62012 Randomised trial of single agent doxorubicin S Rodenhuis III 9-7-2003 19<br />
versus doxorubicin plus ifosfamide in the first<br />
line treatment of advanced or metastatic soft<br />
tissue sarcoma<br />
E62024 Intermediate and high risk localized, completely S Rodenhuis III ‘25/11/2004 13<br />
resected, gastrointestinal stromal tumors (GIST) (20/10/<strong>2008</strong>)’<br />
expressing KIT receptor:a controlled randomized<br />
trial on adjuvant Imatinib mesylate (Glivec)<br />
versus no further therapy after complete surgery<br />
E62061 Randomised phase II study of brostallicin M Kerst II ‘12/03/2007 7<br />
(PNU-166196A) versus doxorubicin as first line (07/08/<strong>2008</strong>)’<br />
chemotherapy in patients with advanced or<br />
metastatic soft tissue sarcoma<br />
E62072 PALETTE A randomized double blind phase III trial of M Kerst III 18-11-<strong>2008</strong> 0<br />
Pazopanib versus placebo in patients with soft<br />
tissue sarcoma whose disease has progressed<br />
during or following prior therapy<br />
M01EUR EURO-EWING EURO-E.W.I.N.G. 99. EUROpean Ewing tumor S Rodenhuis III ‘18/09/2001 13<br />
Working Initiative of National Groups (04/12/<strong>2008</strong>)’<br />
M01ROS Phase II study of rosiglitazone in advanced S Rodenhuis II 24-8-2001 5<br />
liposarcoma<br />
URO-GENITAL<br />
E30983 Randomized phase II/III study of Taxol-BEP M Kerst II/II 7-9-1999 4<br />
versus BEP in patients with intermediate<br />
prognosis germ cell cancer<br />
E30986 Randomized phase II/III study assessing JH Schornagel II/II ‘07/05/2002 21<br />
Gemcitabine/Carboplatin and Methotrexate/ (17/03/<strong>2008</strong>)’<br />
Carboplatin/Vinblastine in previously untreated<br />
patients with advanced urothelial cancer<br />
ineligible for Cisplatinum based chemotherapy<br />
E30994 Randomized phase III trial comparing immediate M Kerst III ‘28/01/2003 26<br />
versus deferred chemotherapy after radical (25/07/<strong>2008</strong>)’<br />
cystectomy in patients with pT3-pT4, and/or<br />
N+ M0 transitional cell carcinoma (TCC) of the<br />
bladder.<br />
* = not registered at trialbureau
176<br />
CLINICAL TRIALS<br />
type of nickname title study coordinator phase activated no pts.<br />
cancer study in <strong>NKI</strong>-AVL (closed) in <strong>NKI</strong>-<br />
AVL per<br />
18/12/<br />
<strong>2008</strong><br />
M00LMT Identification of occult lymph node metastases S Horenblas Pilot 14-11-2000 *<br />
in testicular cancer to select patients for<br />
adjuvant treatment. Feasability of a laparoscopic<br />
selective retroperitoneal lymphadenectomy.<br />
M06HFP HYPRO Hypofractionated irradiation for prostate cancer: F Pos III 8-3-2007 87<br />
a randomized multicenterphase III study<br />
M06LAN Late Adverse effects in Dutch testicular cancer M Kerst other 12-2-2007 *<br />
survivors: a Nationwide case-control study on<br />
Cardiovascular Events<br />
M06SIL Phase I dose-escalation trial for the combination JBAG Haanen I 20-11-2006 3<br />
of sorafenib with interleukin-2 treatment in<br />
patients with clear cell renal carcinoma<br />
M07ILS An open label, dose escalation safety and JBAG Haanen I/II ‘23/07/2007 2<br />
tolerability trial of the combination of s.c. (18/04/<strong>2008</strong>)’<br />
recombinant human IL-21 (rIL-21) and sunitinib<br />
(phase I) followed by an open label stratified<br />
randomized 2-arm trial of rIL-21 + sunitinib<br />
versus sunitinib alone (phas<br />
M07PGC Phase II trial of paclitaxel, gemcitabine and M Kerst II 8-1-<strong>2008</strong> 0<br />
cisplatin in patients with relapsing germ cell<br />
cancer after first line chemotherapy<br />
M08ZDP ENTHUSE M1 A phase III randomized double blind study to HG van der Poel III 8-9-<strong>2008</strong> 4<br />
assess the efficacy and safety of 10 mg ZD4054<br />
versus placebo in patients with hormone<br />
resistant prostate cancer and bone metastasis<br />
who are painfree and mildly symptomatic<br />
M94SAL Salvage regimen incorporating repeated ablative S Rodenhuis II 4-7-1994 24<br />
chemotherapy with autologous PSCT, a phase II<br />
study<br />
N06MPM Samarium-153-EDTMP (QUADRIMET) versus HG van der Poel II 14-3-2007 7<br />
docetaxel for multiple painful oseous<br />
metastases in prostate cancer<br />
N06SNR Feasability of lymphoscintigraphy and sentinel<br />
node biopsy in patients with renal cell carcinoma A Bex other 9-10-2007 *<br />
N06SUN Sunitinib prior to nephrectomy in patients with A Bex II 16-5-2007 10<br />
metastatic renal cell carcinoma and the primary<br />
in situ<br />
N07TAP TAP-block Evaluation of the Transversus Abdominis Plane – DR Buitelaar other 8-1-<strong>2008</strong> 84<br />
block (TAP-block) in preventing painful urine<br />
bladder spasm in patients undergoing Robot<br />
Assisted Laparoscopic Radical Prostatectomy<br />
under general anesthesia, a prospective<br />
randomized controlled trial<br />
* = not registered at trialbureau
INVITED SPEAKERS <strong>2008</strong><br />
Fabrizio d’Adda di Fagagna, Milan, Italy<br />
Molecular bases of cellular senescence<br />
Bruce Alberts, San Francisco, CA, USA<br />
Biology past and biology future: where have we been and<br />
where are we going?<br />
Haico van Attikum, Leiden, The Netherlands<br />
Chromatin and the DNA damage response<br />
Bradley Bernstein, Boston, MA, USA<br />
Genome-wide maps of chromatin state in pluripotent<br />
and multipotent cells<br />
Eveline Bleiker, Amsterdam, The Netherlands<br />
Psycho-oncogenetics: opportunities for a center of<br />
excellence<br />
Thijn Brummelkamp, Boston, MA, USA<br />
Organ size, stem cells and loss of function genetics in<br />
mammals<br />
Luc Brunsveld, Dortmund, Germany<br />
Chemical biology approaches for the nuclear receptor –<br />
cofactor interaction<br />
Dmitry Bulavin, Singapore<br />
Wip1 phosphatase to trace adult stem cells and wip-out<br />
diseases<br />
Harmen Bussemaker, New York, NY, USA<br />
Predicting expression from sequence using data-driven<br />
models of (post-)transcriptional networks<br />
Anthony Chalmers, Brighton, UK<br />
Radiation plus PARP inhibition in the treatment of high<br />
grade glioma: enhancing the therapeutic ratio<br />
Gerhard Christofori, Basel, Switserland<br />
Distinct mechanisms of tumor cell invasion and<br />
metastasis<br />
Sarah Darby, Oxford, UK<br />
Effects of breast radiotherapy on cardiovascular disease<br />
Heran Darwin, New York, NY, USA<br />
Proteasomes, a new ubiquitin-like protein, and<br />
tuberculosis<br />
Marileen Dogterom, Amsterdam, The Netherlands<br />
Dynamics and force generation of cytoskeletal systems<br />
in vitro and in vivo<br />
Anindya Dutta, Charlottesville, VA, USA<br />
Genomic instability of mammalian cells from disorders<br />
of replication<br />
177<br />
INVITED SPEAKERS<br />
Ger van den Engh, Seattle, WA, USA<br />
Cell sorters for advanced research: how standard flow<br />
cytometers can be adapted for microbiology, large, fragile<br />
cell analysis and single-cell genomics<br />
Ype Elgersma, Rotterdam, The Netherlands<br />
Oncogenes on my mind: the role of the RAS/ERK/<br />
mTOR pathways in neuronal function<br />
Emer S. Ferro, São Paulo, Brazil<br />
Intracellular peptides as natural regulators of cell<br />
signaling<br />
Alan Fersht, Cambridge, UK<br />
The tumour suppressor p53: structure, function-rescue<br />
Pier Paolo di Fiore, Milan, Italy<br />
Integration of EGFR signaling and traffic<br />
Judy Garber, Boston, MA, USA<br />
Triple negative breast cancer: An update<br />
Kenneth Gilhuijs, Amsterdam, The Netherlands<br />
Multi-modality image analysis to guide diagnosis and<br />
therapy of cancer<br />
Ernesto Guccione, Milan, Italy<br />
Histone Methylation: Regulator of chromatin activity<br />
and transcription factor binding<br />
Peter van Haastert, Groningen, The Netherlands<br />
Navigation of amoeboid cells in chemical gradients<br />
William Hahn, Boston, MA, USA<br />
Functional genomics, experimental models and cancer<br />
genes<br />
Benjamin Haibe-Kains, Brussels, Belgium<br />
Gene modules, breast cancer subtypes and prognosis<br />
Samir Hanash, Seattle, WA, USA<br />
Mining the cancer proteome for diagnostic markers and<br />
therapeutic targets<br />
Kristian Helin, Copenhagen, Denmark<br />
Epigenetics, stem cells and cancer<br />
Thomas Helleday, Oxford, UK<br />
Homologous recombination in mammalian cells<br />
Jeroen den Hertog, Utrecht, The Netherlands<br />
Phosphotyrosine signaling in gastrulation cell<br />
movements: modeling Noonan syndrome in zebrafish<br />
Ian Hickson, Oxford, UK<br />
Genome instability and cancer: lessons from analysis of<br />
Bloom’s syndrome
178<br />
INVITED SPEAKERS<br />
Tan Ince, Boston, MA, USA<br />
Cell of origin and neoplastic phenotype<br />
Metello Innocenti, Frankfurt, Germany<br />
Actin dynamics in membrane protrusion and<br />
endocytosis<br />
Stefan Jakobs, Goettingen, Germany<br />
Fluorescence nanoscopy: Taking a closer look at cellular<br />
structures<br />
Stefan Jentsch, Martinsried, Germany<br />
Role of Ubiquitin and Sumo in DNA transactions<br />
Joseph Jiricny, Zürich, Switzerland<br />
When DNA repair kills: cytotoxic processing of<br />
methylation damage and 5-fluorouracil<br />
Jagath Reddy Junutula, San Francisco, CA, USA<br />
Targeted cancer chemotherapy: new insights into<br />
antibody drug conjugates<br />
Sander van Kasteren, Dundee, Scotland<br />
Imaging inflammation – novel probes for detecting<br />
vascular selectin upregulation<br />
Patrick Kemmeren, Utrecht, The Netherlands<br />
An accurate physical interactome map of saccharomyces<br />
cerevisiae<br />
Batsheva Kerem, Jerusalem, Israel<br />
Regulation of chromosomal instability in response to<br />
replication stress<br />
Nevan Krogan, San Francisco, CA, USA<br />
Biology without bias: Functional insights from genetic<br />
and physical interaction maps<br />
Peter Kuhn, La Jolla, CA, USA<br />
Circulating tumor cells in human blood – a point of<br />
entry for cancer diagnosis, prognosis and therapy<br />
management<br />
Alan Lehmann, Brighton, UK<br />
Replication of DNA damage and translesion synthesis<br />
Susanne Lens, Utrecht, The Netherlands<br />
The chromosomal passenger complex and the control of<br />
chromosomal stability<br />
Daniel Low, St. Louis, MO, USA<br />
Modeling human breathing motion for radiation<br />
therapy and dose response assessments<br />
Umar Mahmood, Charlestown, MA, USA<br />
Oncological applications of optical molecular imaging<br />
Tomi Mäkelä, Helsinki, Finland<br />
Mesenchymal LKB1 is required for suppression of<br />
polyposis and TGFß signaling<br />
Elisabetta Marangoni, Paris, France<br />
Cancer stem cells and residual disease<br />
Eric May, Baden-Baden, Germany<br />
Communicating difficult topics effectively<br />
Martin McMahon, San Francisco, CA, USA<br />
BRaf-induced tumor initiation, progression and<br />
senescence in mouse models of lung cancer and<br />
melanoma<br />
Tom Muir, New York, NY, USA<br />
The histone code explored through protein<br />
semisynthesis<br />
Alexander van Oudenaarden, Cambridge, MA, USA<br />
Signal processing, perfect adaptation and evolvability in<br />
the yeast osmo-sensing network<br />
KJ Patel, University of Cambridge, UK<br />
The FA pathway DNA repair pathway in model<br />
organisms<br />
Jan-Michael Peters, Vienna, Austria<br />
How chromosomes are segregated in mitosis<br />
Richard Peto, Oxford, UK<br />
Changing cancer mortality<br />
Ruth Pfeiffer, Bethesda, MA, USA<br />
Probability of detecting disease-associated Single<br />
Nucleotide Polymorphisms (SNPs) in single stage or<br />
multi-stage case-control genome-wide association studies<br />
Hidde Ploegh, Cambridge, MA, USA<br />
Ubiquitin and a ubiquitin-like modifier that targets RNA<br />
Marie France Poupon, Paris, France<br />
Patient derived breast cancer xenografts a tool for drugefficacy<br />
evaluation<br />
Oliver Rando, Worcester, MA, USA<br />
Static and dynamics genome-wide views of yeast<br />
chromatin<br />
Caetano Reis e Sousa, London, UK<br />
Innate regulation of dendritic cell unction<br />
Chris Reutelingsperger, Maastricht, The Netherlands<br />
Molecular imaging of the biomarker phosphatidylserine<br />
with Annexin A5. Shifting from a diagnostic towards a<br />
therapeutic arena<br />
Sjoerd Rodenhuis, Amsterdam, The Netherlands<br />
Response prediction and response-monitoring in the<br />
neoadjuvant treatment of breast cancer<br />
Rinat Rotem-Jehudar, Yavne, Israel<br />
CT-011, a humanized PD-1-interacting antibody, for the<br />
treatment of hematological malignancies and solid<br />
tumors
Sanne Schagen, Amsterdam, The Netherlands<br />
Cognitive problems following cancer and cancer<br />
treatment<br />
Ben Scheres, Utrecht, The Netherlands<br />
Stem cells: tales from another kingdom<br />
Eran Segal, Rehovot, Israel<br />
Cracking the code of gene regulation<br />
David Sherwood, Durham, NC, USA<br />
Breaching the basement membrane: Anchor cell<br />
invasion in C. elegans<br />
Jan Jakob Sonke, Amsterdam, The Netherlands<br />
Sharpening the soft-knife<br />
Hergen Spits, San Francisco, CA, USA<br />
The role of STAT5 in development of normal and<br />
malignant human B cells<br />
John Sundberg, Bar Harbor, MA, USA<br />
Getting old at the Jackson Laboratory: an overview of the<br />
Jackson Aging Center<br />
Jack Szostak, Boston, MA, USA<br />
What can we learn about the origin of life from efforts to<br />
design an artificial cell<br />
Stepen Taylor, Manchester, UK<br />
How do anti-mitotic drugs kill cancer cells?<br />
Heiddis Valdimarsdottir, New York, NY<br />
Emotion, cognition, and biology in cancer: Theories and<br />
interventions<br />
Richard Vallee, New York, NY, USA<br />
Live in vivo imaging of molecular motor-driven brain<br />
stem cell division and migration<br />
Lidia Vasilieva, Boston, MA, USA<br />
Role of transcription-coupled RNA degradation in<br />
heterochromatic silencing<br />
Karin de Visser, Amsterdam, The Netherlands<br />
The inflammatory tumor microenvironment:<br />
tumor-protective or tumor-promoting?<br />
Robert Weinberg, Cambridge, MA, USA<br />
Mechanisms of malignant progression<br />
Katarina Wolf, Nijmegen, The Netherlands<br />
Molecular determinants of tumor cell invasion and<br />
plasticity<br />
179<br />
INVITED SPEAKERS
180<br />
PROJECTS<br />
PROJECTS SUPPORTED<br />
BY THE DUTCH CANCER SOCIETY<br />
Principal Number of Title Started Ended/Ends<br />
investigator project<br />
Aaronson, Neil KWF 2003-2977 Psychological and behavioral issues in cancer genetics 1-10-2003 1-12-2009<br />
Aaronson, Neil KWF 2006-3470 Cognitive behavioral therapy (CBT) and physical exercise for<br />
climacteric symptoms in breast cancer patients experiencing<br />
treatment-indiced menopause<br />
1-9-2006 1-9-2010<br />
Agami, Reuven KWF 2005-3387 Identification and characterization of human tumor suppressor<br />
genes in the p53 pathway<br />
1-9-2005 1-9-2009<br />
Agami, Reuven KWF 2006-3558 Genetic screens to identify cancer related functions of human<br />
microRNAs<br />
1-11-2006 1-11-2010<br />
Agami, Reuven KWF 2007-3881 Role of miRNA genes in etiology of malignant germ cell tumors 1-3-2007 1-3-2011<br />
Agami, Reuven KWF 2007-3908 In vivo functional screens for oncogenic and tumorsuppressive<br />
microRNAs<br />
1-8-<strong>2008</strong> 1-8-2012<br />
Begg, Adrian KWF 2005-3420 Expression profiling to predict outcome after combined radiotherapy 1-3-2005<br />
and chemotherapy in head and neck tumors<br />
1-5-<strong>2008</strong><br />
Bernards, Rene KWF 2005-3375 The Role of PRAME in oncogenesis 1-8-2005 31-7-2009<br />
Bernards, Rene KWF <strong>2008</strong>-4042 Identification of enzymes involved in regulatory ubiquitination in<br />
TNF a--stimulated signalling by loss-of-function screens<br />
1-1-<strong>2008</strong> 1-1-2012<br />
Berns, Ton KWF 2004-3086 Dissecting the role of the pp21 INK4a genes in development and<br />
tumor suppression in mouse models<br />
1-12-2004 1-12-<strong>2008</strong><br />
Berns, Ton KWF 2005-3390 Mouse models for Small Cell Lung Cancer. Genotype-phenotype<br />
correlations as a basis to design better therapeutic intervention<br />
strategies<br />
1-5-2005 30-4-2009<br />
Berns, Katrien KWF <strong>2008</strong>-4027 Understanding resistance to HER2-targeting therapy in human<br />
breast cancer through functional genetics<br />
1-1-<strong>2008</strong> 1-1-2012<br />
Blank, Christian KWF<strong>2008</strong>-3988 Blockade of PD-1/D-L1 interaction to improve T cell mediated<br />
immunotherapy of cancer<br />
1-1-<strong>2008</strong> 1-1-2012<br />
Bleiker, Eveline KWF 2004-2987 Long-term physosocial impact of genetic testing among familial<br />
adenomatous polyposis(fap) families<br />
1-11-2004 1-11-<strong>2008</strong><br />
Bleiker, Eveline KWF 2005-3209 Psychosocial aspects of genetic testing in families at high risk of<br />
multiple tumors at various sites and ages<br />
1-11-2005 1-11-2009<br />
Bleiker, Eveline KWF <strong>2008</strong>-4016 Identification of psychosocial problems and perceived need for<br />
support in cancer genetics<br />
1-1-<strong>2008</strong> 1-1-2012<br />
Blitterswijk, Wim van KWF 2005-3377 Lipid rafts as novel targets for anti-cancer therapy and induction<br />
of apoptosis<br />
1-1-2005 1-1-2009<br />
Borst, Jannie KWF 2003-2859 Improving anti-tumor immunity via TNF receptor family member<br />
CD27 and its ligand<br />
1-8-2003 1-3-<strong>2008</strong><br />
PROJECTS
181 PROJECTS<br />
Principal Number of Title Started Ended/Ends<br />
investigator project<br />
Borst, Jannie KWF 2004-3087 Manipulation of the CD70 locus to monitor and modulate the<br />
anti-tumor immune response and tumor development<br />
14-6-2004 13-6-<strong>2008</strong><br />
Borst, Jannie KWF 2004-3079 Tumor therapy with ionizing radiation and death ligand Trail:<br />
efficacy and key molecular determinants<br />
1-7-2004 14-5-2009<br />
Borst, Jannie KWF<strong>2008</strong>-4028 T cell programming at the dendritic cell interface: impact on<br />
anti-tumor immunity<br />
1-1-<strong>2008</strong> 1-1-2012<br />
Borst, Jannie KWF<strong>2008</strong>-4110 Impact of TRAIL death receptor trafficking on pro-apoptotic signaling 1-3-<strong>2008</strong> 1-3-2012<br />
Borst, Piet KWF 2005-3379 A study of drug resistance mechanisms using large scale RNA<br />
interference screens<br />
13-6-2005 13-6-2009<br />
Borst, Piet KWF 2006-3566 Mechanisms of chemotherapy resistance in spontaneous tumors<br />
of genetically modified mice<br />
21-6-2006 21-6-2010<br />
Brekel, Michiel van den KWF 2007-3941 Prediction of local control after radiotherapy in head and neck cancer 1-11-2007 1-11-2009<br />
Collard, John KWF 2004-3062 The role of Tiam1 and Rac in Wnt/beta-catenin-induced tumorigenicity 1-12-2004 1-12-<strong>2008</strong><br />
Collard, John KWF 2007-3753 The par complex in cell polarity and tumor progression 1-7-2007 1-7-2011<br />
Dalesio, Otilia KWF Datamanagement KWF Datamanagement 1-1-1982 1-1-2009<br />
Dannenberg, KWF 2007-3978 Genetic analysis of class I HDACs in development and treatment 1-9-2007 1-9-2011<br />
Jan-Hermen of cancer<br />
Driel, Willemien van KWF CKTO 2006-16 Phase III randomised clinical trial for stage III ovarian carcinoma<br />
randomising between secondary debulking surgery with or without<br />
hyperthermic intraperiotoneal chemotherapy (OVHIPEC-1)<br />
14-3-2007 14-3-2010<br />
Fornerod, Maarten KWF 2004-3080 The role of RanBP3 in beta-catenin dependent nuclear signalling and 1-9-2004<br />
oncogenesis<br />
1-9-<strong>2008</strong><br />
Gilhuijs, Kenneth KWF 2004-3082 Application of Pathology-correlated Imaging to Improve the Accuracy 18-10-2004<br />
of Surgery and Radiotherapy in Breast-conserving Therapy<br />
17-10-<strong>2008</strong><br />
Haanen, John KWF 2007-3943 HPV 16 E6/E7 DNA prime and E6/E7 long peptide boost vaccination 1-1-<strong>2008</strong><br />
for multifocal Vulvar Intraepithelial Neoplasia (VIN)<br />
1-1-2010<br />
Herk, Marcel van KWF 2007-3895 Probability based treatment planning with biological objectives for<br />
high-dose high-precision radiotherapy of prostate cancer<br />
1-11-2007 1-11-2011<br />
Herk, Marcel van KWF 2007-3751 High precision image-guided radiotherapy for bladder cancer 1-1-<strong>2008</strong> 1-1-2012<br />
Hilkens, John KWF 2001-2489 Cloning of novel mammary tumor progression and metastasis genes 1-7-2001 1-8-<strong>2008</strong><br />
Jacobs, Heinz KWF <strong>2008</strong>-4112 A cancer genome atlas of the activation-induced cytidine deaminase: 1-3-<strong>2008</strong><br />
novel insights into the pathogenesis and prognosis of b cell lymphoma<br />
1-3-2012<br />
Jacobs, Jacqueline KWF 2005-3458 Novel factors involved in chromosome end protection by telomeres 1-1-2006 1-1-2010<br />
Jacobs, Jacqueline KWF 2007-3907 Dissecting the telomere damage response 1-5-<strong>2008</strong> 1-5-2010<br />
Jalink, Kees KWF 2007-3733 TRMP7, a novel regulator of cytoskeletal tension: implications for<br />
cancer progression, invasion and metastasis<br />
1-9-2007 1-9-2011<br />
Jonkers, Jos KWF 2006-3486 Dissection of the role of E-cadherin loss-of-function in breast cancer 13-2-2006<br />
development and metastasis<br />
12-2-2010
182<br />
PROJECTS<br />
Principal Number of Title Started Ended/Ends<br />
investigator project<br />
Jonkers, Jos KWF 2006-3715 Functional assessment of innate and adaptive immunity in breast<br />
cancer development<br />
1-1-2007 1-1-2011<br />
Jonkers, Jos KWF 2007-3772 Preclinical validation of chemical inhibitors of poly (ADP-ribose)<br />
polymerase (PARP) in conditional mouse models for BRCAassociated<br />
breast cancer<br />
1-2-2007 1-2-2011<br />
Jonkers, Jos KWF<strong>2008</strong>-4116 Impact of BRCA muntations on breast tumorigenesis and treatment 1-1-<strong>2008</strong><br />
response: functional analysist of defined truncation mutants and<br />
unclassified variants<br />
1-1-2012<br />
Leeuwen, Floor van KWF 2004-3068 Long-term risk of second cancers and cardiovascular disease<br />
following treatment of Hodgkiâ€s disease<br />
1-11-2004 1-11-<strong>2008</strong><br />
Leeuwen, Floor van KWF 2006-3631 Long-term risk of cancer after ovarian stimulation for in vitro<br />
fertilization<br />
1-4-2007 1-4-2011<br />
Leeuwen, Floor van KWF <strong>2008</strong>-3994 Cardiovascular morbidity and mortality in breast cancer survivors 1-10-<strong>2008</strong> 1-10-2012<br />
Linn, Sabine KWF 2006-3706 Towards patient-tailored systemic therapy in breast cancer:<br />
a combined approached of translational research and mouse<br />
model systems<br />
1-1-2007 1-1-2013<br />
Lohuizen, Maarten van KWF 2005-3376 Investigating the role of the Polycomb- group gene BMi in<br />
medullosblastomas and in neural cancer stem cell maintenance<br />
1-7-2005 1-7-2009<br />
Lohuizen, Maarten van KWF 2007-3877 Epigenetic regulation by the deubiquitinating enzyme USP3:<br />
impact on genome stability and tumorgenesis<br />
1-3-2007 1-3-2010<br />
Lohuizen, Maarten van KWF 2007-3803 Cancer stem cells and breast cancer: a polycomb connection 1-4-2007 1-4-2011<br />
Marijnen, Corrie KWF 2007-3945 Avoidance of overtreatment with radiotherapy in rectal cancer patients 15-9-2007 15-9-2009<br />
Medema, Rene KWF 2004-3063 Structure/function analysis of the role of polo-like kinase-1 in<br />
cellular transformation<br />
1-4-2004 31-3-<strong>2008</strong><br />
Michalides, Rob KWF 2005-3388 Molecular basis of resistance to anti-estrogens in estrogen-receptor<br />
positive breast cancer<br />
1-10-2005 1-10-2009<br />
Moolenaar, Wouter KWF 2005-3392 Autotaxin, a secreted lysophospholipase D: role in tumor progression 1-1-2005 1-1-2009<br />
Moolenaar, Wouter KWF 2005-3383 PIP4K-beta/PtdIns5P/p53: A cellular stress regulated pathway and its 1-11-2005<br />
implication in oncogenesis<br />
31-10-2009<br />
Nederlof, Petra KWF 2007-3749 Specific chromosomal imbalance in breast carcinomas as a marker<br />
for breast cancer susceptibility<br />
15-8-2007 15-8-2011<br />
Neefjes, Jacques KWF 2005-3391 TPPII and the role in antigen presentation by MHC Class I molecules 1-10-2005 1-10-2009<br />
Neefjes, Jacques KWF 2007-3883 Anti-cancer drugs and their effects on the ubiquitin cycle 1-4-2007 1-4-2011<br />
Ovaa, Huib KWF 2005-3368 Activity and distribution of proteasome inhibitors 1-7-2005 30-6-2009<br />
Peeper, Daniel KWF 2007-3957 Towards improved melanoma treatment: gene identifcation, in vivo<br />
modeling and drug target discovery<br />
15-4-<strong>2008</strong> 15-4-2014<br />
PROJECTS<br />
Peeper, Daniel KWF 2005-3382 The role of the neurotrophic tyrosine kinase receptor TrkB in anoikis 11-7-2005<br />
resistance, inavsion and metastasis of human tumors<br />
10-7-2009
183 PROJECTS<br />
Principal Number of Title Started Ended/Ends<br />
investigator project<br />
Peeper, Daniel KWF 2006-3505 Human melanocytic nevi: oncogene-induced senescence as a<br />
potential tumor-suppressing mechanism<br />
1-8-2006 1-8-2010<br />
Remeijer, Peter KWF <strong>2008</strong>-4024 Image guided radiotherapy for breast cancer 20-10-<strong>2008</strong> 20-10-2012<br />
Riele, Hein te KWF 2004-3084 Subtle gene modification to study the role of the mismatch repair<br />
complex MSH2/MSH6 in mutation avoidance and tumor suppression<br />
15-11-2004 14-11-<strong>2008</strong><br />
Riele, Hein te KWF 2006-3589 Significance of the Fanconi anemia caretaker pathway in development 1-1-2007<br />
and treatment of cancer<br />
1-1-2011<br />
Riele, Hein te KWF 2007-3790 Role of the G2 restriction point in tumor development and behavior 1-4-2007 1-4-2011<br />
Rookus, Matti KWF 2004-3088 Gene-environment interactions in BRCA1/2 breast/ovarian cancer<br />
families, a project of the Netherlands Collaborative Group on<br />
Hereditary Breast Cancer<br />
6-9-2004 5-9-<strong>2008</strong><br />
Rookus, Matti KWF 2007-3756 Heterogeneity of risk of breast and ovarian cancer in BRCA1 and<br />
BRCA2 mutayion carriers<br />
15-11-2007 15-11-2009<br />
Schagen, Sanne KWF 2002-2771 The incidence, nature and etiology of cognitive problems following<br />
chemotherapy for cancer<br />
18-11-2002 17-11-<strong>2008</strong><br />
Schagen, Sanne KWF 2007-3797 Late effects of chemotherapy on brain functioning in the elderly 1-1-2007 1-1-2010<br />
Schinkel, Alfred KWF 2003-2940 Compound Mrp2(Abcc2) and other drug transporter knock-out mice 1-11-2003<br />
to study determinants of pharmacokinetics, toxicity risks and possible<br />
therapy optimization for anticancer drugs<br />
1-4-<strong>2008</strong><br />
Schinkel, Alfred KWF 2007-3764 ‘OATP1A/1B (SLCO1A/1B) drug uptake transporters in anticancer<br />
drug pharmacokinetics and toxicity risks; possible strategies for<br />
therapy optimization’<br />
1-12-2007 1-12-2011<br />
Schmidt, Marjanka KWF 2007-3839 Genetics determinants of survival and second breast cancer<br />
development in premenopausal breast cancer patients<br />
1-1-2007 1-1-2011<br />
Schumacher, Ton KWF 2003-2860 Feasibility and limitations of T cell receptor gene transfer 1-8-2004 1-1-2009<br />
Schumacher, Ton KWF 2006-3530 Generation of human tumor specific T cells with high affinity<br />
T cell receptors<br />
1-12-2006 1-12-2010<br />
Schumacher, Ton KWF 2007-3825 SNP-based genome-wide identification of hematopoiesis-restricted<br />
minor histocompatibility antigens<br />
1-7-2007 1-7-2011<br />
Sixma, Titia KWF 2006-3476 Structural and functional analysis of a novel E3 ubiquitin ligase<br />
consisting of the polycomb proteins BMI1 and Ring1b/RNF2<br />
1-8-2006 1-8-2010<br />
Sixma, Titia KWF <strong>2008</strong>-4014 Comparative study of Ubiqutin-specific proteases 1-2-<strong>2008</strong> 1-2-2012<br />
Sonke, Jan Jakob KWF 2005-3378 High precision radiotherapy in the presence of anatomical changes 14-11-2005 14-11-2009<br />
Sonke, Jan Jakob KWF 2006-3545 Imaged guided correction strategies to optimize the precision of<br />
radiotherapy<br />
1-8-2006 1-8-2010<br />
Sonnenberg, Arnoud KWF 2006-3714 Tumor metastatis: the role of acto-myosin contraction in the<br />
downregulation of E-cadherinmediated cell-cell adhesion<br />
15-2-2007 1-2-<strong>2008</strong><br />
Sonnenberg, Arnoud KWF 2004-3067 Function of integrin-CD151 complexes in tumor invasion<br />
and metastasis<br />
1-11-2004 31-10-<strong>2008</strong>
184<br />
PROJECTS<br />
Principal Number of Title Started Ended/Ends<br />
investigator project<br />
Sonnenberg, Arnoud KWF <strong>2008</strong>-3996 A mouse stem cell model for skin cancer: Dual role of the integrin<br />
a6B4 in tumor initiation and progression<br />
1-8-<strong>2008</strong> 1-8-2012<br />
Stewart, Fiona KWF 2005-3370 The role of TGFb and Notch signaling in the development of<br />
radiation-induced telangiectasia<br />
1-4-2005 1-4-2009<br />
Stewart, Fiona KWF 2005-3373 KWF 2005-3373 Radiation-induced atherosclerosis: mechanisms and 1-6-2005<br />
preventive intervention strategies<br />
1-6-2009<br />
Stewart, Fiona KWF <strong>2008</strong>-3993 The role of endoglin in induction and repair of cardiovascular damage 1-2-<strong>2008</strong> 1-2-2012<br />
after radiation alone or in combinatione with trastuzumab (Herceptin)<br />
Tan, Bing KWF Ontwikkelingssamenwerkingsprogramma<br />
KWF Ontwikkelingssamenwerkingsprogramma 1-1-<strong>2008</strong> 1-1-2012<br />
Tan, Bing KWF <strong>2008</strong>-4233 Early detection of primary and recurrent nasopharyngeal carcinoma<br />
(NPC) using (anti-)EBV based tumor markers and options for using<br />
photodynamic therapy (PDT) in the treatment of local disease<br />
1-9-<strong>2008</strong> 1-9-2013<br />
Verheij, Marcel KWF 2007-3939 Improvement of chemoradiation response in head and neck cancer<br />
by (-)Gossypol (AT-101), a small molecule inhibitor of Bcl-XL/Bcl-2<br />
1-2-<strong>2008</strong> 1-2-2010<br />
Verheij, Marcel KWF <strong>2008</strong>-4113 Short-chain sphingolipids for enhanced cellular uptake of<br />
amphiphilic anti-cancer drugs<br />
1-1-<strong>2008</strong> 1-1-2012<br />
Vijver, Marc van de KWF 2002-2575 Microarray analysis of breast cancer as a diagnostic tool to guide<br />
optimal treatment<br />
1-2-2002 1-11-2009<br />
Wolthuis, Rob KWF 2007-3789 Targeting Mitotic Protein Destruction as an approach for cancer<br />
therapy<br />
1-9-2007 1-9-2011<br />
Wolthuis, Rob KWF <strong>2008</strong>-4135 Divergent Control of Cyclin B1-Cdk1 in cancer cells: a key role in<br />
therapy<br />
1-9-<strong>2008</strong> 1-9-2012
MAJOR PROJECTS<br />
SUPPORTED BY OTHER ORGANIZATIONS<br />
185 PROJECTS<br />
Principal Granting agency Title Started Ended/Ends<br />
investigator<br />
Agami, Reuven Dr. Josef Steiner<br />
Krebsstiftung<br />
Dr Josef Steiner Cancer Award 2007 1-5-2007 1-5-2011<br />
Agami, Reuven EEG-CEC / EU Mutant p53 as target for improved cancer treatment (mutp53) 1-2-2004 1-2-2009<br />
Agami, Reuven EEG-CEC / EU Indentifying novel regulatory mechanisms of miRNA functions 1-10-<strong>2008</strong> 1-10-2013<br />
Agami, Reuven NWO Genome wide search for DNA damage checkpoint functions in human<br />
cells (Euryi-award) 15-2-2005 14-2-2010<br />
Agami, Reuven NWO Combined miRNA profiling and genetic screens to identify and<br />
characterize cancer-related miRNAs<br />
1-10-2007 1-10-2012<br />
Balm, Fons Maurits en Anna<br />
de Kock Stichting<br />
Acute en late ototoxiciteit na chemoradiatie van hoofd-halstumoren 1-7-2007 31-12-<strong>2008</strong><br />
Bartelink, Harry EEG-CEC / EU Molecular imaging for Biologically Optimized Cancer Therapy (BioCare) 1-3-2004 1-9-2009<br />
Beijersbergen, Roderick Johnson Johnson en Johnson 1-12-2006 1-12-<strong>2008</strong><br />
Bernards, Rene Astra Identification of novel activity modulators of small molecule mTOR<br />
kinase inhibitos in breast cancer<br />
7-12-2007 7-12-2009<br />
Bernards, Rene CBG CBG 2e fase: Identification of cancer-relevant genes using a functional<br />
genetic approach 1-1-2004 31-12-<strong>2008</strong><br />
Bernards, Rene CGC CGC <strong>2008</strong>-2012 1-1-<strong>2008</strong> 1-1-2013<br />
Bernards, Rene EEG-CEC / EU Identification of Novel Targets for Cancer Therapy (INTACT) 1-1-2004 1-7-<strong>2008</strong><br />
Bernards, Rene EEG-CEC / EU Translational and Functional Onco-Genomics: from cancer-oriented<br />
genomic screenings to new diagnostic tools and improved cancer<br />
treatment. (Transfog)<br />
1-6-2004 1-12-<strong>2008</strong><br />
Bernards, Rene EEG-CEC / EU Breast Cancer Biomarkers and Functional mediators : harnessing the<br />
New Wealth of Omic Data (Target Breast)<br />
13-2-2006 12-2-2010<br />
Bernards, Rene EEG-CEC / EU EG Rubicon Annex I EC 1-1-2006 1-1-2011<br />
Bernards, Rene NIH System-based predications of responses to cancer therapy 1-9-2005 1-9-2009<br />
Bernards, Rene NWO Spinozapremie 26-9-2005 26-9-2009<br />
Bernards, Rene TI Pharma Kinases in cancer (TI Pharma) 1-7-2007 1-7-2011<br />
Berns, Ton EEG-CEC / EU “Lung stem cells and small-cell carcinoma; Marie Curie International<br />
Incoming Fellowships”<br />
1-1-2007 1-1-2009<br />
Berns, Ton EEG-CEC / EU MUGEN: Identification of Novel Targets for Cancer Therapy.<br />
Translating basic knowledge of functional oncogenomics into cancer<br />
diagnosis and treatment<br />
1-1-2005 1-1-2010
186<br />
PROJECTS<br />
Principal Granting agency Title Started Ended/Ends<br />
investigator<br />
Berns, Ton Merck Merck Res Coll LKR 25006 1-2-2006 1-2-2009<br />
Berns, Ton TI Pharma Kinases in Cancer (TI Pharma) 1-11-2007 1-11-2011<br />
Bleiker, Eveline Dutch Gastr Res<br />
Foundation<br />
Pancreas project (Rotterdam) 1-11-<strong>2008</strong> 1-11-2009<br />
Boersma, Menno Maastro Accurate target definition of non-small cell lung tumors using<br />
pathology-validated PET and CT imaging for the optimization of<br />
radiotion treatment<br />
1-9-2006 1-9-2010<br />
Borst, Jannie NWO A novel type of ubiquitination regulates apoptosis signaling by<br />
BH3-only protein Bid<br />
1-11-<strong>2008</strong> 1-11-2012<br />
Borst, Jannie SFN SFN OIO Hendriks SFR 2.1.25 1-11-2002 1-1-2040<br />
Borst, Jannie TI Pharma TNF ligands in cancer 1-3-2006 1-3-2010<br />
Borst, Jannie ZonMw The role of CD27/CD70-regulated cell survival in shaping effector and<br />
memory T cell responses<br />
1-5-2004 1-5-<strong>2008</strong><br />
Borst, Piet EEG-CEC / EU Molecular mechanisms underlying chemotherapy resistance,<br />
therapeutic escape, efficacy and toxicity (CHEMORES)<br />
1-2-2007 1-2-2012<br />
Borst, Piet ZonMw Characterization of the physiological roles of Multidrug Resistance<br />
Protein (MRP) 1-6 by in vivo screening for their substrates (TOP subsidie)<br />
1-1-<strong>2008</strong> 1-1-2012<br />
Cats, Annemieke Roche Ondersteuning junior medical trials coordinator. CRITICS-studie en<br />
rectum-carcinoom projecten. ML20482<br />
1-10-2007 1-10-2009<br />
Coevorden, Frits van SFN Liposarcoom project 1-1-2007 1-12-<strong>2008</strong><br />
Cohen, Serge NWO NWO Veni 700,55,405 1-2-2006 31-1-2009<br />
Collard, John EEG-CEC / EU An integrated concept of tumor metastasis: implications for therapy 1-4-<strong>2008</strong> 1-4-2011<br />
Dalesio, Otilia EEG-CEC / EU ‘eTEN’ Programme - Call identifier: 05/1 ETEN-029334 - ‘TENALEA-ID’ 1-11-2006 1-5-2010<br />
Dalesio, Otilia IKA IKA kankerregistratie 1-9-1990 1-1-2009<br />
Dalesio, Otilia NVALT Statistical Center NVALT Clinical Trials in oncology 1-5-2004 1-1-2010<br />
Dalesio, Otilia VIKC Verdeling voor CKTO projecten VIKC 1-7-2007 1-1-2011<br />
Dam, Frits van Vereniging tegen<br />
de Kwakzalverij<br />
Vereniging tegen de Kwakzalverij 1-1-2007 1-1-2009<br />
Dannenberg, Jan-Hermen NWO-ALW The role of class I HDACs in normal physiology, tumorigenesis and<br />
transcriptional regulation<br />
1-8-2007 1-8-2012<br />
Elsakkers, Peter AMC Poli Familiare Tumoren (vervolg PFT) 1-1-<strong>2008</strong> 1-1-2009<br />
Filippo, Ronald VU medisch<br />
centrum<br />
Cytofys (EVA project) 1-9-2007 1-9-2011<br />
Fornerod, Maarten NWO-ALW Connecting chromatin to the plasma membrane 1-1-2007 1-4-2010<br />
Gilhuijs, Kenneth EEG-CEC / EU HYPERImage: Hybrid PET-MR system for concurrent<br />
ultra-sensitive imaging<br />
1-4-<strong>2008</strong> 1-4-2011
Principal<br />
investigator<br />
Granting agency Title Started 187 PROJECTS<br />
Ended/Ends<br />
Haanen, John NWO ZonMW 432-00-001 1-7-2006 1-7-2009<br />
Haramis, Anna Pavlina NWO NWO VIDI Haramis 917-56-322 1-1-2005 31-12-2009<br />
Harten, Wim van CvZ Operations/Lean Management 1-1-2002 1-12-2009<br />
Harten, Wim van CvZ CvZ Array Raster 3 (fase 2) 1-1-2006 31-12-2009<br />
Herk, Marcel van CTMM Personalized chemo-radiation of lung and head and neck cancer<br />
(aiming at the relief of suffering of patients with tumors of the upper<br />
aero-digestive tract using innovative navigation tools in high precision<br />
tumor targeting by chemo-radiation<br />
1-10-<strong>2008</strong> 1-10-2013<br />
Herk, Marcel van EOS /<br />
voorheen Philips<br />
EOS 1-5-2000 1-5-2010<br />
Hilgers, Frans ATOS Development and evaluation of a third generation ProvoxTM voice<br />
prosthesis<br />
1-6-2003 1-2-2009<br />
Hilgers, Frans ATOS ATOS Medical Collaboration 30-6-2003 1-1-2009<br />
Hofland, Linda Biolitec PDT Biolitec: Photodynamic Therapy 1-1-2005 1-1-2009<br />
Holtkamp, Christine EEG-CEC / EU ESF (Europees Sociaal Fonds) 1-6-2007 1-6-2009<br />
Jacobs, Heinz ZonMw Role of translesion DNA synthesis in immunity and cancer development 1-1-2005 1-1-2010<br />
Jacobs, Jacqueline NWO NWO Vidi Jacobs 1-2-2007 1-2-2012<br />
Jalink, Kees ZonMw ZonMW 9120.6110 1-6-2006 1-6-2010<br />
Jonkers, Jos AICR Genome-wide insertional mutagenesis screens to identify cancer genes<br />
that collaborate with E-cadherin loss in mammary tumourigenesis<br />
1-10-2007 1-10-2010<br />
Jonkers, Jos EEG-CEC / EU Eurosystem 1-3-<strong>2008</strong> 1-3-2011<br />
Jonkers, Jos Kudos <strong>NKI</strong>-Kudos Collaborative Research 1-7-2006 1-7-2009<br />
Jonkers, Jos Merck Merck Mouse Model License 1-12-2006 1-12-<strong>2008</strong><br />
Jonkers, Jos Organon <strong>NKI</strong>-Organon 1-8-2006 1-8-<strong>2008</strong><br />
Jonkers, Jos TI Pharma Kinases in cancer (TI Pharma) 1-11-2007 1-11-2011<br />
Klomp, Houke Roche Roche HQ Neo adjuvant Tarceva: Tumor remission rate with neoadjuvant 1-7-2006<br />
erlotinib in operable patients with clinical stage I/II non-small cell lung<br />
cancer (NSCLC) (M06NEL)<br />
1-1-2009<br />
Klomp, Houke Vrolijk Aanstelling voor arts-onderzoeker 4-8-2006 1-7-2009<br />
Leeuwen, Fijs van SFN Multifunctional labels for translational in vitro - in vivo annexin V<br />
diagnostics<br />
1-3-<strong>2008</strong> 1-3-2010<br />
Leeuwen, Flora van AZG Cardiovascular morbidity in testicular cancer survivors: case-control<br />
study of risk factors and assessment of pharmacogenomic determinants<br />
of toxicity<br />
18-2-2005 31-12-2009<br />
Leeuwen, Flora van CGC The benefits and risks of cancer genomics for society 1-1-2006 1-3-<strong>2008</strong>
188<br />
PROJECTS<br />
Principal Granting agency Title Started Ended/Ends<br />
investigator<br />
Leeuwen, Flora van EEG-CEC / EU EG GENE-Rad-Risk: Radiation exposures at an early age: impact of<br />
genotype on breast cancer risk<br />
1-6-2005 1-6-2009<br />
Leeuwen, Flora van NIH Risk and prognosis of uterine corpus cancer after tamoxifen treatment<br />
for breast cancer<br />
25-9-2007 1-9-2009<br />
Leeuwen, Flora van Pink Ribbon Invloed van lichaamsbeweging en andere leefstijlaspecten op kwaliteit<br />
van leven en prognose bij borstkanker<br />
1-8-2007 1-1-2009<br />
Leeuwen, Flora van SFN SFN 2.4.54. NO 1-3-<strong>2008</strong> 1-9-<strong>2008</strong><br />
Leeuwen, Flora van SFN Ondersteuning epidemiologisch onderzoek 1-7-2007 1-1-2009<br />
Leeuwen, Fred van NPC Chromatin dynamics and epigenetic changes 1-1-2009 1-1-2013<br />
Leeuwen, Fred van NWO The role of histone methylation in silencing and transcriptional memory 1-2-2005 1-2-2009<br />
Leeuwen, Fred van NWO A multifunctional genome-wide screen to unravel the roles of the<br />
conserved epigenetic regulator Dot1 in chromosome organization<br />
(ECHO subsidieronde 2006-2007)<br />
1-7-2007 1-1-2010<br />
Lohuizen, Maarten van Dijkzigt Stem cells in Development and Disease 1-1-2004 31-12-<strong>2008</strong><br />
Lohuizen, Maarten van EEG-CEC / EU Platforms for biomedical discovery with human ES cells 1-8-2006 1-8-2010<br />
Lohuizen, Maarten van EEG-CEC / EU Eurosystem 1-3-<strong>2008</strong> 1-3-2012<br />
Lohuizen, Maarten van Ernst Schering Regulation of histone H2A monoubiquitination byRing1b/Bmi1 and 1-9-2007 1-9-2009<br />
Foundation USP3 in DNA damage response and oncogenic transformation<br />
Lohuizen, Maarten van Merck Merck LKR #40778 PO X21000363 2-6-2006 31-1-2010<br />
Lohuizen, Maarten van NWO SFN 2.2.33 Startgeld 1-1-2005 31-12-2009<br />
Lohuizen, Maarten van NWO-ALW Genome-wide Spatial Profiling of Polycomb Domains in<br />
Drosophila melanogaster<br />
1-12-2007 1-12-2010<br />
Lohuizen, Maarten van NWO-ALW Maintenance of genome integrity by histone (de)ubiquitinating enzymes 1-11-<strong>2008</strong> 1-11-2013<br />
Luenen, Henri van NWO L1 and Alu elements: Structural and functional analysis of human<br />
non-LTR retrotransposons<br />
1-10-2004 1-10-2009<br />
Luenen, Henri van Roche <strong>AvL</strong>-prijs 2006 Voorhoeve 1-1-2007 1-1-2009<br />
Luenen, Henri van Roche <strong>AvL</strong>-prijs 2007 Bruggeman 1-1-<strong>2008</strong> 1-1-2010<br />
Michalides, Rob TI Pharma Nuclear receptors in targeted cancer therapy: improved methods for<br />
candidate selection<br />
1-2-2007 1-2-2011<br />
Neefjes, Jacques EEG-CEC / EU High throughput development of drugs for immunotherapy of<br />
autoimmune diseases. (Drugs for Therapy)<br />
1-11-2004 1-11-<strong>2008</strong><br />
Neefjes, Jacques EEG-CEC / EU The role of the intracellular peptidases in the immune system and in the<br />
cellular metabolism<br />
1-12-2006 1-12-<strong>2008</strong><br />
Neefjes, Jacques EEG-CEC / EU Microban 1-11-2005 1-11-2009<br />
Neefjes, Jacques EEG-CEC / EU Systems biology of phagosome formation and maturation - modulation<br />
by intracellular pathogens<br />
1-12-<strong>2008</strong> 1-12-2012
189 PROJECTS<br />
Principal Granting agency Title Started Ended/Ends<br />
investigator<br />
Neefjes, Jacques KNAW Tissue responses to radiotherapy and development of immunotherapy<br />
for successful radioimmunotherapy of human melanoma<br />
15-11-2007 15-11-2011<br />
Neefjes, Jacques NWO Manipulating the MHC class II system to control immune responses in<br />
autoimmunity 1-10-2007 1-10-2012<br />
Neefjes, Jacques NWO-ALW Defining the MHC class II loading microdomain to understand<br />
immune responses<br />
1-11-2005 1-11-2009<br />
Nieweg, Omgo SFN SFN 2.5.6 IKR Lymphatic mapping 1-5-2004 1-12-<strong>2008</strong><br />
Oldenburg, Hester Pink Ribbon Werkhervatting en seksualiteit na borstkanker 1-9-<strong>2008</strong> 1-9-2011<br />
Ovaa, Huib NWO Modulation of autotaxin activity by small molecules 1-11-2006 1-11-2009<br />
Ovaa, Huib NWO Chemical biology of ubiquitin-like modifications 1-9-2005 1-9-2010<br />
Ovaa, Huib NWO “Development of conditional protein-ligand exchange applied to<br />
immune technology; Onderzoeksprogramma: Integration of Biosynthesis<br />
& Organic Synthesis (IBOS)”<br />
1-9-<strong>2008</strong> 1-9-2012<br />
Ovaa, Huib SFN SFN: 2.4.58 Cye Dye 11-8-<strong>2008</strong> 11-8-2009<br />
Ovaa, Huib ZonMw Immunoproteomics: antigen mapping through MHC epitope<br />
exchange reactions<br />
1-11-<strong>2008</strong> 1-6-2010<br />
Peeper, Daniel EEG-CEC / EU ‘EU FP6 Grant No.037758 ‘ ‘The anoikis suppressor TrkB as a target for<br />
novel anti-cancer agents’<br />
1-1-2007 1-1-2010<br />
Peeper, Daniel EMBO EMBO Young Investigators 1-9-2006 1-9-2009<br />
Peeper, Daniel NWO Novel functional genomic screens to identify pathways that protect<br />
mouse and human cells against oncogenic transformation by mutant RAS<br />
2-12-2002 5-1-<strong>2008</strong><br />
Peeper, Daniel NWO Synthetic lethality: a novel tactic for selective anticancer drug target<br />
discovery<br />
1-6-2007 1-6-2012<br />
Peeper, Daniel NWO-ALW Genome-wide identification and functional characterization of novel<br />
tumor suppressor genes<br />
1-1-2007 31-12-2009<br />
Perrakis, Anastassis EEG-CEC / EU EG Biocrystallography Bioxhit 1-1-2004 1-7-<strong>2008</strong><br />
Perrakis, Anastassis EEG-CEC / EU BIOXHIT Management 1-1-2004 1-7-<strong>2008</strong><br />
Perrakis, Anastassis EEG-CEC / EU Integrating and strengthening the European Research Area FP6-2005<br />
Lifescihealth 6 Proposal NO 037198<br />
1-1-2007 1-7-2009<br />
Perrakis, Anastassis EEG-CEC / EU A multidisciplinary approach to determine the structures of protein<br />
complexes in a model organism<br />
1-2-2005 31-1-2010<br />
Perrakis, Anastassis EEG-CEC / EU From Receptor to Gene: structures of complexes from signalling<br />
pathways linking immunology, neurobiology and cancer (SPINE 2)<br />
1-7-2006 1-7-2010<br />
Perrakis, Anastassis NIH NIH grant 2R01 GM062612-05, EMBL Heidelberg 1-5-2006 1-5-2010<br />
Peters, Peter Aeras Global TB Public Private Partnership for research into and development of 1-5-2007 1-5-2011<br />
Vaccine Foundation medicines, vaccines and diagnostic aids in the domain of AIDS,<br />
tuberculosis and malaria<br />
Peters, Peter EEG-CEC / EU EG AntePrion CT2006-019090 1-6-2006 1-6-2009
190<br />
PROJECTS<br />
Principal<br />
investigator<br />
Granting agency Title Started Ended/Ends<br />
Peters, Peter EEG-CEC / EU EG AntePrion CT2006-019090 (M) Development of a pre-clinical blood<br />
test for prion diseases Coordiantie<br />
1-6-2006 1-6-2009<br />
Peters, Peter EEG-CEC / EU EG ImmunoPrion Immunological and structural studies of prion diversity 1-6-2006 1-6-2009<br />
Peters, Peter EEG-CEC / EU Understanding prion strains and species barriers and devising novel<br />
diagnostic approaches (strainbarrier)<br />
1-11-2006 1-5-2010<br />
Peters, Peter MIN OCW ‘E-cadhering and cadhering-11 and their role in cancer migration’<br />
‘binnen het SmartMix programma NIMIC’<br />
1-2-<strong>2008</strong> 1-2-2012<br />
Peters, Peter NIH Pathways of antigen presentation by CD 1 a, b and c 1-1-2005 31-12-2009<br />
Riele, Hein te AFM Cellular factors that determine somatic (CTG)n hypermutability in DM1:<br />
targets for therapy<br />
1-7-<strong>2008</strong> 1-7-2009<br />
Riele, Hein te NWO Horizon 050-71-051 1-12-2006 1-12-2011<br />
Rodenhuis, Sjoerd CTMM Neoadjuvant drug treatment for breast cancer-response-prediction and<br />
response monitoring<br />
1-10-<strong>2008</strong> 1-10-2013<br />
Rookus, Matti DES ‘DES Schadefonds; Projectleiderschap: Matti Rookus en<br />
Floor van Leeuwen’<br />
15-6-2006 1-1-2010<br />
Rookus, Matti SFN ‘startgeld’ ‘setting up an IBCCS coordination centre at <strong>NKI</strong>’ 1-1-2009 1-1-2010<br />
Roos, Ed Johnson Sponsorcontract Johnson & Johnson 1-11-<strong>2008</strong> 1-11-2011<br />
Roos, Ed SFN SFN nieuwe ontwikkelingen <strong>2008</strong> 2.4.52 1-3-<strong>2008</strong> 1-3-2009<br />
Rutgers, Emiel EORTC EORTC breast group 1-1-2000 1-1-2010<br />
Rutgers, Emiel EORTC EORTC Breast Group werkvergadering 1-3-2002 1-1-2010<br />
Sandick, Johanna van Vrolijk Slokdarmkankeronderzoek Stichting Vrolijk 1-1-<strong>2008</strong> 31-12-2009<br />
Schagen, Sanne Pfizer Assessment of neuropsychological sequelae of tamoxifen and<br />
exemestane in postmenopausal women with early breast cancer.<br />
TEAM-neuropsychologiestudie<br />
1-6-2003 1-1-2009<br />
Schagen, Sanne SFN Nature and mechanism of cognitive deficits following chemotherapy:<br />
an (f)MRI study SFN 2.2.40<br />
1-4-2007 1-1-2009<br />
Schellens, Jan aCBG Conv. <strong>NKI</strong>-aCBG (Geneesm.) 1-8-2006 1-1-2009<br />
Schellens, Jan Astra AstraZeneca Affinity of AZD1152 24-3-2005 1-1-2009<br />
Schellens, Jan Astra Preclinical in vivo testing of AZD1152 to determine the effect of the<br />
ABC-drug transporters P-glycoprotein (Pgp, Mdr1) and BCRP (Bcrp1,<br />
Abcg2) on tissue distribution and clearance of AZD1152<br />
1-9-2007 1-1-2009<br />
Schellens, Jan Fonds NutsOhra Veiligheid- & kostenbesparingstudie voor DPYD2A genotypering bij<br />
fluoropyrimidinetherapie<br />
1-1-<strong>2008</strong> 1-1-2010<br />
Schellens, Jan Novartis Novartis evaluation gimatecan 1-3-2005 1-3-<strong>2008</strong><br />
Schinkel, Alfred GLAXO An evaluation of organic anion transporters on the pharmacokinetics<br />
and hepatic disposition of drugs using in vitro and in vivo techniques<br />
13-12-2004 12-12-<strong>2008</strong>
191 PROJECTS<br />
Principal Granting agency Title Started Ended/Ends<br />
investigator<br />
Schinkel, Alfred SLVRT Imapct of the multidrug transportes P-glycoprotein, MRP2 and BCRP on<br />
the in vivo behaviour of experimental anticancer drugs<br />
1-10-2004 1-10-<strong>2008</strong><br />
Schinkel, Alfred STW Generation and validation of cyctochrome P450 3A knockout and<br />
transgenic mice as tools for improved drug development and research<br />
1-6-2004 1-10-2009<br />
Schumacher, Ton ACTS (NWO) Development of conditional protein-ligand exchange applied to immune 1-9-<strong>2008</strong><br />
technology<br />
1-9-2012<br />
Schumacher, Ton EEG-CEC / EU MIF1-CT-2006-039477 (naik) Determining the relationships between<br />
haematopoletic lineages using genetic barcoding<br />
1-12-2006 1-12-<strong>2008</strong><br />
Schumacher, Ton EEG-CEC / EU Attack: Adoptive engineered T-cell targeting to activate cancer killing 1-11-2005 1-11-2010<br />
Schumacher, Ton Landsteiner Stichting MHC multimer-based adoptive T cell therapy 1-5-2006 1-1-2009<br />
Schumacher, Ton Landsteiner Stichting Graft versus Host disease by TCR-engineered T cells: mechanisms and<br />
means for prevention<br />
1-11-<strong>2008</strong> 1-11-2011<br />
Schumacher, Ton Melanoma<br />
Research Alliance<br />
Platform For MHC-Exchange Based T-Cell therapy For Melanoma 1-9-<strong>2008</strong> 1-9-2010<br />
Schumacher, Ton University College LRF New Grant Award 06037 toegekend aan Dr Gavin Bendle: 1-3-2007 1-3-2009<br />
London Adjuvant strategies in T cell receptor gene therapy<br />
Schumacher, Ton ZonMw Molecular dissection of AIRE-driven ectopic antigen expression 1-6-2005 1-6-<strong>2008</strong><br />
Schumacher, Ton ZonMw T cell receptor gene therapy of metastatic melanoma: a phase I clinical trial 1-4-2005 31-3-2010<br />
Sixma, Titia AZVU Onderzoekschool Oncologie 1-1-2000 1-1-2009<br />
Sixma, Titia EEG-CEC / EU MCRTN Ubiregulators: Signal Transduction by Ubiquitination, a Matter<br />
of Location<br />
1-1-2007 31-1-2011<br />
Sixma, Titia EEG-CEC / EU Neurotransmitter Cys-loop recptors: structure, function and disease 1-2-<strong>2008</strong> 1-2-2012<br />
Sixma, Titia EEG-CEC / EU Mismatch2model-Characterization and quantitative modeling of DNA 1-10-<strong>2008</strong> 1-10-2012<br />
mismatch repair and its role in the maintenance of genomic stability and<br />
cancer avoidance<br />
Sixma, Titia INTAS Selective ligands of nicotinic acetylcholine receptor subtypes designed on 1-6-2006<br />
the basis of X -ray structures of complexes of acetylcholine-binding<br />
proteins with agonists and antagonists<br />
31-12-<strong>2008</strong><br />
Sixma, Titia NWO NWO CW PIO 700.51.401 1-2-2003 1-1-2009<br />
Sixma, Titia SFN Protein purification facility 1-11-2007 1-11-2011<br />
Sixma, Titia STW Exploring new uses of ChhBP as model for ligand-gated ion channel<br />
research<br />
1-12-2003 1-5-<strong>2008</strong><br />
Sixma, Titia TI Pharma New approaches for Ligand-Gated Ion Channel (LGIC) drug discovery 1-9-2007 1-9-2011<br />
Smit, Linda AICR Uncovering the molecular pathways that regulate the self-renewal of<br />
breast cancer initiating cells<br />
1-4-<strong>2008</strong> 1-5-<strong>2008</strong><br />
Sonnenberg, Arnoud AICR AICR Grant 06-528 1-10-2006 1-2-<strong>2008</strong><br />
Sonnenberg, Arnoud DEBRA Generation of a mouse model for EBS-MD 1-12-2005 1-12-2009
192<br />
PROJECTS<br />
Sonnenberg, Arnoud nierstichting Function of tetraspanin-integrin complex Cd151- a3B1 in development and 1-10-<strong>2008</strong><br />
maintenance of the glomerular filtration barrier<br />
1-10-2012<br />
Sonnenberg, Arnoud NWO NWO 815.02.002 1-11-2005 1-11-2009<br />
Sonnenberg, Arnoud NWO-ALW Controlling hemidesmosome dynamics by phosphorylation of residues<br />
on the integrin b4 subunit<br />
1-3-2007 1-4-2011<br />
Steensel, Bas van EEG-CEC / EU NET member of The Epigenome Network of Excellence: Epigenome,<br />
epigenetic plasticity of the genome<br />
1-6-2005 1-6-<strong>2008</strong><br />
Steensel, Bas van NWO Whole-genome analysis of the regulation of chromatin structure and<br />
gene expression<br />
1-3-2005 1-3-2010<br />
Steensel, Bas van NWO Multi-gene chromatin domains 1-12-2006 1-12-2010<br />
Steensel, Bas van NWO Chromatin Protein Discovery Project 1-2-<strong>2008</strong> 1-2-2012<br />
Stewart, Fiona EEG-CEC / EU Cardiorisk - The mechanisms of cardiovascular risks after low<br />
radiation doses<br />
1-2-<strong>2008</strong> 1-2-2011<br />
Valdes Olmes, Renato ONCO Vision Dection of Deep Located Sentinel Nodes using Preoperative SPECT-CT<br />
with an Hybrid System and Peroperative Real Time Imaging with a<br />
Portable Small Camera<br />
1-5-<strong>2008</strong> 1-5-2010<br />
Valdes Olmes, Renato EEG-CEC / EU Mammography with molecular imaging (MAMMI) Specific Targeted<br />
project<br />
1-1-2007 1-1-2011<br />
Veer, Laura van ‘t EEG-CEC / EU Molecular signatures as diagnostic and therapeutic targets for<br />
disseminated epithelial malignancies<br />
1-11-2005 1-5-2009<br />
Veer, Laura van ‘t EEG-CEC / EU Translating molecular knowlegde into early breast cancer management:<br />
building on the BIG (Breast International Group) network for improved<br />
treatment tailoring. TRANSBIG<br />
1-3-2004 1-3-2011<br />
Veer, Laura van ‘t EEG-CEC / EU Collaborative Oncological Gene-environment Study 1-10-<strong>2008</strong> 1-10-2012<br />
Veer, Laura van ‘t TI Pharma Predicting drug responses in breast cancer. TI Pharma T3-108 1-7-2007 1-7-2011<br />
Vijver, Marc van de Astra ASTRA/Kudos/Vijver/Linn 1-1-2006 1-1-2009<br />
Visser, Karin de ZonMw The inflammatory tumor microenvironment and its impact on<br />
breast cancer development and therapy<br />
1-1-2009 1-1-2014<br />
Vyth-Dreese, Florry AMC MR UVA 2006-3606 1-7-2006 1-7-2010<br />
Vyth-Dreese, Florry LUMC ‘KWF 2003-2804 (UL) Ex vivo in situ analyses of Graft versus Host<br />
mechanisms by Minor Histocompatibility antigen specific T cells;<br />
relevance for stem cell transplantation’<br />
1-12-2003 31-5-<strong>2008</strong><br />
Wessels, Lodewyk MIN OCW NBIC Biorange 1-1-2004 1-1-2010<br />
Wessels, Lodewyk NBIC NBIC / BioAssist / HTHC Programmeur 1-1-<strong>2008</strong> 1-1-2011<br />
Wolthuis, Rob NWO-ALW Coordination of cell division by regulated protein destruction 1-1-2007 31-12-2011
PERSONNEL INDEX<br />
Aaronson, Neil 90<br />
Aarts, Marieke 62<br />
Achachah, Mohamed 101<br />
Ackerstaff, Annemieke 143<br />
Adriaansz, Sandra 117<br />
Agami, Reuven 46<br />
Ahmed, Kalam 144<br />
Ait Moha, Daoud 101<br />
Ajouaou, Abderrahim 101<br />
Albers, Harald 34<br />
Alderden, Carolien 117<br />
Alderliesten, Maaike 30<br />
Alderliesten, Tanja 101<br />
Aleman, Berthe 95, 126<br />
Alendar, Andrej 82<br />
Alfieri, Andrea 18<br />
Anirudhan, Gireesh 84<br />
Annabel, Zwaagstra 86<br />
Antonini, Ninja 156<br />
Ariotti, Silvia 57<br />
Armstrong, Nicola 72<br />
Atsma, Douwe 100<br />
Aukema, Tjeerd 100<br />
Axwijk, Priscilla 101<br />
Baank, Saskia 100<br />
Baars, Danny 156<br />
Baars, Joke 116<br />
Baars, Philippe 100<br />
Baas, Paul 116<br />
Baas-Vrancken Peeters, Marie-Jeanne 143<br />
Bakker, Martine 100<br />
Bakker, Sietske 62<br />
Balague Ponz, Olga 100<br />
Balm, Alfons 143<br />
Bartelink, Harry 126<br />
Batteau, Lukas 101<br />
Beaudry, Jean Bernard 80<br />
Beelen, Karin 116<br />
Beers-Bauhuis, Carolien 100<br />
Begg, Adrian 40<br />
Beijersbergen, Roderick 78<br />
Beijnen, Jos 44, 116<br />
Bejaoui, Inès 87<br />
Belderbos, Jose 126<br />
Bendle, Gavin 55<br />
Bentin Toaldo, Cristiane 33<br />
Bergman, André 116<br />
Berkers, Celia 34<br />
Bernards, René 76<br />
Berns, Anton 82<br />
Berns, Katrien 76<br />
Besnard, Peter 101<br />
Bessels, Frauwkje 156<br />
Betgen, Anja 127<br />
Bex, Axel 144<br />
Bin Ali, Rahmen 82<br />
Blank, Christian 61, 116<br />
Bleiker, Eveline 94, 101<br />
Blitz, Johanna 82<br />
Bloemers, Monique 126<br />
Blom, Marleen 80<br />
Blom, Marie-José 95<br />
Boekel, Naomi 95<br />
Boekhout, Annelies 117<br />
Boerrigter-Barendsen, Lucie 100<br />
Bonfrer, Hans 100<br />
Boogerd, Willem 92, 116<br />
Boot, Henk 116<br />
Borkner, Lisa 61<br />
Borst, Jannie 53<br />
Borst, Gerben 126<br />
Borst, Piet 64<br />
Bos, Arnold 46<br />
Bosma, Astrid 38<br />
Boss, David 116<br />
Boutmy-de Lange, Majella 101<br />
Bouwman, Peter 66<br />
Braaf, Linde 38<br />
Brandsma, Dieta 92, 116<br />
Brandwijk, Kim 38<br />
Braumuller, Tanya 66<br />
Braunschweig, Ulrich 48<br />
Broeks, Annegien 38, 95<br />
Brohet, Richard 95<br />
Brouwers, Elke 116<br />
Bruggeman, Sophia 80<br />
Bruin, Sjoerd 38, 143<br />
Bruin, Natascha 100<br />
Buckle, Tessa 100, 101<br />
Bueno de Mesquita, Jolien 68, 99,100<br />
Buikhuisen, Wieneke 116<br />
Buitelaar, Dirk 144<br />
Bulut, Tomas 144<br />
Buning-Kager, Marian 100<br />
Burgers, Sjaak 116<br />
Calbo, Joaquim 82<br />
Cardous-Ubbink, Mathilde 95<br />
Carreno, Monique 156<br />
Cats, Annemieke 116<br />
Cavalli, Silvia 34<br />
Celie, Patrick 20<br />
Chin, Patrick 101<br />
Christodoulou, Evangelos 20<br />
Citterio, Elisabetta 80<br />
Clijsters, Linda 74<br />
Cohen, Serge 20<br />
Collard, John 26<br />
Copper, Marcel 143<br />
Coquet, Jonathan 53<br />
Cornelissen, Sten 38<br />
Cornelissen, Paulien 80<br />
Courrech Staal, Ewout 143<br />
Daems, Daphne 156<br />
Dahmen, Rutger 99<br />
Dalesio, Otilia 156<br />
Damen, Carola 116<br />
Damen, Eugène 126<br />
Dannenberg, Jan-Hermen 87<br />
De Boer, Jan Paul 116<br />
De Boer, Johan 127<br />
De Boer, Roel 126<br />
De Bois, Josine 127<br />
De Bruin, Michiel 66<br />
De Bruin, Chiel 68<br />
De Bruin, Marieke 95<br />
De Castro Genebra de Jesus, Inês 48<br />
De Haas, Marcel 64<br />
De Jong, Annemieke 34<br />
De Jong, Monique 40<br />
De Jong, Daphne 100<br />
De Jong, Gerda 156<br />
De Leeuw, Renée 33<br />
De Marco, Valeria 20<br />
De Punder, Karin 36<br />
De Ridder, Jeroen 72<br />
De Ronde, Jorma 72<br />
De Ruiter, Michiel 92<br />
De Visser, Karin 66<br />
De Vreeze, Ronald 143<br />
De Vries, Diederick 20<br />
De Vries, Evert 53<br />
De Vries, Hilda 82<br />
De Vries, Sandra 62<br />
De Waal, Marjolijn 156<br />
De Widt, John 30<br />
Deenen, Maarten 44, 116<br />
Deetman, Joost 99<br />
Dekker, Marleen 62<br />
Dekker, Rob 62<br />
Dellemijn, Trees 57<br />
Delzenne-Goette, Elly 62<br />
Desmet, Christophe 84<br />
Devriese, Lot 44<br />
Dewit, Luc 126<br />
Dirac, Annette 76<br />
Doodeman, Petra 100<br />
Doodeman, Valerie 44<br />
Dorlo, Thomas 116<br />
Douma, Sirith 84<br />
Douma, Kirsten 90, 94<br />
Doumont, Gilles 66<br />
Drost, Brigit 144<br />
Drost, Jarno 46<br />
Drost, Rinske 66<br />
Dubbelman, Anne Charlotte 116<br />
Dubbelman, Ria 117<br />
Dufournij, Brigitte 156<br />
Duijts, Saskia 90<br />
Duppen, Joop 127<br />
Eenink, Martijn 126<br />
Efthymiou, Katina 144<br />
Egan, David 78<br />
Eijzenga, Willem 94<br />
El Oualid, Farid 34<br />
Elkon, Rani 46<br />
Elkuizen, Paula 126<br />
Ellenbroek, Saskia 26<br />
193<br />
PERSONNEL INDEX
194<br />
PERSONNEL INDEX<br />
Elouarrat, Dalila 24<br />
Engelsma, Dieuwke 52<br />
Epping, Mirjam 76<br />
Evers, Bastiaan 66<br />
Fabius, Armida 78<br />
Faesen, Alex 18<br />
Fazalalikhan, Arifa 100<br />
Feddema, Wouter 64<br />
Feenstra, Christel 100<br />
Fernandez Salcedo, Ernesto 156<br />
Filion, Guillaume 48<br />
Fish, Alex 18<br />
Fles, Renske 38<br />
Floot, Ben 40, 42<br />
Foekema, Joke 117<br />
Fornerod, Maarten 52<br />
Frederiks, Floor 50<br />
Frijns, Evelyne 22<br />
Gadiot, Jules 61<br />
Gallenne, Tristan 84<br />
Gast, Marie-Christine 116<br />
Gebretensae, Abadi 117<br />
Gehring, Karin 90<br />
Geiger, Thomas 84<br />
Genest, Paul-André 88<br />
Gerard, Audrey 26<br />
Gerlach, Carmen 55<br />
Gerritsen, Bram 72, 78<br />
Gerritsma, Miranda 90<br />
Geurts, Tom 143<br />
Geutjes, Ernst-Jan 76<br />
Gilhuijs, Kenneth 101<br />
Gilles, Rozan 99<br />
Godsave, Sue 36<br />
Gomez, Raquel 57<br />
Gomez Benito, Maria 46<br />
Gonggrijp, Maaike 64<br />
Graafland, Niels 144<br />
Gundy, Chad 90, 92, 94<br />
Haanen, John 57, 116<br />
Haas, Rick 126<br />
Haenen, Inge 99<br />
Hage, Joris 144<br />
Hagenaars, Christiane 156<br />
Hahn, Daniela 94, 101<br />
Hajdo-Milasinovic, Amra 26<br />
Hamming-Vrieze, Olga 126<br />
Haramis, Anna-Pavlina 89<br />
Harms, Emmy 117<br />
Hart, Guus 126<br />
Hau, Song-Hieng 156<br />
Hauptmann, Michael 72<br />
Hausmann, Jens 20<br />
He, Youji 38<br />
Heemsbergen, Wilma 126<br />
Heemskerk, Bianca 55, 57<br />
Heessen, Stijn 52<br />
Heidebrecht, Tatjana 20<br />
Heideman, Marinus 59<br />
Heideman, Wieke 95<br />
Heijens, Claudia 116<br />
Hengeveld, Trudi 24<br />
Hesselink, Gijs 99<br />
Heynen, Guus 76<br />
Hibbert, Rick 18<br />
Hiemstra, Annelies 156<br />
Hijmans, Marielle 76<br />
Hilarius, Doranne 90<br />
Hilgers, Frans 143<br />
Hilkmann, Henk 34<br />
Hillebrand, Michel 117<br />
Hoebers, Frank 126<br />
Hoefnagel, Cornelis 100<br />
Hoekstra, Paula 156<br />
Hofland, Ingrid 40<br />
Hogenbirk, Marc 59<br />
Hogervorst, Frans 38, 100, 101<br />
Hollmann, Birgit 126<br />
Holstege, Henne 66<br />
Holtkamp, Marjo 117<br />
Hölzel, Michael 76<br />
Hömig, Cornelia 84<br />
Hoopman, Rianne 90<br />
Hoppes, Rieuwert 34<br />
Houben, Anna 24<br />
Houben, Diane 36<br />
Hoving, Saske 42<br />
Huang, Sidong 76<br />
Huitema, Alwin 116<br />
Hulsman, Danielle 80<br />
Hummel, Marian 99<br />
Iden, Sandra 26<br />
Iri-Kiraz, Hasibe 38<br />
Iusuf, Dilek 70<br />
Ivanov, Eduard 95<br />
Iyer, Jayasree 64<br />
Jacobs, Heinz 59<br />
Jacobs, Jacqueline 88<br />
Jalink, Kees 29<br />
Jansen, Edwin 126<br />
Jansen, Hans 36<br />
Jansen, Robert 116<br />
Janssen, Esther 95<br />
Janssen, Lennert 31<br />
Jaspers, Janneke 66<br />
Jeanson, Kiki 95<br />
John, Hilkens 86<br />
Jongmans, Petra 143<br />
Jongsma, Maikel 24<br />
Jongsma, Marlieke 31<br />
Jongsma, Johan 82<br />
Jonkers, Jos 66<br />
Jonkman, Joop 156<br />
Joosse, Simon 38<br />
Joosten, Krista 20<br />
Jorritsma, Annelies 57<br />
Joshi, Niel 144<br />
Kaiser, Andrew 55<br />
Kalisvaart, Robin 127<br />
Kalverda, Bernike 52<br />
Kant, Josien 156<br />
Kaplon, Joanna 84<br />
Kappers, Ingrid 143<br />
Kasiem, Mobien 101<br />
Kedde, Martijn 46<br />
Keessen, Marianne 117<br />
Keijzer, Christiaan 144<br />
Keizer, Ron 116<br />
Keller, Anna 53<br />
Kerkdijk, Desiree 143<br />
Kersbergen, Ariena 64<br />
Kerst, Martijn 116<br />
Kerver, Emile 116<br />
Ketema, Mirjam 22<br />
Kim, Yeung-Hyen 57<br />
Kimmings, Nikola 143<br />
Kind, Jop 48<br />
Klarenbeek, Jeffrey 29, 30<br />
Klarenbeek, Sjoerd 66<br />
Klerkx, Edwin 156<br />
Klijn, Christiaan 66, 72<br />
Klokman, Willem 95<br />
Klomp, Houke 143<br />
Klop, Martin 143<br />
Klous, Marjolein 116<br />
Kluijt, Irma 94, 101<br />
Knauer, Michael 68, 100<br />
Knegjens, Joost 126<br />
Knols, Ruud 90<br />
Kok, Marleen 38, 68<br />
Kolmschate, Lies 156<br />
Kolodziej, Katy 48<br />
Kool, Jaap 80, 82<br />
Koolen, Stijn 116<br />
Koopman-Kroon, Ciska 117<br />
Koops, Wim 101<br />
Koorn, Jenneke 156<br />
Koornstra, Rutger 68<br />
Koppelmans, Vincent 92<br />
Korse, Tiny 100<br />
Kortlever, Roderik 76<br />
Kortmann, Jan 144<br />
Koseoglu, Murat 46<br />
Koudijs, Marco 66<br />
Kranenburg, Andor 82<br />
Krap, Menno 143<br />
Kreeft, A 143<br />
Kreft, Maaike 22<br />
Kregel, Eva 66<br />
Kreike, Bas 126<br />
Kremmler, Lukas 61<br />
Krewinkel, Han 126<br />
Krijger, Peter 59<br />
Krimpenfort, Paul 82<br />
Kröger, Robert 101<br />
Kross, Kenneth 143<br />
Kuenen, Marianne 90, 95<br />
Kuijl, Coen 31<br />
Kuiken, Johan 78<br />
Kuikman, Ingrid 22<br />
Kuilman, Thomas 84<br />
Kuiper, Maria 117<br />
Kujala, Pekka 36
Kunst, Peter 116<br />
Kwint, Margriet 127<br />
Lagas, Jurjen 70<br />
Lambooij, Jan Paul 82<br />
Lammens, Chantal 90, 94<br />
Lange, Charlotte 101<br />
Lange, Jan 144<br />
Langerak, Petra 59<br />
Langeslag, Michiel 29<br />
Lankheet, Nienke 117<br />
Le Duc, Quint 22<br />
Le Sage, Carlos 46<br />
Lebesque, Joos 126<br />
Leijen, Suzanne 44<br />
Leijte, Joris 144<br />
Lenain, Christelle 84<br />
Léveillé, Nicolas 46<br />
Lieftink, Cor 78<br />
Linders, Dorothé 100<br />
Linn, Sabine 38, 68, 92, 116<br />
Linnemann, Carsten 55<br />
Littler, Dene 20<br />
Liu, Xiaoling 66<br />
Lohuis, Peter 143<br />
Loo, Claudette 101<br />
Lorist, Lissette 156<br />
Lubsen–Brandsma, Lotti 144<br />
Lucas, Luc 117<br />
Lukas, Anne 116<br />
Maas, Chiel 53<br />
Maas, Marnix 126<br />
Madiredjo, Mandy 76<br />
Mager, Alet 116<br />
Mahn, Marianne 156<br />
Mallo, Henk 117<br />
Manders, Peggy 95<br />
Mandjes, Ingrid 156<br />
Mandy, Boer 86<br />
Mans, Anton 127<br />
Marchetti, Serena 44<br />
Marees, Tamara 95<br />
Margadant, Coert 22<br />
Marijnen, Corrie 126<br />
Masselink, Harry 126<br />
Mattiroli, Francesca 18<br />
Mazgani, Mona 144<br />
McDermott, Leah 127<br />
Meijer, Joost 28<br />
Meijerman, Irma 44<br />
Meijnen, Philip 126<br />
Meinhardt, Wim 144<br />
Meissl, Katrin 84<br />
Mencarelli, Angelo 127<br />
Menendez, Victoria 31<br />
Mertens, Sander 26<br />
Meuleman, Wouter 72<br />
Meulenaar, Jelte 117<br />
Mexner, Vanessa 127<br />
Michalak, Ewa 66<br />
Michalides, Rob 33<br />
Michaloglou, Chrysiis 84<br />
Middendorp, Sabine 53<br />
Mijnheer, Ben 126<br />
Mikolajewska, Izabela 38<br />
Minderhoud, Tom 127<br />
Mitsiki, Eirini 20<br />
Modder, Carla 156<br />
Moerman, Esther 144<br />
Moes, Johannes 117<br />
Molloy, Tim 38<br />
Monserrat, Veronica 31<br />
Mooij, Wijnand 20<br />
Mooij, Thea 95<br />
Mook, Stella 38, 101<br />
Moolenaar, Wouter 24<br />
Moonen, Luc 126<br />
Mulder, Ina 95<br />
Mulder, Lennart 38<br />
Mulder, Renée 95<br />
Mullenders, Jasper 76<br />
Muller, Saar 100, 101<br />
Muller, Pietje 156<br />
Muusers, Rick 100<br />
Nacerdine, Karim 80<br />
Nagel, Remco 46<br />
Nagtegaal, Tanja 90, 94<br />
Naik, Shalin 55<br />
Nan-Offeringa, Lianda 117<br />
Nederlof, Petra 38, 95, 100, 101<br />
Neefjes, Jacques 31<br />
Neijenhuis, Sari 40<br />
Nieuweboer-Krobotova, Inka 144<br />
Nieuwenhuis, Lotte 143<br />
Nieweg, Omgo 143<br />
Nijholt, Juup 127<br />
Nijkamp, Jasper 127<br />
Nijkamp, Wouter 78<br />
Nijwening, Jeroen 78<br />
Nol, Annemarie 117<br />
Nooijen, Willem 100<br />
Nota, Ingrid 99<br />
Numan, Rachel 143<br />
Nuyen, Bastiaan 116<br />
Nuyten, Dimitry 126<br />
Nyst, Heike 143<br />
Ogink, Janneke 74<br />
Oldenburg, Hester 90, 143<br />
Olijve, Albert 117<br />
Olsthoorn-Ooms, Marije 101<br />
Olszewska, Agnieszka 127<br />
Ong, Nico 36<br />
Oostendorp, Roos 44, 117<br />
Oosterhuis, Koen 57<br />
Oosterkamp, Rianne 76<br />
Oostveen, Lida 156<br />
Oude Vrielink, Joachim 46<br />
Ouwens, Gabey 95<br />
Ovaa, Huib 34<br />
Paape, Anita 101<br />
Pagie, Ludo 48<br />
Pajic, Marina 64<br />
Pameijer, Frank 101<br />
Pang, Baoxu 31<br />
Panneman, Carmen 127<br />
Paul, Petra 31<br />
Paulus van Pauwvliet, Cecile 156<br />
Pawlitzky, Inka 80<br />
Peeper, Daniel 84<br />
Pengel, Kenneth 101, 127<br />
Peperzak, Victor 53<br />
Peric-Hupkes, Daniel 48<br />
Perrakis, Anastassis 20<br />
Peters, Carolien 127<br />
Peters, Peter 36<br />
Peuscher, Marieke 88<br />
Piek-den Hartog, Marianne 143<br />
Pierson, Jason 36<br />
Pieterse, Mark 66<br />
Pietersen, Alexandra 80<br />
Pijpe, Anouk 95<br />
Pindyurin, Alexey 48<br />
Ploeger, Lennert 127<br />
Plug, Rob 101<br />
Pluim, Dick 44<br />
Ponsioen, Bas 24, 29<br />
Pool, Bert 100<br />
Pos, Floris 126<br />
Postel, Ruben 22<br />
Pramana, Jimmy 143<br />
Prevoo, Warner 101<br />
Prieur, Alexandre 84<br />
Pritchard, Colin 82<br />
Pronk, Loes 156<br />
Proost, Natalie 82<br />
Pruntel, Roelof 101<br />
Puppe, Julian 80<br />
Qiao, Xiaohang 31<br />
Quaak, Susanne 117<br />
Rasch, Coen 126<br />
Rehorst, Harriet 156<br />
Reichgelt, Babs 126<br />
Reinders, Anneke 156<br />
Reker Hadrup, Sine 55<br />
Remeijer, Peter 126<br />
Remmelzwaal, Jolanda 156<br />
Retel, Valesca 99<br />
Reumer, Annet 18<br />
Richard, Wietske 143<br />
Rijkhorst, Erik-Jan 127<br />
Rinia, Bas 143<br />
Rit, Simon 127<br />
Roberts, Daniel 156<br />
Rodenhuis, Sjoerd 116<br />
Rodenko, Boris 34<br />
Roelvink, Marja 117<br />
Rondaij, Mariska 33<br />
Ronday, May 144<br />
Rookus, Matti 95<br />
Roos, Ed 28<br />
Roosjen, Edwin 127<br />
Rooswinkel, Rogier 53<br />
Rooze, Lyandra 100<br />
Rosado, Aranzazu 84<br />
195<br />
PERSONNEL INDEX
196<br />
PERSONNEL INDEX<br />
Rosenberg, Efraim 100, 101<br />
Rosing, Hilde 116<br />
Roskam, Marielle 156<br />
Rossi, Maddalena 127<br />
Rottenberg, Sven 64<br />
Rubio, Miguel 76<br />
Rucktooa, Prakash 18<br />
Ruers, Theo 143<br />
Ruijter-Schippers, Henrique 101<br />
Russell, Nicola 42, 95, 126<br />
Rutgers, Emiel 90, 95, 143<br />
Rzadkowski, Adrian 24<br />
Sachs, Norman 22<br />
Salomon, Izhar 31<br />
Salverda, Govert 126<br />
Sander, Saskia Amra 26<br />
Sani, Musa 36<br />
Scanu, Tiziana 31<br />
Schaap, Jeffrey 143<br />
Schaefer, Henning 46<br />
Schagen, Sanne 92<br />
Scharpfenecker, Marion 42<br />
Scheenstra, Renske 143<br />
Scheerman, Esther 101<br />
Schellens, Jan 44, 116<br />
Schilder, Christien 92<br />
Schinkel, Alfred 70<br />
Schippers-Gillissen, Carla 101<br />
Schmidt, Marjanka 38, 95, 101<br />
Schmitz, Alexander 101<br />
Schmitz, Annemarie 101<br />
Schneider, Christoph 126<br />
Schol, Joke 117<br />
Schornagel, Jan 116<br />
Schot, Margaret 117<br />
Schotte, Remko 55<br />
Schrier, Mariette 46<br />
Schumacher, Ton 55<br />
Schuster, Kerstin 61<br />
Schutte, Peter 144<br />
Schuurman, Karianne 34<br />
Secades, Pablo 22<br />
Seemann, Ingar 42<br />
Seigers, Riejanne 92<br />
Sein, Johan 57<br />
Shanmugham, Anitha 34<br />
Shu, Jenny 55<br />
Siedschlag, Christian 101<br />
Simon, Horenblas 144<br />
Sinaasappel, Michiel 100, 101<br />
Sivro Prndelj, Ferida 100<br />
Sixma, Titia 18<br />
Smeele, Ludi 143<br />
Smit, Judith 18<br />
Smit, Linda 76<br />
Smit, Marjon 84<br />
Smits, Marianne 116<br />
Smitsmans, Monique 127<br />
Snoek, Margriet 82<br />
Sonke, Gabe 116<br />
Sonke, Jan-Jakob 127<br />
Sonneborn, Mariska 100<br />
Sonnenberg, Arnoud 22<br />
Sparmann, Anke 80<br />
Speijer, Gabriele 126<br />
Speksnijder, Ewoud 66<br />
Srámek, Michael 144<br />
Sriram, Jincey 101<br />
Steenbakkers, Roel 126<br />
Stewart, Fiona 42<br />
Stoppa, Tino 144<br />
Storm, Dea 156<br />
Straver, Marieke 143<br />
Stroom, Joep 126<br />
Stulemeijer, Iris 50<br />
Sutherland, Kate 82<br />
Swart, Erwin 55<br />
Taal, Babs 116<br />
Taghavi, Panthea 80<br />
Talhout, Wendy 48<br />
Tan, Bing 143<br />
Tanger, Ellen 80<br />
Te Poele, Johannes 42<br />
Te Riele, Hein 62<br />
Te Velde, Lisette 143<br />
Teertstra, Jelle 101<br />
Temurhan, Mine 127<br />
Ten Bokkel Huinink, Wim 116<br />
Ten Cate, Julia 144<br />
Ter Brugge, Petra 66<br />
Ter Heine, Rob 117<br />
Ter Riet, Bas 64, 74<br />
Tesselaar, Margot 116<br />
Teunissen, Bas 117<br />
Teunissen, Hans 80<br />
Teunissen, Jaap 100<br />
Thijssen, Bas 117<br />
Tibben, Matthijs 117<br />
Tick, Lidwine 116<br />
Tielen, Ivon 101<br />
Tielenburg, René 127<br />
Tilgenkamp, Ria 156<br />
Timmers, Adriaan 144<br />
Tjin, Esther 57<br />
Toebes, Mireille 55<br />
Tolhuis, Bas 80<br />
Topolnjak, Rajko 127<br />
Torres Acosta, Alex 156<br />
Tran, Ly 117<br />
Tromp-van Driel, Catrien 116<br />
Udo, Renate 95<br />
Urbanus, Jos 55<br />
Uren, Anthony 82<br />
Uyterlinde, Wilma 117<br />
Vaessen, Heleen 156<br />
Vainchtein, Liia 117<br />
Vainio, Saara 64<br />
Valdés Olmos, Renato 100<br />
Valkenet, Ludy 156<br />
Van ’t Veer, Laura 38, 95, 100, 101<br />
Van As-Brooks, Corina 144<br />
Van Bemmel, Joke 48<br />
Van Berkel, Peter 99<br />
Van Beurden, Marc 90, 143, 144<br />
Van Blitterswijk, Wim 30<br />
Van Boven, Hester 100<br />
Van Bunningen, Bart 126<br />
Van Coevorden, Frits 143<br />
Van Dam, Frits 92<br />
Van de Ahé, Fina 82<br />
Van de Kasteele, Willeke 57<br />
Van de Meij, Suzan 143<br />
Van de Poel, Henk 144<br />
Van de Steeg, Evita 70<br />
Van de Velde, Tony 156<br />
Van de Vijver, Marc 100<br />
Van de Wetering, Koen 64<br />
Van Deemter, Liesbeth 64<br />
Van den Belt-Dusebout, Sandra 95<br />
Van den Berg, Joost 57, 117<br />
Van den Berk, Paul 59<br />
Van den Bongard, Desiree 126<br />
Van den Brekel, Michiel 143, 144<br />
Van den Brink-de Vries, Nienke 100<br />
Van den Haak, Marjolijn 156<br />
Van den Heuvel, Michel 116<br />
Van den Hoorn, Tineke 31<br />
Van den Hoven, Jolanda 117<br />
Van der Berg, Marieke 144<br />
Van der Burg, Eline 66<br />
Van der Donk, Emile 156<br />
Van der Gulden, Hanneke 66<br />
Van der Heijden, Ingrid 66, 68<br />
Van der Horst, Gerda 53<br />
Van der Kammen, Rob 26<br />
Van der Kant, Rik 31<br />
Van der Kruijssen, Corina 70<br />
Van der Maas, Martin 57<br />
Van der Molen, Lisette 143<br />
Van der Ploeg, Iris 143<br />
Van der Pot, Wouter 144<br />
Van der Sar, Jana 117<br />
Van der Torre, Jaco 88<br />
Van der Veen, Wietze 144<br />
Van der Velde, Hella 52<br />
Van der Velden, Yme 89<br />
Van der Velden, Sophie 101<br />
Van der Wal, Anja 62<br />
Van der Weide, Marchien 116<br />
Van der Wel, Nicole 36<br />
Van Dijk, Pim 18<br />
Van Dongen, Miranda 76<br />
Van Driel, Willemien 144<br />
Van Duijn, Miranda 99<br />
Van Esch, Anita 70<br />
Van Gijn, Roel 116<br />
Van Haaften, Gijs 46<br />
Van Harten, Wim 99<br />
Van Heijst, Jeroen 55<br />
Van Hell, Albert 30<br />
Van Herk, Marcel 126<br />
Van Hien, Richard 38<br />
Van Huizum, Martine 144
Van Kouwenhove, Marieke 46<br />
Van Kranen, Simon 127<br />
Van Leeuwen, Flora 38, 95<br />
Van Leeuwen, Fred 50<br />
Van Leeuwen, Fijs 42, 100, 101<br />
Van Leeuwen, Anne 101<br />
Van Lent, Wineke 99<br />
Van Lohuizen, Maarten 80<br />
Van Luenen, Henri 64<br />
Van Meer, Oene 126<br />
Van Montfort, Erwin 82<br />
Van Mourik, Anke 127<br />
Van Netten, Gabry 156<br />
Van Noord, Janet 88<br />
Van Oosterwijk, Els 156<br />
Van Pel, Renée 100<br />
Van Rens, Anja 101<br />
Van Sandick, Johanna 143<br />
Van Seters, Manon 144<br />
Van Steensel, Bas 48<br />
Van Tellingen, Olaf 100<br />
Van Tinteren, Harm 156<br />
Van Tongeren, Joost 144<br />
Van Triest, Baukelien 126<br />
Van Velthuysen, Loes 100<br />
Van Vliet, Martin 72<br />
Van Vliet-Vroegindeweij, Corine 126<br />
Van Vugt, Huub 80<br />
Van Waardenberg, Wil 156<br />
Van Waterschoot, Robert 70<br />
Van Welsem, Tibor 50<br />
Van Werkhoven, Erik 156<br />
Van Winden, Annemieke 117<br />
Van Zandwijk, Nico 116<br />
Van Zeijl, Leonie 24<br />
Van Zon, Maaike 36<br />
Van Zon, Wouter 74<br />
Van Zwienen, Marieke 126<br />
Vanneste, Ben 126<br />
Vargas, Mark 18<br />
Veltkamp, Sander 44<br />
Vens, Conchita 40<br />
Veraar, Elise 53<br />
Verbrugge, Inge 53<br />
Vergouwe, Ingeborg 144<br />
Verhagen, Caroline 40<br />
Verhagen, C 144<br />
Verheij, Marcel 30, 126<br />
Verhoef, Senno 38, 90, 94, 95, 101<br />
Verhoeven, Els 80<br />
Verloop, Janneke 95<br />
Vermeeren, Lenka 100<br />
Vermeulen, Eric 90, 95<br />
Verschueren, Karijn 126<br />
Verwaal, Vic 143<br />
Verwijs, Manon 40<br />
Verwoerd, Désirée 33<br />
Verzijlbergen, Kitty 50<br />
Vincent, Andrew 156<br />
Visser, Daan 29<br />
Visser, Nils 42<br />
Vissers, Joep 80<br />
Vlaming, Marijn 70<br />
Vlaskamp, Marcel 156<br />
Voets, Erik 74<br />
Vogel, Maartje 48<br />
Vogel, Celia 84<br />
Vogel, Wouter 100<br />
Vollebergh, Marieke 68<br />
Voncken, Francine 126<br />
Vormer, Tinke 62<br />
Vos, Matthijn 36<br />
Vos, Sanne 144<br />
Voskuil, Dorien 95<br />
Vredeveld, Liesbeth 84<br />
Vroonland, Colinda 100<br />
Vuist, Ilona 29<br />
Vyth-Dreese, Florry 57<br />
Wagenaar, Els 70<br />
Wals, Anneke 156<br />
Wang, Liqin 89<br />
Wendling, Markus 127<br />
Wesseling, Jelle 100<br />
Wessels, Lodewyk 72<br />
Westerman, Bart 80<br />
Wever, Lidwina 156<br />
Wevers, Marijke 90<br />
Wielders, Camiel 62<br />
Wielders, Eva 62<br />
Wientjens, Ellen 66<br />
Wieringa-Ariaens, Aafke 101<br />
Wigbout, Gea 101<br />
Wijnands, Yvonne 28<br />
Willemse, Els 156<br />
Winterwerp, Herrie 18<br />
Wit, Niek 59<br />
Witte, Marnix 127<br />
Wittkämper, Frits 126<br />
Woerdeman, Leonie 94, 144<br />
Wolthaus, Jochem 127<br />
Wolthuis, Rob 74<br />
Wouters, Michel 143<br />
Xiao, Yanling 53<br />
Xylourgidis, Nikos 52<br />
Yang, Jonathan 127<br />
Yanover, Eva 87<br />
Yüksel, Gül 127<br />
Zandbergen, Jeroen 156<br />
Zander, Serge 64<br />
Zerp, Shuraila 30<br />
Zevenhoven, John 82<br />
Ziblat, Lonny 156<br />
Zijp, Lambert 127<br />
Zlotorynski, Eitan 46<br />
Zupan-Kajcovski, Biljana 144<br />
Zuur, Karel 144<br />
Zwart, Wilbert 33<br />
197<br />
PERSONNEL INDEX
THE<br />
NETHERLANDS<br />
CANCER<br />
INSTITUTE<br />
SCIENTIFIC<br />
ANNUAL<br />
REPORT <strong>2008</strong><br />
The Netherlands Cancer Institute<br />
Antoni van Leeuwenhoek Hospital<br />
Plesmanlaan 121<br />
1066 CX Amsterdam<br />
www.nki.nl<br />
p26.1<br />
p25.3<br />
p24.3<br />
p24.2<br />
p24.1<br />
p22.3<br />
p22.1<br />
p21.31<br />
p14.3<br />
p14.2<br />
p14.1<br />
p13<br />
p12.3<br />
p12.2<br />
p12.1<br />
q11.2<br />
q13.11<br />
q13.13<br />
q13.31<br />
q21.3<br />
q22.1<br />
q23<br />
q24<br />
q26.1<br />
q26.2<br />
q26.31<br />
q26.32<br />
p26.33<br />
p28<br />
p29<br />
p15.33<br />
p15.31<br />
p15.2<br />
p15.1<br />
p14.3<br />
p14.1<br />
p13.3<br />
p13.2<br />
p13.1<br />
p12<br />
q11.2<br />
q12.1<br />
q12.3<br />
q13.2<br />
q13.3<br />
q14.1<br />
q14.3<br />
q15<br />
q21.1<br />
q21.3<br />
q23.1<br />
q23.2<br />
q23.3<br />
q31.1<br />
q31.3<br />
q32<br />
q33.1<br />
q33.2<br />
q33.3<br />
q34<br />
q35.1<br />
q35.2<br />
q35.3