huhhhhuh - NKI / AvL

Patron HM The Queen Beatrix

SCIENTIFIC ANNUAL REPORT 2003 

THE NETHERLANDS CANCER INSTITUTE 

CANCER RESEARCH LABORATORY AND CANCER HOSPITAL 

www.nki.nl

COPYRIGHT 

Scientific Annual Report 2003 

Illustrations and unpublished data in these reports may not be used without permission of the author. 

Copyright ©: 

The Netherlands Cancer Institute 

Antoni van Leeuwenhoek Hospital 

PIesmanlaan 121 

1066 CX Amsterdam 

The N etherlands 

Phone +31 20 512 9II1 

Fax +31 20 617 2625 

ISSN 1387-86II

CONTENTS 

Board Members 8 

Research Divisions 9 

Introduction 

Il 

Education in Oncology 16 

I Division of Cell Biology 19 

11 Division of Molecular Carcinogenesis 27 

111 Division of Cellular Biochemistry 35 

IV Division of Immunology 43 

V Division of Molecular Biology 52 

VI Division of Tumor Biology 60 

VI I Division of Molecular Genetics 70 

VIII Division of Experimental Therapy 78 

IX Division of Radiotherapy 89 

X Division of Medical Oncology 100 

XI Division of Surgical Oncology IlO 

XII Division of Psychosocial Research and Epidemiology Il9 

XIII Division of Diagnostic Oncology 126 

Biometrics Department 134 

Research Facilities 139 

Clinical Trials 146 

Invited Speakers 158 

Projects 160 

Funding 171 

Personnel Index 172

Board of Oirectors 

AJM Berns, chairman and director of research 

S Rodenhuis, director clinical research and development 

WH Van Harten, director organization and 

management 

BOARD MEMBERS 

International Scientific Advisory Board 

JR Bertino, Professor of Medicine and Pharrnacology, 

The Cancer Institute ofNew Jersey, New Brunswick, 

USA 

RA Flavell, Professor of Immunobiology, 

Yale University School of Medicine, New Haven, USA 

S Hellman, AN Pritzker Distinguished Service 

Professor, The University of Chicago, Chicago, USA 

WGJ Hol, Professor of Biochemistry and Biological 

Structure, University of Washington, Seattle, USA 

J Mendelsohn, President, MD Anderson Cancer 

Center, The University ofTexas, Houston, USA 

P Nurse, Professor of Microbiology, Director-Generalof 

the Imperial Cancer Research Fund, London, UK 

R Nusse, Professor of Developmental Biology, 

Stanford University, Stanford, USA 

HL Ploegh, Edward Mallinckrodt Jr Professor of 

Immunopathology, Harvard Medical School, Boston, 

USA 

RA Wei n berg, Professor of Biology, Massachusetts 

Institute ofTechnology, Whitehead Institute, 

Cambridge, USA 

C Weissman, Professor of Neurogenetics, Imperial 

College School of Medicine at St. Mary's, Londen, UK 

Board of Governors 

WF Duisenberg, president 

HCJ Van der Wielen, vice-president 

PJ Kalff, treasurer 

WKok 

D Sinninghe Damsté 

JHMTemmink 

GNJ Tytgat 

PF Van der Heijden 

J Van der Meer 

GP Vooijs 

MWM Vos-van Gortel 

Scientific Advisory Council 

AJM Berns, president 

W Mooienaar, secretary 

RMedema 

J Neefjes 

S Rodenhuis 

L Van 't Veer 

National Scientific Advisory Board 

LA Aarden, Professor of Molecular Immunology, 

Amsterdam 

SWJ Lamberts, Professor ofInternal Medicine, 

Rotterdam 

B Löwenberg, Professor of Hematology, Rotterdam 

Cl LM Meijer, Professor of Pathological Anatomy, 

Amsterdam 

Cl M Melief, Professor ofImmunohematology, Leiden 

H M Pinedo, Professor of Clinical Oncology, 

Amsterdam 

FH Schröder, Professor ofUrology, Rotterdam 

G N J Tytgat, Professor of Gastroenterology, Amsterdam 

AJ Van der Eb , Professor of Fundamental Virology, 

Leiden 

PC Van der Vliet, Professor of Physiological Chemistry, 

Utrecht

RESEARCH DIVISIONS 

I Cell Biology 

Arnoud Sonnenberg (head) 

J ero Calafat 

J ohn Collard 

Kees Jalink 

Ed Roos 

Natascha Borsje (secretary) 

II Molecular Carcinogenesis 

René Bernards (he ad) 

Roderick Beijersbergen 

Anastassis Perrakis 

Titia Sixma 

Marlijn Sonne (secretary) 

111 Cellular Biochemistry 

Peter Ten Dijke (head) 

Nullin Divecha 

Wouter MooIenaar 

Wim Van Blitterswijk 

Marcel Verheij 

Suzanne Corsetto (secretary) 

IV I mmunology 

J annie Borst (head) 

John Haanen 

Heinz Jacobs 

Ton Schumacher 

Florry Vyth-Dreese 

José Overwater (secretary) 

V Molecular Biology 

Hein Te Riele (he ad) 

Piet Borst (honorary staff member) 

René Medema 

Henri Van Luenen 

Jos Jonkers 

Tom De Knegt (secretary) 

Linda Römer (secretary) 

VI Tumor Biology 

Jacques Neefjes (head) 

Reuven Agami 

Maarten Fomerod 

J ohn Hilkens 

Rob Michalides 

Peter Peters 

Marieke Van der Velde (secretary) 

VII Molecular Genetics 

Maarten Van Lohuizen (he ad) 

Anton Berns 

Paul Krimpenfort 

Daniel Peeper 

Margriet Snoek 

Gerdien De Kuijer (secretary) 

Marie Anne Van Halem (secretary) 

VIII Experimental Therapy 

Adrian Begg (he ad) 

Petra Nederlof 

Jan Schellens 

Alfred Schinkel 

Fiona Stewart 

Marc Van de Vijver 

Bas Van Steensel 

Laura Van 't Veer 

Thea Eggenhuizen (secretary) 

IX Radiotherapy 

Harry Bartelink 

Berthe Aleman 

Jose BeIderbos 

Liesbeth Boersma 

Eugenè Damen 

RoeI De Boer 

Katrien De Jaeger 

Luc Dewit 

Rick Haas 

Guus Hart 

Wilma Heemsbergen 

Edwin Jansen 

Joos Lebesque 

Harry Masselink 

Ben Mijnheer 

Luc Moonen 

Coen Rasch 

Peter Remeijer 

Nicola Russell 

Govert Salverda 

Christoph Schneider 

Joep Stroom 

Bart Van Bunningen 

Marcel Van Herk 


Fritts Wittkämper 

Patricia Haye-Fewer, Secretary 

Elly Hofmann, Secretary 

Desiree Van Ham, Secretary 

X Medical Oncology 

Sjoerd Rodenhuis (head) 

Joke Baars 

Paul Baas 

Evert Bais 

Jos Beijnen 

Willem Boogerd 

Henk Boot 

Annemieke Cats 

Jan Paul De Boer 

Bert De Gast 

John Haanen 

Martijn Kerst 

Sabine Linn 

Herman Neering 

Jan Schellens 

Jan Schornagel 

Babs Taal 

Wim Ten Bokkel Huinink

Jaap Van der Sande 

Marchien Van der Weide 

Nico Van Zandwijk 

Ans Vielvoye-Kerkmeer 

Mariëlle De Kwant (secretary) 

Annemieke Hoogland (secretary) 

XI Surgical Oncology 

Bin Kroon (board; head) 

Fons Balm (board) 

Omgo Nieweg (board) 

Axel Bex 

Dick Buitelaar 

Matthé Burger 

Marcel Copper 

Myrtille De Vries 

Steven Gonggrijp 

Joris Hage 

Frans Hilgers 

Simon HorenbIas 

Hans Huitink 

Houke Klomp 

Frans Kroon 

Peter Lohuis 

Lottie Lubsen 

Wim Meinhardt 

Hester Oldenburg 

May Ronday 

Emiel Rutgers 

Peter Schutte 

Marian Slot 

Ludi Smeele 

BingTan 

Julia Ten Cate 

Adriaan Timmers 

Marc Van Beurden 

Frits Van Coevorden 

Michiel Van den Brekel 

Henk Van der Poel 

Vic VerwaaI 

Leonie Woerdeman 

Frans Zoetrnulder 

Noëlle Tameris (secretary) 

Marion Van Zuilen (secretary) 

Wim Koops 

Robert Kröger 

Claudette Loo 

Wolter Mooi 

Saar Muller 

Petra Nederlof 

Willem Nooijen 

Frank Pameijer 

Hans Peterse 

Wamer Prevoo 

Ferida Sivro 

Jelle Teertstra 

Renato Valdes Olmos 

Hester Van Boven 

Ol af Van Tellingen 

Laura Van 't Veer 

Loes Van Velthuysen 

Senno Verhoef 

Christine Arkes (secretary) 

Carla Van Tiggelen (secretary) 

Biometncs Department 

Otilia Dalesio (head) 

Harm Van Tinteren 

HEADS OF GENERAL RESEARCH SERVICES 

Financial Administration 

Christel Deckers 

General Facilities 

Petra Tuyp 

Personnel Department 

Eric De Wilde 

Research Coordination 

Wouter MooIenaar, laboratory research coordinator 

Henri Van Luenen, laboratory research manager 

Egbert Vos, clinical research manager 

XI I Psycosocial Research and Epidemiology 

Neil Aaronson (he ad) 

Matti Rookus 

Frits Van Dam 

Flora Van Leeuwen 

Marian Chin-A-Kwie (secretary) 

XI I I Diagnostic Oncology 

Marc Van de Vijver (head) 

Philippe Baars 

Peter Besnard 

Hans Bonfrer 

Daphne De Jong 

Kenneth Gilhuijs 

Cees Hoefnagel 

Frans Hogervorst

':: ~,~>~~-:"'.-:",'" ',' I 

.>.~: : ' , :.:.' :', _~',","'~.'. '''', ::-.: .,_,]", ,'._ ":~":j 

I~-·'::'Q ~ _~j ~ 

Director of Research Ton Berns 

INTRODUCTION 

I am pleased to present our Scientific Annual Report 

2003. It provides an overview of the scientific activities 

at The Netherlands Cancer InstitutejAntoni van 

Leeuwenhoek Hospital (NKljAvL). Additional 

information can be found on the websites of the various 

divisions, as weIl as at www.nki.nl. 

NKljAvL is a Comprehensive Cancer Center, combining 

hospital and research laboratories under one roof in a 

single independent organization. The hospital comprises 

180 beds, and outpatient clinic and a large radiotherapy 

department. Facilities for patient research include a 

large patient database, clinical data management and 

active research groups in epidemiology and psychosocial 

oncology. The laboratory covers all major areas of cancer 

research, with special emphasis on cell-based screens, 

mouse tumor modeis, cell biology, structural biology, 

immunology and translational research. There is close 

collaboration between clinical and basic scientists. This 

scientific report mainly deals with the clinical and basic 

science in the NKlj AvL. Information on patient-care is 

described in our General Report. 

In 2003, our new hospital building and the new 

radiotherapy unit became available and all clinical 

activities were transferred to the new premises. Some 

fine-tuning and small adjustments are still needed, but 

we expect that the hospital will be on line at full capacity 

and to full satisfaction by March 30, 2004, the day of the 

official opening. This will be amemorable milestone. 

The next phase, comprising the renovation of the 

research facilities, represents an even more daunting 

task. We expect that the renovated research facilities 

become available in 2007. 

As was the case in 2002, shortage ofhospital personnel 

has prevented us from substantial growth of our clinical 

activities in 2003, although we did observe a growth in 

productivity in the last quarter of the year. Particular, our 

outpatient clinic and radiotherapy department contribute 

to this increase of productivity. Whereas we we re able to 

fill most critical vacancies of medical specialists, 

shortage of oncology nurses continued to be a 

bottleneck. We expect that this shortage will diminish 

following a range of measures we have already taken. 

The integration of clinical and basic research continues 

to grow. This is not only the result of our long-time 

investment in building tumor banks and patient records 

for expre'ssion profiling and other molecular analyses, 

but basic research leads more of ten than before to direct 

clinical application. An example is mentioned under the 

highlights for the treatment of cylindromatosis. 

We have started to utilize micro-array analysis as part of 

the diagnostic routine for a subset of our breast cancer 

patients. Micro-array analysis of tumors permits a 

considerably more accurate prediction of disease 

progression than has been possible so far. A substantial 

part of early breast cancer patients can now be spared 

chemotherapy as micro-array analysis accurately predicts 

that they do not require it. To facilitate introduction of 

this diagnostic service, two staff members have founded 

a spin-off company, called AGENDIA.. AGENDIA, 

which is occupying labo ra tory space in the nearby 

Slotervaart hospital, will provide this service to our 

patients and aims to expand this service to other 

hospitais. We have obtained a grant of CVZ (a national 

agency supporting introduction of new diagnostic and 

therapeutic procedures) to test if this service can be 

offered on a routine basis to patients elsewhere in the 

country. In addition, the benefits for the patients and 

cost-effectiveness of this service will be evaluated. Based 

on the information gathered, this service might become 

part of the general diagnostic service accessible to all 

patients with breast cancer in The Netherlands. 

Highlights 

The scientific output of the institute is very high for 

a medium-sized Institute; 2003 was a specifically 

succes sf ui year with a substantial increase in the 

number of citations of publications over previous years 

and a significant rise in impact of the scientific reports 

published in 2003. While gratifying, one should realize 

that this is only one measure of scientific productivity. 

Our work can also be gauged by the appreciation of 

colleagues, from the invitations of our staff to 

prestigious scientific meetings, and our competitiveness 

Tabl 1 

Research funding NKljAvL from the different sources in period 1981-2°°3 in million Euros. 

Year 1981 1984 1987 1990 1993 1996 1999 2001 2002 2003 

Cancer Society 3.0 2·5 1.9 2·5 4.1 5.6 7.0 8·7 8.6 9.1 

Ministry of Health 6·9 7.2 6·5 7·3 7·3 8.1 8.8 9·4 9·9 9·9 

Project Grants 2.0 ).0 4·5 6·4 8.1 8.6 12.0 12.6 14.0 15·0'i~ 

Other income 0.6 0.8 1.1 2.6 2·5 2·9 2·4 3.8 3·3 3·5"( 

Total 12·5 1),5 14.0 18.8 22.0 25.2 3°·2 34.0 35.8 37·5* 

.,~ estimates

j'~ • ••:: '11,'7 . -. . 

• • • ': _. . '. • • >. .' .. ' • 

for grants. Staff members of the Institute have also been 

very succes sf ui in these areas in 2003. 

It is always difficult to estimate the impact of research in 

a phase in which the results are still fresh. The research 

highlights summarized here, serve primarily as an 

overview of interesting work currently ongoing in the 

Institute. The results of our most appealing publications 

in 2003 were already mentioned in the SAR of 2002. 

For a more complete and detailed account of specific 

projects I refer to the reports of the individual group 

leader in this SAR. 

J ohn Collard and coworkers (Division of Cell Biology) 

found that Tiaml is a WNT-responsive gene and that 

Tiaml-deficiency affects the initiation and growth of 

intestinal tumors formed in APC mutant mice. 

TIAMr/RAC signaling may thus play a role in the 

formation and progression of tumors induced by various 

oncogenic pathways. 

The group of René Bernards (Division of Molecular 

Carcinogenesis ) succeeded in setting the next step in 

RNAi technology development by constructing a human 

RNAi library in retroviral vectors that allowed to perform 

synthetic lethal screens, providing a direct route to 

pathways that can act as sensitive drug targets only in 

tumor cells carrying specific genetic lesions. In order to 

make this enterprise affordable, René Bemards 

managed to secure the financial support from several 

organizations, such as the Centre of Biomedical 

Genetics, the Cancer Genomics Center and Cancer 

Research UK (CR-UK). A consortium supported by an 

EU-FP6 program has been formed for the generation of 

a mouse RNAi library. 

Thijn Brummelkamp and colleagues performed a 

functional genetic screen to search for members of 

the family of de-ubiquitinating enzymes that regulate 

NF-KB. In this siRNA-based screen, they identified the 

cylindromatosis tumor suppressor gene as a key 

regulator of a protein kinase named IKKbeta, which 

controls NF-KB activity. Importantly, they found that 

aspirin could correct the deregulated activity of this 

kinase, which results from loss of the cylindromatosis 

tumor suppressor. This suggests a simple therapeutic 

intervention strategy to treat familial cylindromatosis. 

A clinical study to test the efficacy of aspirin in the 

treatment of cylindromatosis is currently under way in 

our hospital. This is an example of 'bench to bedside' 

where fundamental research led to a clinical trial in less 

than a year. This is likely to be seen more often, as basic 

research will increasingly identify therapeutic targets 

that are affected by drugs already tested and proven to be 

safe for other disease indications. 

The micro-array analyses ofbreast cancer patients 

performed by Laura Van 't Veer and Marc Van de Vijver 

(Division of Diagnostic Oncology), in collaboration with 

the group of René Bernards and scientists from Rosetta 

Inpharmatics (USA), showed that the molecular 

program established in a primary breast carcinoma is 

not only highly preserved in its regionallymph-node 

metastasis, but also in its distant metastasis. These 

findings suggest that metastatic capability in breast 

cancer is an inherent feature, and challenges the dogma 

of gradual progression of tumors from benign to high 

malignancy. The results further imply that neo-adjuvant 

treatment given to patients based on the response 

expres sion profiles of their primary breast tumor, may 

also prevent the outgrowth of micrometastases. It has 

major consequences for how-and-when to treat patients, 

and will boost the application of expres sion analysis in 

treatment decisions. 

Wim Van Blitterswijk and colleagues (Division of 

Cellular Biochemistry) showed the importance of 

sphingomyelin in lipid rafts for apoptosis induction by 

alkyl-Iysophospholipid. Cellular resistance to this 

anticancer ether-lipid is related to defective 

sphingomyelin synthesis and is accompanied by crossresistance 

to other stress inducers. The group also 

found that certain short-chain sphingolipid analogues 

potentiate doxorubicin cytotoxicity, also when applied in 

liposomal formulation. 

A multidisciplinary study performed by Rob Michalides, 

Alexander Griekspoor and Jacques Neefjes (Division of 

Tumor Biology), in collaboration with Kees Jalink and 

Laura van 't Veer, revealed an important mechanism 

explaining tamoxifen -resistance of breast tumors. 

Phosphorylation of the estrogen receptor converted 

tamoxifen from a growth inhibitor into a stimulator. 

This has important consequences for the clinic, and 

reagents are being made to detect tamoxifen resistance 

at an early phase. 

Eric Reits and colleagues have defined the earliest steps 

in antigen presentation by MHC class I molecules by 

following the fate of intracellular peptides. They 

showed that most peptides are destroyed before being 

considered by MHC class I molecules. As aresult, only 

proteins present in relative high copy numbers will be 

presented by MHC class I molecules to the immune 

system. Reuven Agami and Mathijs Voorhoeve applied 

RNAi technology to separate the function of two genes 

in the INI

self tissues, to which the endogenous mouse T-cell 

repertoire is tolerant. These experiments provide 

conceptual support for the future testing ofT-cell 

receptor gene transfer in clinical trials. 

The group of Alfred Schinkel (Division of Experimental 

Therapy) demonstrated in a knockout mouse model that 

the Breast Cancer Resistance Protein (BCRP jABCG2) is 

a major factor in reducing exposure to the food 

carcinogen 2-amino-r-methyl-6-phenylimidazo[4,s-b] 

pyridine (PhIP). Humans on a normal western diet are 

exposed to PhIP on a daily basis. The results suggest that 

intra- or interindividual differences in BCRP activity in 

humans may likewise affect the exposure to PhIP and 

related food carcinogens, with possible consequences 

for carcinogen susceptibility. 

The group of Bas Van Steensel and collaborators 

published genomic maps of the binding sites of 

7 chromatin proteins and transcription factors in 

Drosophila, thus firmly establishing their Dam 

methyltransferase chromatin profiling technology. 

The Piet Borst group (Division of Molecular Biology) 

found that one of the Multidrug Resistance (associated) 

Proteins, MRP 4, can transport prostaglandins Er and E2, 

signal molecules that are involved in many physiological 

responses, such as the inflammatory response. 

Painkillers often work by blocking the biosynthesis of 

these prostaglandins, and the group showed that some 

of these drugs (e.g. indomethacin) also inhibit transport 

by MRP 4- How important MRP 4 is for the physiological 

effects of prostaglandins is under investigation. 

Hein Te Riele and coworkers found that deficiency for 

the mis match repair protein MSH3 allows the 

introduction of four-nucleotide insertions in 

endogenous genes by single-stranded DNA 

oligonucleotides. As MSH3-deficient ES cells have no 

overt mutator phenotype, this system provides a timesaving 

alternative to current gene knockout procedures. 

The group of René Medema showed that defects in the 

survivinj Aurora B complex specifically affects the 

response of a tumor cell to paclitaxel, while leaving the 

response to vinblastine intact. They currently are 

extending these observations to other components of the 

spindle assembly checkpoint. 

By using tissue-specific inactivation of E-cadherin and 

PS3 in conditional mouse mutants, Jos Jonkers and 

colleagues have established a mouse model for lobular 

breast cancer that mimics not only the histopathologic 

phenotype but also the high metastatic capacity of the 

human disease. 

My own group (Division of Molecular Genetics) 

generated a mouse model for small celllung cancer that 

shows all the features, including metastatic behavior, of 

human Small Cell Lung Cancer. This model will be used 

to delineate pathways that could serve as therapeutic 

intervention targets and to identify factors that make 

these tumors refractory to chemotherapy. 

In a collaborative study, the Maarten Van Lohuizen 

group, together with the group of Silvia Marino in 

Zürich, Switzerland, demonstrated that the oncogene 

and polycomb group gene Bmil is overexpressed in a 

large fraction of primary human medulloblastomas, 

which are the most frequently occurring childhood 

malignant brain tumors. Moreover, they were able to 

show that the Sonic Hedgehog signaling pathway, which 

is implicated in medulloblastoma formation, regulates 

BMIr. These results highlight the emerging role of 

BmiI- containing Polycomb complexes in the 

maintenance and expansion of stem cells or committed 

progenitors and in the pathogenesis of tumors that 

originate or re-gain stem cell characteristics. 

Metastasis represents a principal cause of cancertreatment 

failure and is prevented by several physiologic 

barriers, including 'anoikis': apoptosis resulting from 

lack of adhesion. U sing anoikis suppression as the basis 

for a functional genome-wide screen, Daniel Peeper and 

colleagues have identified the neurotrophic receptor 

TrkB as an oncogene that induces highly invasive 

metastases. As this gene is frequently overexpressed and 

mutated in cancer, it may represent an attractive drug 

target. 

The group of Jan Schellens and J os Beijnen (Divisions of 

Medical Oncology and Pharmacy) continued their work 

on the oral administration of anticancer drugs that are 

not resorbed under norm al conditions. They had 

previously shown that resorption by the bowel could be 

achieved in the clinical situation by co-administration of 

drugs that inhibit P-glycoprotein, MRPI andjor other 

drug transporters. This year, they published results of 

a phase II study in gastric cancer, in which paclitaxel 

was combined with cyclosporin A, an inhibitor ofboth 

P-glycoprotein and CYP-3A mediated drug metabolism. 

Not only were they able to show an 8-fold increase in the 

systemic exposure to oral paclitaxel, they also 

documented a 32% objective response rate in these 

patients, proving the clinical effectiveness of this 

approach. 

The image acquisition and processing group (Division of 

Radiotherapy) has been the first to introduce a linear 

accelerator equipped with a cone-beam CT imaging 

system into clinical practice. This linear accelerator 

allows on-line collection of three and four-dimensional 

patient data prior to treatrnent. Together with the 

development of new intensity-modulated radiotherapy 

techniques for head and neck, breast, lung and bladder 

tumors, the group strives to achieve higher cure rates 

while further lowering complication rates. 

The Epidemiology group of Flora van Leeuwen (Division 

of Psychosocial Research and Epidemiology) investigated 

the effects of radiation dose, chemotherapy, and 

reproductive factors in a cohort of 770 female patients 

who were treated for Hodgkin's disease at age 40 years 

or younger. Breast cancer risk rose strongly with 

increasing radiation dose up to at least 40 Gy. Patients 

who received both chemotherapy and radiotherapy had 

significantly lower risk than those treated with 

radiotherapy alone. The risk reduction from 

chemotherapy could be attributed to high frequency of

early menopause among chemotherapy treated patients. 

These results indicate that ovarian hormones play a 

critical role in promoting tumorigenesis after radiation 

has produced an initiating event. 

Quality of research 

The quality of research can be monitored in several 

ways. Our scientific productivity as based on 

bibliometric parameters (citations and impact of 

scientific articles published by the NKI staff) has shown 

a steady increase since the beginning of the eighties. 

In the last years the number of citations and impact has 

leveled off (Tabie 2) with yearly fluctuations, although 

2002 was a particularly good year. 

Our competitiveness in obtaining grants is another 

measure of quality. We have been quite successful 

during the last decade in obtaining grant support, 

Table 2 

Short-term citations and impact of scientific articles 

published by the NKI research staff 1982 - 2002 

Publication year Citations* Impact~\"~'( 

1982 560 295 

1983 779 365 

1984 1340 616 

1985 1286 549 

1986 1366 650 

1987 1839 765 

1988 1775 742 

1989~\" 1273 764 

1990 2127 854 

1991 21 99 9 10 

1992 2074 9II 

1993 2221 958 

1994 3455 12 9 2 

1995 28 9 6 1415 

1996 3324 1520 

1997,'(,h'( 

3 61 7 18II 

1998 3240 1392 

1999 3727 1766 

2000 3551 1664 

2001 1483 

2002 2455 

,'( 

*,'( 

*,'(,'( 

Citations in the two years af ter publication, 

excluding self-citations. Starting 1989 the citation 

analysis has been carried out on line. This allows 

detection (and elimination) of all self-citations. 

Before 1989 this pruning was limited to first 

authors. 

The impact factor is the average number of 

citations per year of an article in a given journal. 

The total impact is the sum of the impact of all 

articles published that year. 

As from 1997 publication year of articles is the 

criterion, instead of Scientific Report-year listing. 

scoring on average 2-3 fold better than our competitors 

as measured on the basis of successful applications to 

various granting agencies such as the Dutch Cancer 

Society. For 2003 our score in obtaining grants from the 

Dutch Cancer Society was again nearly twice the 

national average. Moreover, we have won a number of 

major grants from other sources, including various EU 

grants (FP6) . The Centre of Biomedical Genetics, a 

consortium in which groups participate from the NKI, 

Hubrecht laboratory, UMC Utrecht, UMC Rotterdam 

and Leiden, was reviewed in 2003 and received very high 

scores which willlikely re sult in a renewal of the support 

for this top research school for a second period of 

5 years. 

The third measure of quality is based on external site 

visits, in which international leaders in a particular field 

of research review the work of a division or research 

groups with a similar theme, on a quinquennial basis. 

These site visits require that group leaders pre pare a 

report and reflect on their research activities. This by 

itself appears very beneficial as it helps focus the 

research. One two-day site was held in 2003 reviewing 

the work of two divisions. The work of the Division of 

Cellular Biochemistry and the Division of Tumor 

Biology was evaluated by Lewis Cantley (Harvard 

Medical School, Boston, USA), Gareth Griffith 

(EMBO Laboratory, Heidelberg, Germany), 

Günther Hämmerling (DKFZ, Heidelberg, Germany) , 

and J oan Massagué (Memorial Sloan Kettering Cancer 

Center, New York, USA) . The site visitors we re overall 

very positive about both divisions, but also indicated 

some weak spots and made a number of suggestions to 

focus research of some of the group leaders. 

Honors and appointments 

The NKIjAvL cannot award university degrees. 

However, many of our staff members hold special parttime 

chairs at Netherlands universities. This facilitates 

the supervision and awarding of degrees to graduate 

students receiving their training at The N etherlands 

Cancer Institute. In 2003, Fons Balm was appointed to 

Professor of Head and Neck Oncology and Surgery, and 

Frans Hilgers to Professor of Oncology-related Voice 

Disorders, both at the University of Amsterdam. 

There we re also changes in our scientific staff. Fred van 

Leeuwen joined the Division of Cellular Biochemistry as 

AvL fellow. He will focus on the epigenetic regulation of 

chromatin function and transcriptional memory. 

Staff of the NKljAvL fulfilled numero us functions in 

national and international organizations, on scientific 

boards of scientific journais, as members of study 

sections, as organizer or co-organizer of scientific 

meetings, workshops and congresses. 

Outlook and acknowledgements 

I am confident that research at the NKljAvL will 

continue to flourish. A number of exciting projects are 

progressing at full speed. The recently completed 

construction of 24,000 human RNAi knockdown 

vectors directed against 8000 genes will enable us to 

perform large-scale loss-of-function genetic screens in

mammalian ceIls in the coming years. Such screens are 

likely to contribute significantly to our understanding of 

the molecular pathways that are deregulated in human 

cancer. The construction of a similar mouse library is 

underway. We have an ideal infrastructure to profit from 

these tools. However, these screens are costly and 

exemplify the steep rise in costs ofbiomedical research 

in genera I as new, often high-throughput techniques 

require expensive equipment, biochemicals and 

disposables to run. The problem is that the financing of 

our research is not following suit. Last year I indicated in 

this introduction my concerns about inadequate support 

from the government. This concern has become more 

tangible now. With a budget cut of 10% for 2004 by the 

ministry we face major problems in maintaining our 

infrastructure in the coming years, especiaIly since the 

contribution of the Dutch Cancer Society which has 

assigned 15% of its income as core funding to the 

Institute wiIl also shrink due to a decIine of its own 

revenues. We will continue fighting the inadequate 

support by the government. Unfortunately, alternatives 

are sparse. Although we have been very successful in 

securing additional grant support, the grants seldom 

reimburse indirect costs and of ten require matching 

funds. This has to be supplied from our core research 

budget. If this diminishes we wiIl have to shrink our 

staff and support for indirect costs. A rough calculation 

teaches that a cut of I million in our core funding wiIl 

result in an additional reduction of 2 million of external 

project funding. This is a painful blow, especiaIly since 

the organization is already very lean, and budget cuts 

cannot be easily absorbed without seriously impairing 

our research capacity. 

The Board of Directors will step up its efforts to find 

new funding sources for our research. Both government 

and the public have an interest in a dedicated 

internationally renowned cancer institute. It should be 

possible to motivate granting bodies to invest in our 

research, especiaIly since there is now a broadly felt 

belief that breakthroughs in cancer treatrnent can be 

realized with approaches in which the Institute holds a 

prominent position worldwide. It would be a sharne if 

we could not capitalize on our investrnents because of a 

deteriorating fmancial situation. 

This only emphasizes how much we are obliged to our 

supporters. For all our research we depend on the 

financial support of the government, the Dutch Cancer 

Society, and of many individuals. 0nly with their help 

can we continue to develop new ideas that williead to 

prevention, early detection, and more effective treatrnent 

of cancer. We hope they will feel encouraged by this 

report. 

Ton Berns 

Director of Research

,. ,." ~. ~ .; . 

r -~":-'. I 

., 

T ~ ', 

• 

'.' , 

• 

.' 

EDUCATION IN ONCOLOGY 

In the Netherlands Cancer Institute, students are being 

trained at various levels, from nurses and technical staff, 

through undergraduate, graduate and post-graduate 

students as wen as medical students through residents 

in training. In basic research this includes theoretical 

courses as wen as practical training, and at the clinical 

level the medical staff is actively involved in various 

training programs. In addition many senior staff 

members have joint appointments as professors at 

Dutch universities and contribute to the regular 

curriculum at these universities and many other staff 

members also te ach courses for undergraduate and 

graduate students, either within the institute or at 

universities. 

The clinical training in the institute includes medical 

and surgical oncology fenowship-programs, teaching 

programs for undergraduate medical students and 

(inter)national postgraduate courses as wen as lectures 

for nurses training for their oncology certificate. The 

departments of Surgical Oncology and Head & Neck 

Oncology participate in the residency-training program 

of the Academic Medical Center Amsterdam. 

The research departments attract graduate students 

from universities throughout the country, who 

contribute to ongoing scientific projects and receive inhouse 

theoretical training. The NKI has a formal 

Table 1 

Course in Experimental Oncology, fall2oo3 

Epidemiology 

Surgery 

Pathology ~(* , 

Molecular Diagnostics>'c 

Gene array>''< 

Conventional chemotherapy 

Radiothera py>b~ 

DNA damage response 

Apoptosis ~< 

Cell cycle>'

Participants ofthe OOA retreat 2003. 

affiliation with the Science Faculty of the University of 

Amsterdam and is committed to contributing to courses 

for graduate and undergraduate students of this 

university. The institute participates in the Oncology 

Graduate School Amsterdam, together with the 

University of Amsterdam (UvA) and the Free University 

(VU). Most graduate students are assigned to this 

graduate school for a four-year period. These graduate 

students make significant contributions to the scientific 

program. In addition they receive practical and 

theoretical training within the institute. 

Undergraduate students 

The undergraduate program in Experimental Oncology 

attracts students of all national universities in partial 

fulfillment of the obligations of their Master's 

curriculum. Students generally have a background in 

(Medical) Biology, Health Sciences, Chemistry, 

Pharmacology, Medicine, or Psychology. The 

undergraduate program offers combined practical and 

theoretical training in various aspects of experimental 

oncology. Practical training includes participation in 

ongoing research projects for a minimum of 4 months, 

af ter which the student delivers oral and written 

accounts of the results obtained. In 2003, 29 university 

students completed a placement of 5-12 months at the 

research divisions of the NKI. The majority (19) of these 

students came from the Amsterdam Universities 

(UvA and VU), others from Wageningen, Utrecht, 

Leiden and Nijmegen. We also had two students from 

abroad. The institute also provides practical training 

opportunities to trainee technicians. In 2003, we 

welcomed for the second time a number of third year 

Medical Biology students of the UvA for a 7-week 

rotation. 

The core element of theoretical training is the course in 

Experimental Oncology, given twice a year by staff 

members of the institute (Tabie I). This course includes 

lectures and tutorials. The book 'Cancer Biology' by 

R. J. King is recommended for background reading. 

The Teaching Committee (Tabie 2) supervises all 

educational activities. Specialized tasks include 

organization of the lecture course, guided tours to 

research departrnents, contact with technician training 

schools, promotional activities at universities. 

Graduate students 

Approximately 120 graduate students at the NKI 

participate in the Graduate School 'Onderzoekschool 

Oncologie Amsterdam' (OOA). The OOA is a 

collaboration between the NKI and the medical faculties 

of the Free University (VUMC) and the University of 

Amsterdam (AMC). The graduate students are scientists 

in-training who receive additional theoretical and 

practical education on various subjects related to cancer. 

Following a successful scientific training, graduate 

students can acquire their PhD degree at one of the 

national universities. 

The OOA organizes various activities. All graduate 

students are tutored by a committee comprised of the 

project leader, the he ad of the division and one or two 

academic staff members from other divisions. They 

meet once a year to evaluate scientific progress and 

problems. 

A graduate student committee with representatives from 

each departrnent, me ets regularly to discuss subject 

matters related to the OOA educational program and 

scientific problems with the Dean of the OOA. As a 

result of these discussions, graduate students now host 

'Special Seminars' and a well-liked course on 'Writing 

and Presentation in English' has become a regular item 

in the program, while more clinical course have been 

planned for the near future. 

The OOA organizes an annual retreat for graduate 

students, which they must attend three times during 

their graduation studies. First year graduate students 

usually present the goal and concept of their project in a 

poster session, graduate students oflater years will give

Table 3 

Courses at the OOA 2003 

January 13,20 Trends in Tumor Immunology Ron Scheper 

March/ April 2003 Writing and pres enting in English Fiona Stewart, Buro Opleidingen 

March 24-28 Anatomy of the mouse WH Lamers 

March 3 I-April 4 Introduction radiobiology/radiotherapy BJ Slotman 

April-May Theoretical course on invasion and metastasis Amoud Sonnenberg, John Collard 

May 22,23 Trends in Microscopy Ron Van Noorden, Peter Peters, Gerard Meijer 

Sept 15-18 Transport in health and disease Ronald Oude Elferink 

Oct 8-10, 20°3 Annual retreat Texel Titia Sixma, Marieke Van der Velde 

Oct 28-Nov 7 Signal transduction Wouter MooIenaar, Peter Ten Dijke 

Nov/Dec Writing and presenting in English Fiona Stewart, Buro Opleidingen 

Nov 27,28 Molecular and clinical genetics of cancer R Verheijen, F Menko 

an oral presentation. This year's retreat was organized 

on Texel, with 96 participating graduate students 

(59 from the NKI). 

The OOA organizes various courses for graduate 

students. Graduate students must attend 4 courses 

during their PhD program, of which one can be outside 

the OOA program. Courses are usually restricted to 

ab out 15-20 graduate students to ensure optimal 

scientific interaction. Courses are advertized on the 

web page of the OOA (www.ooa.vu.nl) and announced 

to every graduate student bye-mail. 

Information Graduate School: 

Secretariat Marieke Van der Velde. 

E-mail m.vd.velde@nki.nl 

Dean Dr Titia K. Sixma. 

E-mail t.sixma@nki.nl

DIVISION OF CELL BIOLOGY 

RECEPTORS FOR MATRIX ADHESION 

Integrins connect the extracellular matrix with the cell interior and trans duce signals 

via interactions of their cytoplasmic domain with cytoskeletal and signaling 

molecules. In our group, we study the mechanisms by which integrins provide 

signals for the regulation of important cellular processes such as cell migration and 

proliferation. In particular, we are interested in how integrins contribute to the 

process of transformation. 

Role of «6fl4 and plectin in hemidesmosome formation Hemidesmosomes are 

stable anchoring structures that provide a tight link between the intracellular 

intermediate filament system and the extracellular matrix. The integrin a6~4 plays a 

central role in the assembly ofhemidesmosomes and the loss of a6~4 results in 

extensive blistering of the skin, both in human patients and in mouse modeis. 

We have shown that the plectin actin-binding domain (ABD) interacts with the 

~4-subunit. We have mapped several residues in the ABD critical for the interaction, 

and in conjunction with the crystal structures, the interaction surface between the 

first two fibronectin type 111 (FNIII) repeats of ~4 and the plectin ABD was calculated 

using a docking program. The predicted binding surface was verified by mutating 

other amine acids that were expected to be involved in the interaction between the 

two fragments. Thus far, the biochemical- and computational data have been 

consistent with each other. 

Furthermore, we have obtained evidence from yeast-two hybrid and biochemical 

assays that the plakin domain of plectin can also mediate binding to ~4 in addition to 

the ABD. The plakin domain specifically interacted with the connecting segment and 

C-tail of ~4. However, in cells these interactions were not sufficient to induce 

hemidesmosome assembly without the Strong interaction of the plectin-ABD to ~4. 

We believe that these additional interactions are necessary to position the ~4- 

cytoplasmic domain for the optimal interaction with other hemidesmosomal 

components, thereby increasing the efficiency ofhemidesmosome assembly. 

Ceneration of conditional «3 and fl4 knockout mice To study the contribution 

of the integrins a3~I and a6~4 in the initiation, maintenance and progression of 

squamous cell carcinomas, we have generated conditional a3 and ~4 knockout 

mouse strains. The conditional knockout mice with the 'floxed' a3 and ~4 alleles 

revealed no obvious abnormalities. Furthermore, when crossed with transgenic 

keratin KI4-Cre mice, the 'floxed' a3 and ~4 DNA segments were efficiently 

removed. Loss of a6~4 from the skin resulted in the formation of blisters 

reminiscent of that seen in ~4 null mice. We have recently established immortalized 

keratinocyte celIlines from the ~4 conditional knockout mice. Furthermore, we have 

induced skin tumors in the conditional ~4 knockout mice by the application of a twostage 

carcinogenesis protocol, and established several independent transformed 

keratinocyte celIlines from the resulting tumors. The capacity of these celIlines, with 

or without ~4 (after transient expres sion of the Cre-recombinase), to grow, 

differentiate and migrate as weIl as to survive when they are deprived of interacting 

with ECM components, is being studied and compared. Furthermore, we will assess 

their tumorigenic potential af ter they have been subcutaneously injected into 

immune-deficient mice. The results should reveal whether one or more of the 

biological responses have been altered and become dependent on the function of 

a6~4 during transformation 

Regulation of gene expression by integrin signalling The ~1 integrin when 

expressed in GE 11 cells was shown previously to negatively regulate the expression of 

a number of genes, including MacMARCKS (MM). MM is a PIP2 binding protein 

that can bind to F-actin and is thought to cross-link actin into fibers. When MM is 

expressed as a GFP fusion protein in GE 11 celIs, it concentrates at the lateral 

membranes along with classic markers such as cadherin and a-actinin. MM is a 

Division head, Group Leader Amoud Sonnenberg 

Arnoud Sonnenberg PhD Group leader 

Erik Danen PhD Post-doe 

Karine Raymond PhD Post-doe 

Certien Smits PhD Post-doe 

Kevin Wilhelmsen PhD Post-doe 

Sandra Van Wilpe PhD Post-doe 

Stephan Huveneers MSc Graduate Student 

Jan Koster MSc Graduate Student 

Sandy HM Litjens MSc Graduate Student 

Iman Van den Bout Graduate Student 

Maaike Kreft Technical staff 

Ingrid Kuikman Technical staff 

Petra Sonneveld Technical staff 

Wille Franken Undergraduate Student 

Alice Ruiter Undergraduate Student 

Norman Sachs Undergraduate Student 

Ntambua Tshimbalanga Undergraduate Student 

1 st pair of FNIII repeats of ~4 

2 nd FNIII i st FNIII 

Fig. I.l: Model ofthe binding surface between the 

pLectin-ABD and the first pair of FNIII repeats of 

(34·

Selected publications 

Danen EHJ, Sonneveld P, Brakebusch C, 

Fässler R, Sonnenberg A. The fibroneetinbinding 

integrins a5(31 and av(33 

differentially modulate RhoA GTP-loading, 

organization of eell-matrix adhesions, and 

fibronectin fibrillogenesis. ] Cell Biol2002; 

159:1071-86. 

Geuijen CAW, Sonnenberg A. Dynamies of 

the a6(34 integrin in keratinocytes. Mol Biol 

Cell2002; 1]:3845-58. 

Cachaço AS, Chuva De Sousa Lopes SM, 

Kuikman I, Bajanca F, Abe K, Baudoin C, 

Sonnenberg A, Mummery CL, 

Thorsteinsdóttir S. Knoek-in ofintegrin 

(31D affeets primary but not seeondary 

myogenesis in miee. Development 2003; 

13°:1659-71. 

Danen EHJ, Sonnenberg A. Integrins in 

regulation of tissue development and 

Junetion. ] Patho12003; 200:471-80. 

Fontao L, Favre B, Riou S, Geerts D, 

Jaunin F, Saurat J-H, Green K, 

Sonnenberg A, Borradori L. Interaction of 

the bullous pemphigoid antigen 1 (BP230) 

and desmoplakin with intermediate 

filaments is mediated by distinet and 

speeifie sequenees within their COOHterminus. 

Mol Biol Cell 2003; 14:1978-92. 

Garcfa-Alvarez B, Bobkov A, 

Sonnenberg A, De Pereda JM. Struetural 

and Junetional analysis of the aetin binding 

domain of pleetin suggest alternative 

meehanisms for binding to F-aetin and 

integrin (34. Structure 2003; 11:615-25. 

Koster J, Geerts D, Favre B, Borradori L, 

Sonnenberg A. Analysis ofthe interactions 

between BP180, BP230, pleetin and the 

integrin a6(34 important for 

hemidesmosome assembly. ] Cell Sei 2003; 

116:387-99. 

Litjens SHM, Koster J, Kuikman I, 

Van Wilpe S, De Pereda JM, 

Sonnenberg A. Speeifieity ofbinding ofthe 

pleetin aetin binding domain to (34 integrin. 

Mol Biol Cell2003; 14:4039-50. 

major substrate for phosphorylation by protein kinase C and PMA stimulation of 

eeUs results in a rapid redistribution ofMM from the membrane into the perinuelear 

region. Subsequently, the aetin eytoskeleton is re-organized and eeU-eeU 

contacts are broken down. AdditionaUy, we were able to show that hepatoeyte growth 

factor (HGF), whieh induees eeU seattering in a variety of epithelial eeUlines, eauses 

a similar redistribution of MM in GE 11 eeUs. The finding that the redistribution of 

MM oeeurs prior to eeU-eeU dissociation supports our hypothesis that this protein 

plays a eritieal role in the ~ l-indueed morphologieal transformation of GE 11 eeUs. 

Currently, we are using siRNA teehniques to knoek down the expres sion of 

endogenous MM to obtain further evidenee for a role ofMM in the ~1-indueed 

morphologieal transformation. The effects of MM on the aetin eytoskeleton may be 

due to a transient inerease in the loeal concentration of PIP2 at the lateral 

membranes. Preliminary studies using the PIP2 sensor PH-mRFP (pleekstrin 

homology domain-monomerie red fluorescent protein) suggest that MM may 

control an important reservoir of PIP2 at the lateral membranes. When eeUs are 

stimulated with PMA, the level ofPH-mRFP was inereased at the membrane while 

that of GFP-MM was deereased, suggesting that PIP2 is released at the later al 

membranes. MM's loealization points to a possible role in the maintenanee of eeUeeU 

contacts by regulating the aetin eytoskeleton in this region, thereby giving 

stability to the lateral membrane. 

Control of cell adhesion, migration, and oncogenic transformation by 

fibronectin-binding integrins We previously reported that adhesion to the 

extraeeUular matrix component fibroneetin strongly stimulates RhoA-mediated aetin 

eytoskeletal organization. Sinee many different integrins ean bind fibroneetin, we 

used eeUlines derived from miee laeking specific integrins, to show that fibroneetin 

stimulates RhoA aetivity only through eertain integrins. In eeUs laeking ~I integrins, 

that bind via aV~3, fibroneetin does not in duce RhoA aetivity. In these eeUs, the 

organization of the aetin eytoskeleton and the distribution of eeU matrix adhesions 

markedly differs from that of eeUs whieh eontain ~I integrins, that bind via a5~I. 

Using ehimerie integrin subunits in whieh the ~I eytoplasmie domain is fused to the 

~3 extraeeUular domain, or vice versa, and ehimeras in whieh the av eytoplasmic 

domain is fused to the a5 extraeeUular domain does not point to an important role 

for the eytoplasmie domains in the above funetions. 

Using GFP-fusions of eeU-matrix adhesion eomponents in living eeUs, we have 

reeently observed that the dynamies of eeU-matrix adhesions and the migratory 

behavior of eeUs are strongly affeeted by the integrins that mediate eeU adhesion. 

This suggests that eeUs ean modulate their migratory behavior by switehing integrin 

expres sion profiles. Indeed, eeUs of ten express aV~3 at inereased levels in wounds 

and in highly invasive tumors. We have observed that this integrin is assoeiated with 

the formation oflamelipodia and persistent, direetional migration, suggesting that 

the migratory capacity of these eeUs is enhaneed by this integrin. 

We have also used eeUlines expressing different fibroneetin-binding integrins to 

investigate their role in eell eyele progression. Our findings indieate that the 

induction of eyelin DI oeeurs in mid-to-Iate GI phase of the eeU eyele in eeUs 

adhering via a5~I, but oeeurs mueh earlier during GI in eeUs laeking bI integrins 

whieh adhere via aV~3- 

FinalIy, we have expressed different oneogenes in these eelIlines and observed a 

strong eooperation between aV~3 and mutationally aetivated e-Sre in the oneogenie 

transformation of epithelioid eeUs. We are eurrently testing if the cross talk between 

integrins and eonstitutively aetivated Sre oeeurs up- or downstream of Stat-3, a 

transcription factor that is a critieal mediator of Sre transformation. Stat-3 

phosphorylation, nuelear translocation, and transeriptional aetivity are being tested in 

Sre-transformed eells expressing or laeking aV~3, and we are testing the ability of 

aetivated forms of Stat-3 to reseue Sre-mediated oncogenie transformation in eeUs 

laeking aV~3-

GENETIC CONTROL OF INVASION AND METASTASIS 

The aim of our research is to identify and characterize genes that play an essential 

role in the acquisition of an invasive and metastatic phenotype of tumorigenic cells. 

Insight into the signaling pathways involved may lead to the development of novel 

diagnostic tools or improved therapeutic strategies. 

Function-based screens for invasion-inducing genes In earlier studies, we have 

identified the invasion-inducing gene Tiamr by retroviral insertional mutagenesis in 

combination with in vitro selection of invasive T-lymphoma cells. Additional 

functional screens for genes involved in T-lymphoma invasion revealed a number of 

novel genes. These genes include the uS- subunit of the usBr integrin and the LPA 2 

receptor, which have been implicated in integrin-mediated cell-matrix adhesion 

andjor signaling pathways mediated by Rho-like GTPases. For the novel screens, 

retroviral cDNA libraries were used in combination with in vitro selection of invasive 

T-lymphoma cells. 

Rho family proteins control dynamic cytoskeletal changes The previously 

identified invasion-inducing Tiamr gene encodes a guanine nucleotide exchange 

factor (G EF) or activator for the Rho-like GTPase Rac. Rho-like GTPases, which 

include CdC42, Rac and RhoA, control a wide range ofbiological processes. In 

particular, they act as key molecules in signaling pathways that regulate the 

reorganization of the actin cytoskeleton in response to receptor stimulation and 

thereby determine the morphology, adhesion, polarity,and motility of cells. The 

principal regulators ofthe activities ofRho proteins are the GEFs and GTPase 

activating proteins (GAPs). GEFs, such as Tiamr, induce activation by exchanging 

GDP for GTP, whereas GAPs enhance the intrinsic rate ofhydrolysis ofbound GTP 

to GDP, resulting in inactivation ofthe GTPases. In addition, the activity ofRho 

proteins is controlled by Rho-GDls. In cells, Rac exists predominantly in its inactive 

GDP-bound form in a complex with RhoGDI. RhoGDI binds and masks the 

hydrophobic C-terminal region of Rac, the same region that is responsible for 

targeting Rac to the plasma membrane. Thus RhoGDI maintains Rac in the 

cytoplasm and must dissociate to allow Rac to translocate to the membrane and 

interact with membrane-associated G EF s. We found that calcium signaling regulates 

the association between RhoGDI and Rac. The increase in intracellular calcium 

[Ca 2 Ji represents a ubiquitous second messenger system, linking receptor activation 

to downstream signaling pathways. Elevation of [Ca 2 +Ji - either artificially or by 

thrombin receptor activation - potently induces Rac activation. Elevation of [Ca 2 +h 

leads to the activation of a conventional protein kinase C (PKC). Both increased 

[Ca 2 Ji and PKC activation induce phosphorylation of RhoGDI leading to dissociation 

of RhoGDI and Rac. As a consequence, dissociated cytosolic Rac translocates to the 

plasma membrane where it can be activated by membrane associated GEFs. The 

importance of RhoG D I in regulating Rac activity has also been demonstrated in 

studies on Racrb. Colorectal tumors express an altematively spliced variant of Racr, 

termed Racrb, containing r9 additional amino acids following the switchII region. 

Although little RaCIb protein is expressed in cells, the amount of activated RaCIb 

protein often exceeds that of activated RaCI. This suggests that RaCIb contributes 

significantly to the downstream signaling of Rac in cells. The difference in activity 

between RaCI and RaCIb is not due to altered dependence on GEFs or GAPs, but 

rather is caused by the inability of Racrb to interact with RhoG DI. As a consequence, 

most Racrb remains bound to the plasma membrane and is not sequestered by Rho 

GDI in the cytoplasm, resulting is high Raclb activity in cells. Unlike RaCI, activated 

Racrb is unable to induce lamellipodia formation, to bind and activate PAK, or to 

activate the downstream protein kinase JNK. However, both Racr and Racrb are able 

to activate NFkB and PIPs-kinase to tbe same extent. These data suggest that 

altemative splicing of Racr leads to a Rac variant that differs in regulation and 

downstream signaling. 

Group leader John Collard 

John G. Collard PhD Group leader 

Saskia Van Es PhD Post-doe 

Irene Hamelers PhD Post-doe 

Angeliki Malliri PhD Post-doe 

Cristina Olivo PhD Post-doe 

Rob C. Roovers PhD Post-doe 

Kristin Strumane PhD Post-doe 

Amra Hajdo-Milasinovic MSc Graduate student 

Sander Mertens MSc Graduate student 

Tomek Rygiel MSc Graduate student 

Rob A. Van der Kammen Teehnieal staff 

Fig. 1.2: Regulation ofthe activity and 

downstream signaling of Rac. For explanation 

see text. 

-eell growth 

-apoptosis 

-E-cadherin 

adhesions 

-eell migrotion 

The activation of GEFs and downstream signalling Apart from increased or 

decreased expression, GEFs that activate Rho-like proteins are basically regulated at 

four different levels in the cello These include intra-molecular inhibition, changes in


Cascone I, Audero E, Giraudo E, 

Napione L, Maniero F, Philips MR, 

Collard JG , Serini G, Bussolino F. 

Tie-2 - dependent activation of RhoA and 

RaC1 participates in endothelial cell motility 

triggered by angiopoietin-1. Blood 2003; 

102:2482-9°. 

Malliri A, Collard JG . Role ofRhoJamily 

proteins in cell adhesion and cancer. 

Curr Opin Celt Bio12003; 15:583-9. 

Price LS, Langeslag M , Ten Klooster J-p, 

Hordijk PL, Jalink K, Collard JG . Calcium 

signalting regulates translocation and 

activation of Rac via PKC-mediated 

phosphorylation of RhoG D J. ] Biol Chem 

2003; 278:39413-21. 

Mertens AE , Roovers RC, Collard JG . 

Regulation ofTiam1-Rac signalling. 

FEBS LeU 200]." 546:11-6. 

Van Leeuwen FN , Olivo C, Grivell S, 

Giepmans BNG, Collard JG, 

Mooienaar W. Rac activation by 

lysophosphatidic acid LP Al receptors: 

a critical role for the guanine nucleotide 

exchange factor Tiam1. ] Biol Chem 2003; 

278:400-6. 

Matos P, Collard JG, Jordan P. Tumor 

related alternative spliced Rac1b is not 

downregulated by RhoGDJ and exhibits 

selective downstream signaling. ] Biol 

Chem; 2003 in press. 

intracellular localization, post-translational modifications and interaction with other 

proteins. All of these regulatory mechanisms seem to be involved in Tiamr 

regulation. In particular, the interaction with various proteins and protein complex 

formation appears to be an important mechanism to determine downstream 

signaling of activated Rac. Oncogenic Ras was shown to mediate Rac signaling 

through the direct interaction of Ras with the RBD domain ofTiamr. The fact that 

Tiamr-deficient mice we re found to be resistant to Ras-induced skin tumors 

underlines the physiological relevance of the connection between Ras, Tiamr and 

Rac. Proteins interacting with Tiamr mayalso determine the downstream signaling 

of the Tiamr-Rac complex. This is demonstrated by the findings that the scaffold 

proteins JIP /IB2 and Spinophilin both bind to Tiamr. JIP2/IB2, a scaffold for the 

MLK3-MKK3-P38-MAP kinase complex, couples Tiamr-induced Rac signaling to P38 

activity. Ectopic expres sion ofTiamr not only stimulates the build-up of a complex of 

JIP2/IB2 with components of the P38 signaling cascade, but also enhanced P38 MAP 

kinase signaling. Spinophilin binds to and enhances the activity of P7oS6-kinase, 

one of the direct downstream effectors of Rac. Interestingly, Spinophilin binding 

suppresses Tiamr's ability to aetivate p2r-activated kinase (PAK) , the downstream 

target of Rac that mediates actin cytoskeletal reorganization. Preliminary data suggest 

that the Tiamr-Rac-P7oS6 kinase pathway plays an important role in regulating 

protein levels in cells by its function in protein translation. Association ofTiamr with 

various proteins may thus determine its localization, activity and Tiamr/Raccontrolled 

downstream signaling. 

Tiaml-Rae signaling and invasion of epithelial eells Metastasis of carcinomas is 

associated with reduced E-cadherin-mediated cell-cell adhesion. Tiamr localizes to 

adherens junctions and ectopic expres sion ofTiamr in epithelial cells inhibits HGFinduced 

cell scattering by increasing E-cadherin-mediated adhesion. Moreover, 

ectopie expression ofTiamr inhibits the formation ofHGF-indueed branehed tubular 

structures in three-dimensional eollagen. Direct proof for a role ofTiamr in the 

formation and maintenanee of E-eadherin-mediated eell-eell adhesions eomes from 

studies using short interferenee RNA. Tiamr-speeifie siRNA introdueed in epithelial 

MDCK eells leads to loss of eell-eell adhesions, a mesenchymal phenotype, and 

migratory eells. Moreover, keratinocytes derived from Tiamr-deficient miee are 

impaired in the formation of adherens junctions when compared with wild-type 

keratinoeytes. The mechanism by whieh Tiamr deficieney affects the formation of 

adherens junction is eurrently being investigated. 

Tumorigenicity in Tiaml mutant mice We have generated Tiamr-deficient miee to 

investigate the eonsequenees of changes in Tiamr/Rae signaling in tumorigenesis. 

Although Tiamr is expressed during embryonie development, Tiam{1- miee develop, 

grow, and repro duce normally. In mouse skin, Tiamr is present in basal and 

suprabasal keratinoeytes of the interfollieular epidermis and in hair follicles. To 

investigate the role ofTiamr in skin tumorigenesis, skin tumors were indueed by 

treatment with the carcinogen DMBA followed by repeated treatments with the 

tumor promoter TP A. Tiam{/- miee displayed a dramatic resistanee to tumor 

formation and their number was on average ro-fold redueed during the promotion 

period. Tiamr -deficieney affeeted also tumor growth and tumor progression. 

Although the number and growth of tumors was strongly redueed in Tiam{/- miee, 

Tiamr -deficieney inereased the frequeney of malignant conversion of Ras-indueed 

skin tumors. Currently we are studying the role ofTiamr in tumorigenesis indueed 

by different oncogenie signaling pathways i.e. Ras, Wnt, Mye, and Neu. Preliminary 

data indieate that Tiamr is a Wnt-responsive gene and that its expression is regulated 

~-catenin-TCF signaling. As a eonsequenee, Tiamr protein levels are inereased in 

intestinal tumors produeed in APC mutant miee. Interestingly, the initiation and 

growth of intestinal tumors is redueed in Tiamr-defieient APC mice when eompared 

with wild-type APC miee. These studies indicate that Tiamr might play a role in 

tumorigenicity indueed by various oncogenie signaling pathways. 

Fig. I.j: Jncreased Tiam1 expression in intestinal 

tumors produced in APC mutant mice.

ADHESION MECHANISMS IN METASTASIS 

Metastasis, the spread of tumor cells to distant sites in the body, is the main cause of 

cancer death. We study the mechanisms of metastasis, with emphasis on lymphomas 

and carcinomas and on the invasion of tissues by blood-borne tumor cells. Our 

recent research on chemokine receptors revealed a role in growth of metastases, 

rather than in invasion as generally assumed. These receptors are therefore potential 

targets for chemotherapy. 

Chemokine receptors in carcinoma metastasis The chemokine receptor CXCR4 

is required for invasion of metastatic lymphoma cells into tissues, as demonstrated 

using an intrakine approach that traps CXCR4 in the endoplasmic reticulum. In the 

same way we showed that CXCR4 is also required for colon carcinoma metastasis. 

This was remarkable since the cells were CXCR4-negative, at least in vitro. In vivo, 

however, CXCR4 expression was induced, and only af ter the cells had established 

themselves in the lungs. Thus, the role of CXCR4 is not in invasion from the blood. 

CXCR4-blocked cens remained present but expanded very slowly, as compared with 

control cells. Ex vivo, CXCR4 expres sion was rapidly lost. The CXCR4ligand SDF-r 

(CXCLr2) is present in most tissues. However, we recently found that the tumor cells 

produce much SDF-r themselves, suggesting autocrine stimulation af ter induction of 

CXCR4 in vivo. 

We also observed a strong induction in vivo of a second chemokine receptor, CXCRS , 

in the colon carcinoma cells. This was not expected since it was so far only detected 

in B- and some T-lymphocytes. Proliferation of CXCRs-transfected cens in vitro, 

especially in serum-free medium, was greatly enhanced by the CXCRsligand BCA-r 

(CXCLr3). BCA-r is present in the lungs but much more in the metastases. Whether 

this is produced by stromal or tumor cells remains to be determined. It is thus 

conceivable that CXCRS signals contribute to proliferation of metastasised tumor 

cells in vivo. 

Group leader Ed Roos 

Ed Roos PhD Group leader 

Belén Alvarez Palomo PhD Post-doe 

Frank Opdam PhD Post-doe 

Peter Stroeken phD Post-doe 

Joost Meijer MSc Graduate student 

Ingrid Zeelenberg MSc Graduate student 

Rosalie De Bruijn Technical staff 

Petra Sonneveld Technical staff 

Yvonne Wijnands Technical staff 

G-protein and integrin signaling during Iymphoma metastasis: role of Jak2 

Invasion by T-lymphoma cens is initiated by CXCR4 signals that trigger migration 

but also activate the integrin LFA-I. This binds ICAM-r or -2 and then elicits 

additional invasion-promoting signaIs. We showed that a Jak kinase, most probably 

Jak2, is involved in invasion and required for T-lymphoma metastasis. A problem 

was that the cells required Jak kinase for proliferation and survival. To circumvent 

this, we expressed active mutants ofSTAT3 and PI3K, which made the cens Jakindependent. 

Next, Jak activity was blocked by expressing the auto-inhibitory Jak2 

JHr 'pseudokinase' domain. This greatly impaired metastasis and in vitro invasion. 

Jak acts in G-protein-induced LFA-r activation and downstream of LFA-r, but not in 

CXCR4-induced migration signals as was claimed by others. 

Blocking desensitization of chemokine migration signals Invasion and 

migration, triggered by the chemokine receptor CXCR4, involves the G-proteins Gi 

and Gq. The G-protein ~y subunit activates PLC and PI3Ky. We blocked ~y, by 

expressing a ~y -binding G RK2 fragment, expecting an inhibition of invasion and 

migration. Surprisingly, however, CXCR4-triggered migration was substantially 

increased and invasion of the ~y-blocked cells was more persistent. We conclude that 

the main ~y function is desensitization, since migration of control cens is blocked 

after a r min exposure to the CXCR4ligand SDF-r (CXCLr2), whereas the ~y-blocked 

cells still migrated after a ro min SDF-r treatrnent. Furthermore, invasion ofT-cell 

hybridoma cells into fibroblast monolayers, which depends on CXCR4, normany 

stops after 30 min, probably due to desensitization. In contrast, the ~y-blocked cens 

continued to invade for at least 4 h. 

This result implies that the ~y dimer is not required to activate PLC or PI3K. Indeed, 

the Gq a-subunit is known to activate PLC and is involved in migration since it is 

inhibited by dominant-negative Gq. Therefore, the ~y subunit is not needed to 

activate PLC. For PI3K the situation is more complicated. PI3K is required, since 

inhibitors block migration. The p8s-regulated PI3K-a, -~ or -6 are probably not 

involved since migration is not affected by dominant-negative p8S or a PI3K-6 

inhibitor. The remaining candidate, PI3K-y, is activated by the ~y dimer, and


Zeelenberg IS, Ruuls-Van Stalle L, Roos E. 

The chemokine receptor CXCR4 is required 

for outgrowth of colon carcinoma 

micro metastases. Cancer Res 2003; 

63:3833-9. 

migration should thus be inhibited in the By-blocked celis, but clearly it is not. In 

fact, surprisingly, the migration of the By-blocked celis was not affected by PI3K 

inhibitors. This rnight be explained if the normal role of PI3K would be the 

amplification of the short CXCR4 signa!. If so, it might not be needed when the 

signal is persistent. In any case, this result shows that PI3K activity is not always 

essential for chemotactic migration as has been claimed for other celi types. 

Desensitization has been a problem, since the celis produce SDF-I themselves, 

resulting in partial desensitization and large variations in migration capacity. The Byblocked 

cens provide a better tooI since their migration and invasion are more 

reproducible and very high. 

VAMP-2 

~~ .... -SNAP-23 

=~~~~~~~~~ Syntaxin 

plasma membrane 

Fig. I+ Docked vesicles are tethered to the plasma 

membrane by a complex which includes the v 

SNARE VAMP-2 and the t-SNARE SNAP-23 . 

Co-immune precipitation ofSNAP-23 and 

VAMP-2from T-lymphoma ceUs shows that 

docked vesicles are present in the cells, in line with 

our hypothesis that synaptotagmin control of 

docked vesicle fusion is essential for chemotaxis 

towards the CXCR4ligand, the chemokine SDF-I 

(CXCL13) 

Synaptotagmin: role in chemotaxis? Lymphoid cens react very rapidly to 

chemokines. We hypothesized that this involves rapid fusion ofvesicles with the 

plasma membrane. In nerve cens, synaptotagmins regulate calcium-triggered fusion 

of docked synaptic vesicles. The isolated synaptotagmin CzB domain can inhibit this 

fusion. T-Iymphoma ceIls express synaptotagmin-3, which is triggered by low calcium 

levels. Transfected CzB completely inhibited CXCR4-triggered migration, in contrast 

to a CzB mutant that binds calcium but does not inhibit fusion. Overexpression of 

full-length synaptotagmin-3 promoted migration, but inhibited when it contained the 

same mutation. The presence of docked vesicles was strongly suggested by the coprecipitation 

ofthe v- and t-SNAREs VAMP-z and SNAP-z3 (see Figure 1-4), and this 

was lost upon Ca 2 + influx and also after CXCR4 activation by SDF-I. Preliminary 

results suggest that docked vesicles are not present in the CzB-expressing cells. 

ICAP-l, ROCK and migration on laminin We became interested in the BI 

integrin-associated protein ICAP-I, because BI mutations that impaired ICAP-I 

binding also impaired BI-dependent metastasis ofESb lymphoma cells. We next 

showed that ICAP-I binds to ROCK kinase. We have now studied this in CZCIZ 

myoblast celis that proved suitable for both RNAi knockdown and overexpression 

approaches as weIl as assays ofBI-dependent migration. ICAP-I RNAi knockdown 

and overexpression led to formation of peripheral and central focal adhesions (FA), 

respectively (see Figure 1.5). These are phenotypes associated with low and high 

migration. Indeed, ICAP-I overexpression enhanced chemotactic migration towards 

HGF on the BI substrate laminin but not on the B3 substrate vitronectin. The HGFinduced 

migration is strongly reduced by a ROCK inhibitor and in preliminary 

experiments, was also inhibited by a ROCK fragment that blocks the interaction 

between ROCK and ICAP-I. These results indicate that the ICAP-ROCK interaction 

plays a role in this migration. Central but not peripheral FA formation depends on 

ROCK activity, so this may involve interaction with ICAP-I. The relevance for 

metastasis is that invasion of carcinoma ceIls into tissues can proceed via two 

mechanisms, one ofwhich is completely dependent on ROCK. We will investigate 

the role ofICAP-I in that mode ofinvasion. 

Fig. IT Staining ofvinculin, a component offocal adhesions (FA), in C2Cl2 myoblasts. 

Left: Overexpression of ICAP-I greatly increases the number of FA in the center of the ce!!, a 

phenotype associated with fast migration. Right: RNAi knockdown of ICAP-I, in the ce!! marked with 

an asterisk, causes a complex loss of centra I FA, whereas peripheral FA at the edges of the ce!!, become 

far more abundant, a phenotype associated with low migration.

BIOPHYSICS IN CELL SIGNALING 

Understanding the often short-lived and local signal transduction events that regulate 

cell behavior requires techniques to study cellular signals with high spatiotemporal 

resolution in single living cells. Our group is engaged in development and 

implementation ofbiophysical techniques that provide this resolution, and in the 

application thereof in ongoing research both within our group and in collaborations 

with other groups in the NKI. 

Group leader Kees jalink 

Spatiotemporal aspects of phosphatidylinositol bisphosphate (PI P 2 ) as a 

membrane-delimited messenger Plasma membrane [PIP 2 ] is a key regulator of 

several cellular responses, induding ion channel gating, receptor internalization and 

the cortical actin rearrangements that underly locomotion. Using the Fluorescence 

Resonance Energy Transfer (FRET)-based assay for PIP 2 that we developed earlier, 

we study the regulation of agonist-induced PIP 2 breakdown. FRET has revealed 

receptor specific and celi type specific differences in the dynamics of PIP 2 hydrolysis 

that are not uncovered by other assays. In collaboration with Nullin Divecha 

(Division of Cellular Biochemistry) we also study the role of the different PIP kinase 

isozyrnes in the subsequent PIP 2 resynthesis phase. 

How can a single pool of a diffusible messenger at the plasma membrane 

independently regulate different cellular responses, apparently with spatial precision? 

We undertook an in-depth analysis of the spatial distribution of this lipid along the 

plasma membrane. We showed that build-up of spatial PIP 2 differences is limited by 

rapid later al diffusion, except when diffusion barriers exist. We are currently 

assessing whether submicroscopical solid phase lipid domains (such as e.g. rafts) 

may effectively act to localize PIP 2 pools at the plasma membrane, using a 

combination ofbiochemistry and biophysical techniques. For this research, highly 

corrected confocal FRET imaging software was developed in our group that should be 

equally valuable to other users. 

Role of the TRPM7 ion channel in cell adhesion TRPM7 is a non-selective cation 

channel with inherent kinase activity that is expressed in many celi types. It has been 

shown to promote agonist-induced myosin heavy chain (MHC) phosphorylation, 

leading to dramatic changes is cell adhesion and morphology. Using patch damp and 

ion imaging techniques to study TRPM7, it was found that it functions as an agonistoperated 

Ca 2 + channel, and that Ca 2 + influx is crucial for MHC-phosphorylation and 

the ensuing morphological changes. We currently study what signaling events lead to 

opening of the channels. 

Towards chemical imaging of cells Mass spectrometry (MS) is sensitive enough to 

detect just a few molecules. In collaboration with the MS group lead by Prof. Ron 

Heeren at AMOLF, we investigate approaches to exploit MS for chemical imaging 

with subcellular resolution. We are currently defining optimized conditions to 

generate ions from rapidly frozen cultured cells, and have started to image single 

cells using a novel so-called stigmatic imaging approach that was developed by our 

collaborators. 

Kees Jalink PhD Group leader 

Gerard Van der Krogt MSc Technician 

Michiel Langeslag MSc Graduate student 

Jacco Van Rheenen MSc Graduate student 

Bas Ponsioen MSc Graduate student 

Andre Djajasaputra U ndergraduate student 


Reits E, Griekspoor A, Neijssen J. 

Groothuis T, Jalink K, Van Veelen P, 

Janssen H, Calafat J. Drijfhout J-W, 

Neefjes J. Peptides in living cells: diffusion, 

protection and degradation in nuclear and 

cytoplasmic compartments before antigen 

presentation by MHC class J. 1mmunity 

2003; 18:1·20. 

Price LS, Langeslag M, Ten Klooster J-p, 

Jalink K, Collard JG. Calcium signalling 

regulates translocation and activation of 

Rac via PKC-mediated phosphorylation of 

RhoGD1. j Biol Chem. 2003; 1278.J9413-21. 

Van Rheenen J. Langeslag M, Jalink K. 

Correcting confocal acquisition to optimize 

imaging of jluorescence resonance energy 

tranifer by sensitized emission. Biophys j , 

in press. 

Lee SB, Várnai P, Balla A, Jalink K, 

Rhee S-G, Balla T. The pleckstrin-homology 

domain of PLC04 is not a major 

determinant ofthe membrane localization 

ofthe enzyme. J. Biol. Chem., in press. 

Scope of cyclic nucleotide messen gers in coupled cells The cydic nudeotides 

cAMP and cGMP are important intracellular messengers involved in regulating 

quiescence and differentiation, among others. It is speculated that these molecules 

also determine the fate of neighboring cells by permeating through gap junctions, 

although direct evidence for this notion is lacking. We utilize FRET sensors for 

cAMP and cGMP in combination with photo-releasable ('caged') cydic nudeotides to 

study the scope of these messengers in cultured cells. 

- Rotlo 

Fig. 1.6: FRET-imaging of cAMP levels in single 

living cello The trace shows the transient increase 

in cAMP following stimulation with isoproterenol, 

contrasting with the sustained effict of forskolin. 

1nset, rat-1fibroblast cells expressing the FRET 

sensor.

IMMUNO-EM FACILITY 

Group leader j ero Calafat 

jero Calafat PhD Group leader 

Hans janssen Technical staff 

Nico Ong Technical staff 


Mollinedo F, Martin-Martin B, Calafat J, 

Nabokina SM, Lazo PA. Role ofvesicleassociated 

membrane protein-2, through 

Q-soluble N-ethylmaleimide-sensitive factor 

attachment protein receptorj R-soluble 

N-ethylmaleimide-sensitive factor 

attachment protein receptor interaction, 

in the exocytosis of specific and tertiary 

granules of human neutrophils. j Jmmunol 

2003; 17°:1°34-42. 

Persson-Dajotoy T, Andersson P, 

Bjartell A, Calafat J, Egesten A. Expression 

and Production ofthe CXC Chemokine 

Growth-Related Oncogene-alpha by 

Human Eosinophils. j Jmmunol 2003; 

17°:53°9-16. 

Gao Y, Rosen H , johnsson E, Cal afat J, 

Tapper H , Olsson I. Sorting of soluble 

TNF-receptor fo r granule storage in 

hematopoietic ce Us as a principle for 

targeting of selected proteins to inflamed 

sites. Blood 2003; 102:682-8. 

Reits E, Griekspoor A, Neijssen J, 

Groothuis T, Jalink K, Van Veelen P, 

Janssen H, Calafat J, Drijfhout JW, 

Neefjes J. Peptide diffusion, protection, and 

degradation in nuclear and cytoplasmic 

compartments before antigen presentation by 

MHC class 1. Jmmunity 2003; 18:97-108. 

Immunoelectron microscopy reveals a great wealth of structural details in the 2 to 

IOO nm range providing information that can be obtained in no other way. This and 

other techniques were used to study the localization and trafficking of molecules in 

the celi i.e. sorting and processing of proteins and localization of molecules in the celi 

during signal transduction events. 

localization and trafficking of molecules in the cell In collaboration with 

Or F Mollinedo from the Universidad de Salamanca, Spain, we have studied the 

subcellular localization of the R-soluble N-ethylmaleimide-sensitive factor attachment 

protein receptor (SNARE) synaptobrevin-2jvesicle-associated membrane protein 

(VAMP)-2 in neutrophils. The results showed that VAMP-2 was present on the 

membranes of specific and gelatinase-containing tertiary granules in resting human 

neutrophils, and, upon activation, translocated to the celI surface. VAMP-2 was also 

found on the external membrane region of granules docking to the plasma 

membrane in activated neutrophils. Activation of neutrophils led to the interaction of 

VAMP-2 with the plasma membrane Q-SNARE syntaxin 4, since anti-syntaxin 4 

antibodies inhibited exocytosis of specific and tertiary granules in electropermeabilized 

neutrophils. Immunoelectron microscopy showed syntaxin 4 on the 

plasma membrane contacting with docked granules in activated neutrophils. These 

data indicate that VAMP-2 mediates exocytosis of specific and tertiary granules, and 

that Q-SNAREjR-SNARE complexes containing VAMP-2 and syntaxin 4 are involved 

in neutrophil exocytosis. In colIaboration with Or A Egesten from Lund University, 

Sweden, we have shown that large amounts of the neutrophil-activating CXC 

chemokine growth-related oncogene (GRO)-a are produced by human eosinophils. 

GRO-a was abundantly present in the crystalloid-containing specific granules 

(Figure r). Tumor-infiltrating eosinophils in Hodgkin's disease of the nodular 

sclerosing type are associated with a poor prognosis. Eosinophils from such tumor 

tissues showed an abundant expression of G RO-a. The G RO-a receptor CXCR2 was 

also detected in tumor tissue, suggesting interactions between eosinophils and the 

tumor. In collaboration with Or. I. Olsson from Lund University, Sweden, we studied 

the sorting and processing of soluble TNF-receptor (sTNFRr) in murine myeloid RBL 

cellline. For successful targeting of sTNFRr to secretory lysosomes, endoplasmic 

reticulum (ER) retention and constitutive secretion have to be prevented. ER export 

was facilitated by using a transmembrane (tm) form of sTNFRr, and constitutive 

secretion was overcome by the incorporation of a cytosolic sorting signal (Y) from 

CD63, a norm al transmembrane component of secretory lysosomes. This signal 

directed the resulting sTNFRr-trn-Y to secretory lysosomes. To confirrn this, we 

perfomed immunoelectron-microscopy with double immunogold labeling. Colocalization 

of sTNFRr-trn-Y was found with RMCP-II, the major constituent of the 

granules ofRBL cells and rat lysosomal glycoprotein r20 (lgpI20). These data are 

consistent with targeting of sTNFRr-trn-Y to secretory lysosomes of RBL cells and 

suggest a potential for using the storage organelles ofhematopoietic cells as vehicles 

for targeting sites of inflammation with therapeutically active agents. 

Collaboration with other groups from N KI/Avl In collaboration with Or E Reits 

(Division of Tumor Biology) we have shown that TAP is excluded from the nuclear 

side of the nuclear envelope. Immunogold electron microscopy using antibodies 

against the cytoplasmic nucleotide binding domain of TAP show gold particle 

staining exclusively on the cytoplasmic side of the nuclear envelope (150 gold 

particles on the cytoplasmic side, none on the nuclear side). TAP is apparently 

excluded from the nuclear face of the nuclear envelope in agreement with the data 

obtained from FLIP analysis. These findings imply that nuclear peptides have to 

enter the cytoplasm for TAP translocation into the ER lumen. 

Fig. J. 7: CXC chemokine growth-related oncogene (G RO )-a labeled by immunogold is abundantly present in 

crystalloid-containing granu!es of eosinophils. Bar, 4°° nm.

11 DIVISION OF MOLECULAR 

CARCINOGENESIS 

FUNCTIONAL GENOMICS 

The focus of this group is on the application of innovative functional genomics tools 

to identify genes having a role in carcinogenesis. We use both high-throughput gainof-function 

genetic screens with retroviral cDNA expression libraries and loss-offunction 

genetic screens with RNA interference libraries to identify novel 

components of cancer-relevant pathways. 

Now that the sequence of the human genome is almost complete, we are presented 

with both unprecedented challenges and opportunities. One of the major remaining 

challenges is that information regarding gene function is available for only some 

6,000 of the approximately 30-40,000 genes in the human genome. Thus, we need 

to functionally annotate the tens of thousands of genes for which this information is 

currently lacking. Our laboratory uses functional genetic approaches to obtain 

information regarding gene function in high-throughput screens. We have 

developed both gain-of-function genetic screens (with retroviral cDNA expres sion 

libraries) and vector-based RNA interference to carry out large-scale loss-of-function 

genetic screens in mammalian cells. We focus on the central growth-regulatory 

pathways that are most frequently deregulated in cancer, such as the pI6INl


Bernards R. Cues for migration. Nature 

2003; 42P47-8. 

Brummelkamp TR. Funtional Genetic 

Screens in Mammalian Cells: Hitting 

Cancer. Thesis 2002, Utrecht. 

Brummelkamp TR, Bernards R. New tools 

for functional mammalian cancer genetics. 

Nature Rev Cancer 2003; 3:781-9. 

Brummelkamp TR, Nijman 5MB, 

Dirac AMG, Bernards R. Loss ofthe 

cylindromatosis tumour suppressor inhibits 

apoptosis by activating NF-KB, Nature 

2003; 424:797-801. 

Das AT, Brummelkamp TR, 

Westerhout EM, Vink M, Madiredjo M, 

Bernards R, Berkhout B. HIV-1 escapes 

from RNA interference-mediated inhibition. 

] Vi rol 2003 (in press). 

Dirac AMG, Bernards R. Reversalof 

senescence in mouse fibroblasts through 

lentiviral suppression of p 53. ] Biol Chem 

2003; 278:11731-4. 

Scheijen B, Bronk M , Van der Meer T, 

Bernards R. Constitutive E2F1 

overexpression delays endochondral bone 

formation by inhibiting chondrocyte 

differentiation. Mol Cell Bio12003; 

2]J656-68. 

Van de Vijver M). He YD, Van 't Veer L). 

Dai H, Hart AAM, Voskuil D, 

Schreiber G). Peterse J L, Roberts C, 

Marton M). Parrish M, Atsma D, 

Witteveen A, Glas A, Delahaye L, 

Van der Velde T. Bartelink H, 

Rodenhuis S, Rutgers ETh, Friend SH , 

Bernards R. A gene expression signature as 

a predictor of survival in breast cancer. New 

England ] Med 2002; 34T1999-2oo9. 

products exists. In simple organisms, such as yeast and C. elegans, many synthetic 

lethal interactions have been identified; in contrast, there are few examples of 

synthetic lethality in mammalian systems. Identification of synthetic lethal 

interactions may be ofkey importance for the identification of novel and more 

powerful classes of cancer drug targets. The problem in identifying siRNA vectors 

that show a synthetic lethal interaction with cancer-specific mutations is that such 

vectors are specifically lost from cells, making it hard to identify such interactions 

when such screens are performed in a polyclonal format. 

To facilitate the identification of synthetic lethal interactions in mammalian cells 

with vector-based siRNA libraries, we have developed a technology that we have 

dubbed 'siRNA bar code screens' (in collaboration with R Beijersbergen, division of 

Molecular Carcinogenesis, Figure II.2). Molecular bar codes we re first used in yeast 

genetic screens, in which large numbers of yeast genes we re inactivated by insertion 

of a DNA segment harboring a unique 20 nucleotide sequence (the 'molecular bar 

code') to mark the individual knock-out yeast strains. The molecular bar code serves 

as a'strain identifier' , which makes it possible to follow the relative abundance of 

each mutant yeast strain by measuring the abundance of the bar codes in the 

population. Quantification of the relative abundance of mutant strains under various 

experimental stress conditions was achieved through PCR amplification of the bar 

codes, followed by hybridization to microarrays containing the bar code sequences. 

By analogy to the yeast bar code approach, we noted that the DNA fragment of each 

siRNA expres sion vector encoding the RNA hairpin transcript has a unique 19 base 

pair target-specific sequence. Therefore, introduction of such a vector into 

mammalian cells results in the creation of a tagged knockdown cell that carries a 

permanent gene-specific identifier (knockdown identifier) (Figure II.2). This 

molecular bar code can be recovered by PCR amplification using vector-derived PCR 

primers that flank the hairpin-encoding DNA sequence. Fluorescent labeling of the 

PCR product allows it to be identified by hybridization to microarrays that contain 

the gene-specific knockdown bar code oligonucleotides (Figure 11.2). The relative 

abundance of each bar-coded DNA fragment in the cell population is influenced by 

the effect that each knockdown vector has on cellular fitness under the experimental 

conditions and can be quantified using a DNA micro array that contains the bar code 

DNA fragments. Comparing the effects ofknockdown vectors on cellular fitness in 

pairs of cells that have defined genetic lesions will make it possible to identifY 

synthetic lethal phenotypes in human cells, as siRNA vectors that are specifically lost 

from one population can be identified in this way. We are currently using siRNA 

bard code screening technology to find genes that are synthetic lethal with known 

anti-cancer drugs (e.g. taxol) and genes whose inactivation is only toxic in cells having 

mutations in oncogenes or tumor suppressor genes. 

."r'-.. 

59·mer oligo 

e 

cloned into 

retrovi ral ve cto r 

NKi library 

mixture 

~ 

i nfe cti 0 n!i nte 9 r ati 0 n 

peR recovery! 

., ULS labeling 

."r'-.. 

."r'-.. 

."r'-.. ------.. 

."r'-""!I array hybridization 

., 

\. DNA micro· array 

r tNith 59-mer oligos 

J 

Figure II.2: siRNA bar code screens. 

DNA microarrays containing the 59-mer oligonucleotides present in a selected set of 990 shRNA vectors 

were generated. Cells infected with pools 1-4 of the library were used for PCR amplification. Fluorescentlylabeled 

bar code fragments were hybridized on micro-arrays that consists of oligonucleotides present in 

pools 1-4 (upper half) or absent from pools 1-4 (lower half).

CONTROL OF TELOMERASE ACTIVATION DURING 

IMMORTALIZATION AND TUMORIGENIC TRANSFORMATION 

The aim of our research is to identify the pathways that control the expression of the 

catalytic subunit of telomerase, hTERT. The extensive proliferative capacity observed 

in malignant tumor cells is directly associated with the activation of telomerase. 

Understanding the mechanism of telomerase activation wiUlead to the identification 

of proteins or pathways, which are deregulated in human cancer and possibly provide 

a therapeutic target. 

The expression hTERT, the catalytic component of telomerase, represents the ratelimiting 

step in process of immortalization and is therefore crucial for human 

tumorigenesis. Forced expression ofhTERT in human primary cells results in 

immortalization, whereas inactivation ofhTERT in human tumor cells leads to 

growth arrest and cell death. Although many proteins have been implicated in the 

regulation ofhTERT expres sion, no significant correlation has been found between 

these factors and the process of immortalization. To date, our attempts at identifying 

genes that, upon over-expression, activate hTERT expression have been unsuccessful. 

However, several studies such as those using cell fusions or micro-cell mediated 

chromosomal transfer have indicated that repression of telomerase is dominant over 

activation of telomerase. They also suggest that activation ofhTERT expression in 

tumor cells is most likely due to the loss of (a) repressor protein(s). In the past year, 

we have generated tools to inactivate a large set ofhuman genes with the aim of 

identifying genes that upon inactivation will result in the activation ofhTERT 

expres sion. 

Group leader Roderiek Beijersbergen 

Roderiek Beijersbergen PhO Group leader 

leone Carlee MSc Graduate student 

Wouter Nijkamp Technical staff 

Corneel Honingh Undergraduate student 

lennart Post Undergraduate student 

Simon Joosse Trainee technician 

Johan Kuiken Trainee technician 

Rebecca Bish Guest 

The NKi shRNA library We have generated aplasmid based RNA interference 

library to inactivate the expres sion of celluiar genes. The recently developed vector 

pRETRO-SUPER mediates stabie suppression of gene expres sion in mammalian 

ceUs through expres sion of a short hairpin RNA (shRNA) with siRNA properties. 

These vectors can be introduced either by transfection or retroviral infection and 

cause permanent loss of expression. In order to identify novel proteins in telomerase 

regulation we have generated a shRNA library that targets more than 7,9°0 genes 

including families of genes that are involved in celluiar processes such as 

transcription, DNA structure and chromatin modelling, DNA damage response 

proteins and proteins involved in signal transduction pathways collaboration with the 

group ofR Bernards). This large collection ofknockdown vectors is generated in a 

single gene per weIl format. Every gene is targeted by three different knock-down 

constructs combined in one weU. This design aUows for the generation of smaU sublibraries 

compromising a gene-family of interest. The knock-down vectors can also be 

combined to create a smaller library in which constructs in each weU target a small 

number of genes at the same time. To identify genes that upon knock-down induce 

telomerase expres sion we are using this collection of shRNA vectors in hTERT 

promoter reporter assays and telomerase activity assays and select those siRNAs that 

activate reporter gene expres sion or siRNAs that induce telomerase enzyme activity. 

Telomerase activity is directly measured in ceU extracts from 96 weU plates and the 

shRNA library is screened in a gene per weIl basis. 

UNTREATEDIUNSELECTED / 

"'-TREATEDISELECTED 

Reeove'Y .hRNA c •••• tte. 

wlll1 PCR ampllflcatlon = = 

Bypass of crisis and immortalization induced by specific siRNAs The most 

elegant way to identify genes that are involved in the repression ofhTERT 

transcription is a model system in which ceUs dep end on the activation ofhTERT 

expres sion for their growth and survival. CeUs that have been transformed with e.g. 

SV 40 T antigen bypass senescence but ultimately cease proliferation due to their 

short telomeres and the resulting genomic instability. During this period, caUed 

crisis, the majority of the cells undergo apoptosis but eventually immortal dones wiU 

appear that have activated hTERT expression. Crisis can be avoided upon 

introduction ofhTERT expres sion. We have generated human embryonic kidney 

cells transformed with SV 40 T antigen (HEKLT ceUs) expressing the ecotropic 

receptor that are dose to crisis and cease proliferation after a finite number of 

population doublings. Infection of these cells with a low titer hTERT encoding 

retrovirus results in the appearance of immortal dones. We use pre-crisis HEKLT 

Figure II.y Schematic outline of the shRNA 

Barcode screen. A population of cells is infected 

with a large pool of shRNA vectors. After 

application of a treatment that will select for a 

specific phenotype, the shRNA inserts are recovered 

by peR and labeled with fluorescent dyes. These 

labeled fragments are used in DNA microarray 

hybridization to arrays containing the 19-mer 

barcode sequences. The relative abundance of a 

specific shRNA can be determined by the ratio of 

the different fluorochromes.

A. 

B .• 

tIJ 

"iL +-. .. 

7 ............. ' ... U . 

, . 

~ 

c .. ,.-------------------, 

Figure II+ 

A. Example of a microarray hybridization 

containing 19-mer barcode sequences with 

PCRfragments labeled with CY3 or Cn. 

E. Ratio of shRNA abundance from mock 

treated ceUs at day 5 compared to day o. 

Ratios are 2log transformed and depicted 

against the 2log of the mean intensity of the 

CY3 and CY5 signal for that particular spot. 

C. Ratios of shRNA abundance of H202 treated 

cells at day 5 compared to day . Ratios are 

depicted as in E. The circled spots contain 

shRNAs targeting the Cylindromatosis 

suppressor gene involved in NF-KB signalling 

and apoptosis. 

cens to perform this functional screen by the introduction of shRNA constructs. The 

cens are infected with library pools of pRETRO-S UPER containing shRNA constructs 

targeting 96 genes per pool. We win infect these pools into HEKLT cells separately 

and culture the infected cens. The advantage of this system is the low complexity per 

infected cen population thereby reducing the contribution of spontaneously arising 

background colonies. Activation ofhTERT win result in immortalization and we are 

screening for those siRNAs that can in duce immortalization in primary or 

transformed mortal human cens. 

ShRNA bar code screens The design ofthe NKi shRNA library anows for the 

identification of individual shRNA vectors in a large pool of shRNA vector containing 

cens. Each hairpin vector contains a unique 19-mer sequence ('bar code') designed to 

specificany target one specific cenular gene. This 19-mer sequence can be used to 

determine the relative abundance of a shRNA vector in a large set of shRNA vectors 

by DNA micro array hybridization to arrays containing the 19-mer bar code 

sequences. Apart from the relative ease in identifying a siRNA that affects the 

phenotypic behaviour of cens, the bar code technique also anows for the 

identification of siRNAs that are selected against in aspecific cen population and 

makes it possible to reveal subtie phenotypes caused by siRNAs that are not detected 

in other types of screening assays. 

In the past year, we have shown that we can use the Nki shRNA library for large-scale 

bar code screens. In the example depicted in Figure II-4, we have identified that 

down-regulation of the Cylindromatosis tumor suppressor gene results in decreased 

cen death upon oxidative stress. We are currently applying the bar code technique in 

several different types of screens. One specific focus is on the identification of genes 

whose inactivation is selectively toxic in telomerase positive post-crisis cens and not 

in telomerase negative pre-crisis cens. The generation of primary human cens with 

and without telomerase in the same genetic background and the transformed 

telomerase negative pre-crisis HEKLT cens and their post-crisis counterparts with 

telomerase expression are very suitable for the identification of genes whose 

inactivation is synthetic lethal with the expression of telomerase. 

With the application of these powerful novel techniques we can potentiany identify 

repressor proteins that win help in the elucidation of the molecular mechanism that 

enables cancer cens to overcome two barriers, senescence and crisis, before they 

become immortal. The identification of the proteins or pathways that are involved in 

regulation of telomerase expression may have implications for interference with the 

process of immortalization and provide us with a molecular event, specific in the 

generation of tumor cens.

STRUCTURAL BIOLOGY 

Development of cancer is generally due to errors that occur in celluiar pathways. 

Structural biology can help to understand these errors at the atomic level, by studying 

the proteins and the DNA involved. We use X-ray crystallography as a tooI to provide 

three-dimensional structures. Subsequently we interpret the structural data using a 

variety ofbiochemical and biophysical techniques. As a member of the European 

SPINE network we aim to implement fa ster methods for structure determination, in 

particular for macromolecular complexes. These studies provide more insight in the 

molecular processes and they can also provide targets for drug design studies. In our 

group we focus mainly on proteins involved in DNA stability (mis match repair), 

transmembrane signaling and cell cycle control. 

DNA mismatch repair Mutations in DNA mismatch repair genes predispose to 

hereditary non-polyposis colon carcinoma (HNPCC). DNA mismatch repair is 

specific for a single mismatch or a small stretch of unpaired bases in DNA. It 

involves a cascade of proteins that is highly conserved from bacteria to humans. The 

initial step of the rep air is the recognition and binding of mismatched DNA by the 

MutS protein (E. coli) or its heterodimeric MSH2jMSH6 or MSH2jMSH3 homologs 

(eukaryotes). This complex is then recognized by the MutL protein or its homologs. 

E. coli MutS recognizes mispaired and unpaired bases and has an intrinsic ATPase 

activity. ATP binding af ter mismatch recognition by MutS serves as a switch that 

enables MutL binding and subsequent initiation of mismatch repair. We have 

studied the effect of ATP binding on the MutS structure. Crystallographic studies of 

ATP soaked crystals of MutS show a trapped intermediate, with ATP in the 

nucleotide binding site (Figure 11.5). Large structural changes are absent in this 

trapped structure, yet rearrangements of several residues around the nucleotide 

binding site suggest a rearrangement of the two ATPase domains of the MutS dimer. 

Analytical ultracentrifugation experiments confirm such a re arrangement, showing 

increased affinity between the ATPase domains upon ATP binding and decreased 

affinity in the presence of ADP. Mutations of specific residues in the nucleotide 

binding domain reduce the dimer-affinity of the ATPase domains. In addition, ATP 

induced release of DNA is strongly reduced in these mutants, suggesting that the two 

activities are coupled. Apparently the rearrangement of the ATPase domains upon 

ATP binding forms the basis for long-range interactions between ATPase domain 

and DNA binding domains, and subsequent binding of MutL and initiation of 

mismatch repair. 

Group leader Titia sixma 

Titia Sixma PhD Group leader 

Patrick Celie PhD Post-doe 

Valerie Notenboom PhD Post-doe 

Joyce lebbink PhD post-doe 

Gretel Buchwald PhD Post-doe 

David Egan PhD Post-doe 

Victor Rucker PhD post-doe 

Puck Knipscheer MSc Graduate student 

Meindert lamers MSc Graduate student 

Ganesh Natrajan MSc Graduate student 

Marc Vargas MSc Technical staff 

Pim Van Dijk Technical staff 

Sari Van Rossum-Fikkert Technical staff 

Fabien Barralon Undergraduate student 

Ester Frische Undergraduate student 

Acetylcholine binding protein (AChBP) Nicotinic acetylcholine receptors respond 

to both carbamylcholine and nicotine binding in their extracellular domain by 

Figure II.5: Changes in the nucleotide binding site of MutS between ADP (lefi) and ATP (right) 

(Lamers et al, in preparation).


Lamers MH. Looking at MutS. A view on 

DNA mismatch repair initiation. Thesis 

2002, Utrecht. 

Lamers MH, Winterwerp HHK, Sixma TK. 

The alternating A TPase domains of the 

DNA mismatch repair enzyme MutS 

control DNA mismatch repair. EMBOJ 

2003; 22746-56. 

Natrajan G, Lamers MH, Enzlin JH , 

Winterwerp HHK, Perrakis A, Sixma TK. 

Structures of Escherichia coli DNA 

mismatch repair enzyme MutS in complex 

with different mismatches: a common 

recognition mode for diverse substrates. Nucl 

Acids Res 2003; 31:4814-21. 

Sixma TK, Smit AB . Acetylcholine binding 

protein (AChBP) : a secreted glial protein 

that provides a high resolution model for the 

extracellular domain of pentameric ligandgated 

ion channels. Annual Reviews of 

Biophysics and Biomolecular Structure 

2003; ]2]11-34· 

Smit AB, Brejc K, Syed N, Sixma TK. 

Structure and function of AChBP, 

homologue ofthe ligand-binding domain of 

the nicotinic acetylcholine receptor. Ann NY 

Acad Sci 2003; 998:81-92. 

Figure II.6: Nicotine binding to AChBP. 

Schematic secondary structure representation of 

AchBP (Celie et al, submittedfor publication). 

opening a transmembrane ion channel. These are pentameric proteins with one 

central pore. The -200 amino-acid extraceilular domain has the ligand binding 

capacity at the interface between two subunits and can be exchanged between family 

members within a superfamily of pentameric ligand-gated ion channels that also 

indudes the GABAA and GABAC, 5HT3 serotonin and glycine receptors. The ligand 

binding domain dosely resembles a water soluble protein from moiluscan glial ceils, 

cailed acetylcholine binding protein (AChBP) . This protein functions in modulating 

the synaptic transmission of acetylcholine. Because of its high sequence similarity 

(15-28% identity) to the nicotinic receptors, our crystal structure of Lymnaea stagnalis 

AchBP has become the established model for the extraceilular do ma in of the 

pentameric LGICs. 

We have solved crystal structures of AChBP bound to carbamylcholine, nicotine 

(Figure n.6) and compared this to a high resolution native data set (2.0 A) and we 

have analysed the ligand binding properties of the AChBP for nicotinic agonists by 

isothermal calorimetry_ Dissociation constants agree weil with those obtained from 

competition studies with bungarotoxin. Ligand binding is dominated by enthalpic 

changes, but entropy also contributes to binding. Carbamylcholine and nicotine bind 

in approxirnately the same position, with their nitrogens almost overlapping. The 

binding is characterized by substantial aromatic and hydrophobic contributions and 

unexpectedly by dose contacts between protein oxygens and carbon atoms in the 

choline portion of carbamylcholine and nitrogen in the pyrrolidine ring of nicotine. 

The largest movement within the binding site is seen in the C-Ioop, which doses in 

around the ligand. The higher affmity of nicotine may be due to a main-chain 

hydrogen bond with the B-Ioop and a closer packing of the aromatic groups. 

Despite the absence of conformational changes that could provide insight in the 

molecular mechanism ofligand-induced gating, these high resolution structures 

reveal agonists binding to apentameric ligand-gated ion channel, one of the most 

important classes of drug targets. These structures will be useful tools for the 

development of new drugs involving nicotinic acetylcholine receptor associated 

diseases. 

Ubiquitin dependent conjugation Ubiquitin conjugation processes are emerging 

as a general addressing system that is essential for cell stability, by controlling 

degradation of short-lived proteins, DNA repair and targeting to specific areas in 

the ceil through endocytosis. Because of its importance for regulating cell cycle, 

apoptosis and DNA rep air deregulation of ubiquitin-dependent processes often leads 

to cancer. 

The process of conjugation by ubiquitin(-like) proteins involves covalent linking of 

one or more 76-arnino-acid ubiquitins to a target protein by an EI/E2/E3 cascade of 

enzymes. Correct ubiquitination requires the complex spatial arrangement of 

ubiquitin, E2, E3 proteins and the target simultaneously in a precise but flexible 

marmer. Although the overall mechanism has been defmed, the atomic details have 

been lacking and the specificity determining factors are unclear. We study several 

E2/E3 complexes_ 

SUMO is a ubiquitin-like protein that plays a variety of regulatory roles in the cello In 

collaboration with the group of Frauke Melchior we study the SUMO pathway at the 

biochemicallevel. We have created E2/E3 complexes as weil as sumoylated targets for 

crystallization purposes. A sumoylated target was identified that could be purified in 

sufficiently large quantities for crystallization_ So far we have solved the crystal 

structure of the sumoylated target to 2.3 Aresolution and refinement is in progress, 

while crystals of the target alone have recently been obtained_ The sumoylation 

prevents the enzymatic activity of the target and therefore the comparison of the 

crystal structures of the target protein with and without Sumo attached will be of 

interest. 

The E2/E3 complex of marnmalian Rad6/RadI8 is involved in error-free postreplicative 

DNA repair. In collaboration with J Hoeijmakers (Rotterdam) we have 

purified the complex from E. eoU to homogeneity_ The complex is stabie through a 

number of purification steps and is currently being submitted to crystallization trials. 

Using in vitro purified protein we can show ubiquitination and sumoylation ofPCNA 

as a target_

STRUCTURAL BIOLOGY 

Our interests for this year remained shared between performing structural biology 

research projects, predominantly in collaboration with in-house groups, and 

developing methods for X-ray crystallography. 

Structural Biology The interests ofthe group on understanding the retroposition 

mechanism, regulation of vesicle transport and transcriptional regulation in 

mitochondria evolved during the year. Two new projects also emerged: The structural 

study of Polo kinase, which is involved in various check points in the cell cycle (with 

R Mederna, Division of Molecular Biology) and ofthe CdtIjgeminin protein complex 

(with Z Lygerou and S Taraviras, University of Patras) which is involved in DNA 

replication licensing. We also initiated (with W Mooienaar, Division of Cellular 

Biochemistry) a project on the study of autotaxin (ATX), a factor secreted by tumor 

cells and recently identified as a serum lysophospholipase D, which hydrolyzes 

circulating lysophosphatidylcholine. 

Structural characterization of the human L 1 retrotransposition machinery 

The human LI endonuclease (LIEN) is encoded by the LI non-LTR retrotransposon 

that is responsible for more than 1.5 million genomic re-integration events in the 

history of the human genome and has resulted in more than a quarter of our 

genomic DNA. Since LIEN infers target specificity for the retrotransposition events it 

is a central determinant of the role of non-LTR retrotransposons for the 'fluidity' and 

evolution of genomes. We determined the first crystal structure ofhuman LIEN 

(Figure 11.7), and based on it we constructed a structure-based alignment that 

explains the role of conserved residues in the DNA nicking mechanism. We were 

also able to provide structural support for the hypothesis that DNA target recognition 

proceeds via the recognition of a flipped-out nucleotide within a pocket of the 

enzyme. Finally, we rationalized existing biochemical data on DNA target recognition 

by LIEN, proposed structural elements and surface side-chains involved in DNA 

binding and discuss the general mode of DNA recognition by retrotransposon 

endonucleases. We anticipate that this work will greatly facilitate attempts to 

modulate the sequence specificity of any given endonuclease, e.g. to convert the 

respective retrotransposon into a genetic tooI for i.e. gene therapy. 

Structural studies of the Polo-like kinase Polo-like kinase-I (PIk-I) plays an 

important but highly controversial role in cell division. Primary cells have a different 

response than tumour cells upon inactivation of Plk-I, raising the question of what 

are the molecular determinants for the different roles of Plk-1. If that can be 

understood at the molecular level and we also determine the structure of the kinase 

domain of PlkI in differently ligated states we can evaluate its potential for the design 

of specific drugs, targeting only Plk-I in tumour cells. Plk-I consists of an N-terminal 

classical kinase domain and two polo box motifs (PBM) towards the C-terminus. The 

polo box motifs form a stabie homodimer, the polo box domain (PBD). The PBD of 

Plk-I is responsible for both the localization and kinase activity regulation of Plk-I, 

possibly being a major determinant of the diverse functional roles of Plk-I in cell 

cycle progression. We aim to gain molecular insight into the function ofPlk-I 

through characterization of its three dimensional structure by x-ray crystallography. 

We have successfully expressed full-Iength human Plk-I and constructed E.coli 

vectors that express different domains ofPlk-I (i.e. kinase domain or Polo boxes, 

either alone, or combined in a bi-cistronic vector). Through the structures we aim to 

study the mechanism of inactivation of Plk-I kinase activity by the preferential 

binding of the Polo-box motif to its non-phosphorylated state and understand the 

preference of Polo-domain for specific phospho-peptides. 

Group leader Anastassis Perrakis 

Anastassis Perrakis PhD Group leader 

Serge Cohen PhD Post-doe 

Valeria De Marco PhD Post-doe 

Francesco Fernandez PhD Post-doe 

Rebecca Persson PhD Post-doe 

Koen Verschueren PhD Post-doe 

Oliver Weichenrieder PhD Post-doe 

Kostas Repanas MSc Graduate student 

Nuno Rocha MSc Graduate student 

Evangelos Christodoulou Technical staff 

Marouane Ben Jelloul MSc Software Engineer 

Mattheos Kakaris MSc Software Engineer 

Thomas Chastaing Undergraduate student 

Xavier Cheval Undergraduate student 

Janet Newman PhD Guest 

Figure IJ. 7: Cartoon representation of the 

structure of the 11 endonuclease. 

The Cdtl/Geminin complex in DNA replication licensing Our main goal is to 

understand the specificity of geminin for inhibiting the action of CdtI, a necessary 

factor for the formation of the pre-replication complex or 'replication license', which 

is necessary to be assembied prior to DNA replication in eukaryotic cells. 

Understanding the details of this interaction at the molecularJstructurallevel will 

firstly allow the construction of follow-up experiments that will further clarifY the role


Morris Rl. Zwart PH, Cohen S, 

Fernandez Fl. Kakaris M, Kirillova 0 , 

Vonrhein C, Perrakis A, Lamzin VS. 

Breaking good resolutions with AR/wARP. 

] Synchrotron Rad (in press). 

Natrajan G, Lamers MH, Enzlin JH , 

Winterwerp HHK, Perrakis A, Sixma TK. 

Structures of Escherichia coli DNA 

mismatch repair enzyme MutS in complex 

with different mismatches: a common 

recognition mode for diverse substrates. 

Nucl Acids Res 2003: 31:4814-21. 

Xiao B, Spencer l. Clements A, 

Ali-Khan N, Mittnacht S, Broceno C, 

Burghammer M, Perrakis A, 

Marmorstein R, Gamblin SJ. Crystal 

structure of the retinoblastoma tumor 

suppressor protein bound to E2F and the 

molecular basis of its reguLation. Proc Natl 

Acad Sci USA 2003: 100:2363-8. 

and importance of the specific interaction of Cdtr and geminin and how this 

interaction prevents Cdtr from recruiting the minichromosome-maintenance (MCM) 

proteins to DNA. Cdtr has been recently demonstrated to have an oncogenic potential 

that wiil set the groundwork for the design of Cdtr inhibitors that mimic it's 

interaction with geminin and might provide the basis for anti-cancer drugs. We have 

generated E.coli over-expres sion clones for full-length geminin and Cdtr and an N 

and C- terminal truncated conserved domain of CdtI. We have also generated a clone 

for di-cistronic expres sion of the Cdtrjgeminin complex in E.coli. We were able to 

over-express and purify large quantities of soluble geminin and Cdtr alone and a 

stabie complex between the conserved domain of Cdtr and geminin. Characterisation 

and crystallisation studies are underway. 

Regulation of Iysosomal motility by RAB7/RI LP Intraceilular vesicle trafficking is 

a tightly controlled process by the RAB family of proteins that interact with aspecific 

subset of effectors, enhancing the selectivity, as weil as spatial and temporal 

regulation of vesicle trafficking. Knowledge of the structural basis by which RAB 

proteins selectïvely interact with their specific set of effectors is critical for 

understanding the specificity of membrane trafficking. RAB7 has been identified to 

be involved in governing aggregation and function on late endocytic transport. The 

activated, GTP-bound form of RAB7 interacts specifically at least with the effector 

protein RILP (RAB-Interacting Lysosomal Protein) which may be involved in 

vectorial vesicle transport by recruiting locally functional dynein-dynactin motors. 

The attempts to purify soluble recombinant RILP (expressed in E. eaU) under native 

conditions are still mostly unsuccessful and yield low amount of protein and no 

crystals. Co-expression ofboth RAB7 (wild type and GTP-form mutant) and its 

effector protein RILP in E.eoli did not yield a soluble complex. We have however 

managed to clone and purify a few deletion constructs of RILP that appear to be 

soluble. Microinjection of the purified protein andjor transfection with vectors 

carrying the corresponding domains (done by I Jordens and J Neefjes, Division of 

Tumor Biology) showed the deletion constructs to interact with RAB7 but not to 

recruit the translocation machinery. We are setting up a GTP-ase assay to check the 

activity of RAB7 and its modulation by RILP and the deletion constructs. If that is 

confrrmed, we plan to co-crystallize RAB7 with these deletion constructs, which 

range from 20-50 residues in length. 

A monomeric transcription factor: the mitochondrial O-Ioop binding protein 

(mtOBP) One of the nuclear encoded components of mitochondria is the 

mitochondrial D-loop binding protein (mtDBP), a monomeric 40kDa transcription 

termination factor (348 residues as mature protein) which binds to two -25bp long 

homologous sequences on the mitochondrial DNA. The protein has crystallized, but 

we we re unable to obtain diffraction quality crystals. We have made a serial of short 

N- and C- terminal deletion constructs and we try to crystallise them complexed with 

several oligonucleotides comprising the DNA binding site, a procedure that is now 

efficient utilising the robotics setup. 

Methods for X-ray crystallography ARP jwARP version 6 continued to be a highly 

valuable software in the field of macromolecular crystailography. Current licensees of 

version 6 include about 7°0 academic downloads and 40 industriallaboratories. 

We are finishing the preparation of ARP jwARP 7.0 that includes a new structure 

rebuilding module that was developed at the NKI. The functionality of that software 

wiil also be available through the CCP 4 Molecular Graphic package, a European 

collaborative project that is developing brand new interactive crystallographic 

modelling and illustration software for Windows PCs, OSX MACs and UnixjLinux 

platforms. Now that the software engineering 'exercise' is ne ar to conclusion we have 

in our hands not only reengineered software but also new software libraries that 

allows us to exploit more advanced algorithms and test new scientific ideas. Our 

effort is two-fold. Firstly, to develop new better 'features' for pattern recognition of 

protein structure in electron density maps, by exploiting various mathematical ideas. 

Secondly, to develop an 'expert system' that will make 'crystallographic decisions' and 

eventually airns to true 'user free' automation of the package.

III DIVISION OF CELLULAR 

BIOCH EM ISTRY 

TGF-fl SIGNAL TRANSDUCTION 

Transforming growth factor-~ (TGF-~) superfamily members, which include TGF-~s , 

activins and bone morphogenetic proteins (BMPs), regulate a broad spectrum of 

developmental processes. Perturbation of their activity has been implicated in a large 

variety ofhuman diseases, including cancer, fibrosis and vascular disorders. The 

aims of our research are to elucidate the molecular mechanisms by which TGF-~ 

family members elicit their cellular effects and to generate animal models for human 

diseases that are caused by subverted TG F-~ family signaling. Our long-term goal is 

to apply newly obtained insights in the development of novel therapeutic protocols to 

target these diseases. 

TGF-~ family members trans duce their signals via specific complexes of type land 

type II serine/threonine kinase receptors. The type I receptor (also termed activin 

receptor-like kinase or ALK) acts downstream of the type II receptor, and propagates 

the signal by phosphorylating specific receptor-regulated (R-)Smads. Whereas Smad2 

and Smad3 are phosphorylated byTGF-~ type I receptor (ALK5) and activin type I 

receptor (ALl

Selected publications (continued) 

Goumans M-J, Lebrin F, 

Valdimarsdottir G. Controlling the 

angiogenic switch: a balance between two 

distinct TG F f3 receptor signaling pathways. 

Trends Cardiovasc Med. 2003; 13:]01-307. 

Goumans M-J, Valdimarsdottir G, Itoh S, 

Lebrin F, Larsson J, Mummery C, 

Karlsson S, Ten Dijke P. Activin receptorlike 

kinase (ALK)1 is an antagonistic 

mediator oflateral TGFf3/ALK5 signaling. 

Mol Ce1l2003; 12:817-828. 

Itoh F, Itoh S, Goumans M-J, 

Valdimarsdottir G, lso T, Dotto GP, 

Hamamori Y, Kedes L, Kato M, 

Ten Dijke P. Synergy and antagonism 

between Notch and BMP receptor signaling 

pathways in endothelial ceUs. EMBO J 

(in press). 

Itoh S, Thorikay M, Kowanetz M, 

Moustakas A, Itoh F, Heldin C-H, 

Ten Dijke P. Elucidation ofSmad 

requirement in transJorming growth 

factor-f3 type I receptor induced responses. 

J Biol Chem. 2003; 278:]751-3761 . 

opposite effects on endothelial behavior; ALKS inhibits EC migration and 

proliferation whereas ALKr stimulates both processes. We identified genes that are 

specifically induced by TGF-~ via ALKS or AL Kr pathway. The activation state of the 

endothelium may depend on the level ofTGF-~-induced ALKr or ALKS signaling. 

Interestingly, we recently demonstrated that ligand binding and intracellular kinase 

domains of ALKS are required for TGF-~/ALKr activation. TGF-~/ALKS pathway can 

elicit an antagonistic signal via direct interaction and activation of ALK!. Activated 

AL Kr not only induced a biological response opposite from ALKS, but also directly 

inhibited ALKs/Smad signaling. The requirement for ALKS in ALKr activation and 

the counteractive interplay between ALKS and ALKr provides the endothelial cell with 

an intricately regulated TGF-~ controlled switch, which will determine whether its 

fate is quiescence or active migration and proliferation (Figure 111.2). 

An important target downstream ofTGF-~/ALKr pathway in promoting endothelial 

cell migration (and most likely also proliferation) is Idr, a naturally occurring 

inhibitor of basic helix-Ioop-helix transcription factors. We recently observed that 

ectopic expres sion of constitutively active ALKr (but not ALKS) or Idr strongly 

promotes the invasive properties of endothelial cells. This effect could be blocked by 

neutralizing antibodies against integrins or metallo-proteinase inhibitors. 

Endoglin, an accessory TGF-~ receptor, is highly expressed on proliferating 

endothelial cells in culture and blood vessels in vivo. Mutations in endoglin and ALKr 

have been linked to vascular disorders HHTr and HHT2, respectively. We found that 

endothelial cells without endoglin do not grow. Ectopie expres sion of endoglin 

promoted endothelial cell proliferation and inhibited TGF-~/ALKs-induced growth 

arrest. Knockdown of endoglin expression using siRNA in endothelial cells abrogated 

TGF-~/ALKr-induced responses, e.g. Smadr/s phosphorylation and stimulation of 

cell proliferation and migration. Thus, our results indicate a pivotal role for endoglin 

in TGF-~/ALKr signaling. The molecular mechanism by which this occurs is under 

investigation. 

Korchynskyi 0 , Dechering KJ, Sijbers A, 

Olijve W, Ten Dijke P. Gene array analysis 

ofbone morphogenetic protein type I 

receptor-induced osteoblast differentiation. 

J Bone Min Res 2003; 18:1177-1185. 

Roeien BAJ, Ten Dijke P. Controlting 

mesenchymal stem ceU differentiation by 

TG F-f3 family members. J Orthopeadic 

Science 2003; 874°-748. 

Shiraishi K, Kurisaki A, Valcourt U, 

Terentiev AA, Pardali K, Ten Dijke P, 

Heldin C-H, Ericsson J, Moustakis A. 

Nuclear factor YY1 inhibits transJorming 

growth factor f3 and bone morphogenetic 

protein-induced cell differentiation. Mol Celt 

Biol. 2003; 23:4494-4510. 

BM P-induced osteoblast differentiation BMPs have an important role in 

controlling mesenchymal cell fate and mediate these effects by regulating gene 

expression. The critical target genes by which BMPs mediate changes in cell fate are 

poorly characterized. When C2CI2 myoblasts are stimulated with BMPs their 

myoblast differentiation is inhibited and differentiation in osteoblast-like cells is 

promoted. We performed transcriptional profiling of C2Cr2 cells up on ectopie 

expression of one of three distinct BMP type I receptors in a constitutively active 

mutant form. Expression analysis was performed using an array of approximately 

8700 unique sequences. Each of the three constitutively active BMP type I receptors 

stimulated equallevels of R-Smad phosphorylation and alkaline phosphatase, an 

early marker for osteoblast differentiation. Interestingly, all three type I receptors 

induced identical transcriptional profiles. Many extracellular matrix genes we re 

upregulated, muscle-related genes downregulated and transcription factors/signaling 

components modulated. These target genes, some of them unexpected, may offer 

new insights into how BMPs elicit biological effects. 

The expression of Runx2 and Idr were found to be potently induced upon BMP 

receptor activation in C2Cr2 cells. When both genes we re ectopically expressed in 

C2Cr2 cells a synergistic response was induced that mimicked BMP-induced 

osteoblast differentiation. Whether Runx2 and Idr also cooperate in in vivo osteogenic 

activity is currently being explored. 

TGFfl 

~ 

TflR-1i 

~ 

ALK5 

Smad2l3 

~ 

Inhibition of endothelial cell 

proliferation and migration 

ALK5 

TGFfl 

~ 

T,~R-II 

I \. 

-. ALK1 

~ ~ 

Smad2l3 I- Smad1/5 

~ 

Stimulation of endothelial cell 

proliferation and migration 

Figure IIU: Schematic model by which TGF-f3 switches endothelial celt behavior via two distinct TGF-f3 

type I receptor /Smad pathways. TG F-f3 first binds to Tf3R- IJ, which subsequently recruits ALK5. ALK5 is 

phosphorylated and activated by Tf3R-II kinase. ALK5 can recruit ALK1 into the complex, and for its 

activation both ALK5 and Tf3R-II are required. Activated ALK1 and activated ALK5 induce the 

phosphorylation of Smad1/5 and Smad2/3, respectively. ALK1 and ALK5 have opposite effects on 

EC migration and proliferation. In addition, ALK1 can indirectly inhibit ALK5/Smad2/3 signaling.

L1PID METABOLISM IN SIGNAL TRANSDUCTION 

We study the role of membrane lipids and their metabolic conversions in signaling 

proeesses related to eell growth, death and differentiation. Our eurrent research 

foeuses on sphingolipids as modulators of signal transduction, as key struetural 

molecules of membrane mierodomains (lipid rafts and eaveolae) and as modulators 

of raft function and membrane permeability for traditional antieaneer drugs, sueh as 

doxorubiein. Alternative antieaneer agents include some unnatural alkyllysophospholipids 

(ALPs), which readily ineorporate in eell membranes, thereby 

interfering with signal transduction and intracellular trafficking processes, which 

may lead to apoptosis in tumor eells. Finally, a traditionalline of our research 

involves diacylglyeerol kinases that attenuate diacylglyeerol seeond messenger 

function and produce the putative messenger phosphatidie acid. 

Alkyl-Iysophospholipid-induced apoptosis: Role of sphingolipids and lipid 

rafts Synthetic alkyl-lysophospholipids (ALPs) such as I-O-oetadecyl-2-0-methylglyeero-3-phosphoeholine 

(Edelfosine), hexadecylphosphoeholine (Miltefosine) and 

its piperidine analog D-21266 (Perifosine) induee apoptosis in many tumor eens and 

are used as anti-eaneer agents in the clinic. We reported that Edelfosine, the ALP 

prototype, ins erts into the plasma membrane and is intemalized by endocytosis to 

inhibit new phosphatidylcholine biosynthesis in the endoplasmie retieulum, which 

triggers apoptosis in S49lymphoma and Hela eells. The apoptotie signaling 

mechanism initiated by this trigger remains unknown, but may be related to 

inhibition of the MAPkinase/ERK and Akt/PKB pathways as wen as activation of 

JNK/stress-aetivated protein kinase pathway, as we previously reported. 

We found that ALP accumulates in sphingolipid- and cholesterol-enriehed 

membrane mierodomains (lipid rafts). Artificial disruption of these rafts prevents 

ALP intemalization and apoptosis. We made S49 eells resistant to ALP (S49 AR eens), 

and this resistanee was due to defeetive raft-mediated endocytosis, and was 

associated with a defect in sphingomyelin (SM) synthesis. A similar defect in SM 

synthesis, ALP uptake and apoptosis induction was elieited upon inhibition of 

intracellular vesieular transport of cholesterol, by using an inhibitor of the NPC 1 

(Niemann-Pick Cl) protein. This makes sense since SM and cholesterol are 

simultaneously required for new raft forrnation in the trans-Golgi network, a proeess 

apparently impaired in the ALP-resistant eens. 

The defect in lipid rafts in the S49 AR eells not only prevents intemalization of ALP 

but also the lateral redistribution at the een surf ace of raft constituents such as the 

glycosphingolipid GMI, indueed by its eross-linking ligand, cholera-toxin-B 

(Figure lII.3)· 

Most intriguingly, ALP-resistant eells are eross-resistant to other stress 

agents/treatments, sueh as ionizing radiation, H 2 

0 2 

, the DNA-damaging agent 

etoposide, and stimulation of the death-reeeptor Fas/CD95. The mechanism behind 

this cross-resistanee may relate to the described defect in rafts and is subject of our 

current research. (More aspects of anti-eaneer activities of ALP are reported by 

M Verheij, Division IX, Radiotherapy) 

Group leader Wim Van Blitterswijk 

Wim Van Blitterswijk PhD Group leader 

Marcel Verheij MD PhD Academic staff 

Arnold Van der luit PhD Postdoc 

Robert Jan Veld man PhD Postdoc 

Jurgen Van Baal PhD Postdoc 

Alrik los MSc Graduate student 

Albert Van Heli Undergraduate student 

Marianne Budde Technical staff 

John De Widt Technical staff 

Shuraila Zerp Technical staff 

Potentiation of cellular uptake and toxicity of doxorubicin by short-chain 

sphingolipids Cellular uptake of classical antieancer drugs such as the anthracyclins 

is believed to oecur through passive diffusion over the plasma membrane. We 

investigated whether pre-insertion of exogenous membrane-perrneable lipid 

analogues into the plasma membrane improve this drug influx. We identified eertain 

short-chain sphingolipids, such as N-hexanoyl-sphingomyelin (C6-SM), N-octanoylglucosylceramide 

(C8-GlcCer) and -lactosylceramide (C8-LacCer), as potent 

enhaneers of drug uptake by cultured endothelial cells and various tumor eenlines. 

Low micromolar amounts of these short-ehain sphingolipids, being not toxie itself, 

enhaneed the uptake of doxorubicin up to 3-fold and decreased their EC 50 toxicity 

values 7 to 14-fold. The underlying mechanism has not been disclosed, but we could 

exclude the involvement oflipid rafts, the endocytic machinery, membrane leakage, 

or ABC-transporter-mediated efflux. Furthermore, we found a correlation between 

the degree of drug lipophilicity, as defined by partitioning in a two-phase octanolwater 

system, and the susceptibility of the drug towards the uptake-enhancing effect 

Figure lIl.]: Cholera-toxin-B-induced lateral 

redistribution (capping) of the raft ganglioside 

GMl on the surface of549 ceUs (lift). ALPresistant 

549 AR ceUs (right) show no GMl 

capping.


Van Blitterswijk Wj, Van der Luit AH, 

Veldman RJ, Verheij M, Borst J. Ceramide: 

second messenger or modulator of 

membrane structure and dynamics? 

BiochemJ 2003; 369:199-211. 

Ruiter GA, Zerp SF, Bartelink H, 

Van Blitterswijk Wj, Verheij M. Anti-cancer 

alkyl-lysophospholipids inhibit the 

phosphatidylinositol3-kinase - AktjPKB 

survival pathway. Anticancer drugs 2003; 

14:167-73. 

Van der Luit AH, Budde M, Verheij M, 

Van Blitterswijk WJ . Different modes of 

intemalization of apoptotic alkyllysophospholipid 

and cell-rescuing 

lysophosphatidylcholine. BiochemJ 2003; 

374:747-53· 

Ruiter GA. Effects of anticancer alkyllysophospholipids 

on cell death and survival. 

Thesis 20°3, University of Amsterdam. 

Los AP, Van Baal j, De Widt j, Divecha N, 

Van Blitterswijk WJ . Structure-activity 

relationship of diacylglycerol kinase O. 

Biochim Biophys Acta 2003 (in press). 

Veldman Rj, Zerp S, Van Blitterswijk Wj, 

Verheij M. N-hexanoyl-sphingomyelin 

potentiates in vitro doxorubicin cytotoxicity 

by enhancing its cellular influx. Brit] 

Cancer 2003 (in press). 

Van der Luit AH, Verheij M, 

Van Blitterswijk WJ. Raft lipid metabolism 

in relation to alkyl-lysophospholipid-induced 

apoptosis. In: Membrane Microdomain 

Signaling: Lipid Rafts in Biology and 

Medicine, Mattson MP (Editor), Humana 

Press Inc. , 2003 (in press). 

of the sphingolipid. A clear optimum was found for amphiphilic drugs such as 

doxorubicin, epirubicin and topotecan, indicating that the sphingolipids might act by 

modulating the average degree of plasma membrane lipophilicity, in turn facilitating 

transbilayer drug diffusion. 

We further employed this potentiating effect of short-chain sphingolipids in the 

context ofliposomal drug delivery. In this technology, doxorubicin is administered as 

a sterically stabilized and long-circulating liposomal formulation (Figure III.4). The 

liposomes are not taken up directly by tumor cells but release the doxorubicin 

gradually. Subsequent uptake by the tumor cells is a limiting step. To enhance this 

uptake, we supplied C8-GlcCer co-formulated within the liposomal complex. With 

these adapted liposomes, doxorubicin accumulation in A43! carcinoma ceUs and 

consequent cytotoxicity proved superior, as compared to controlliposomal 

doxorubicin. The same results were obtained when C8-GlcCer was post-inserted into 

Caelyx®, a commercialliposomal doxorubicin preparation. Advantages of co-delivery 

of doxorubicin and the lipid analogue (within the same liposomal complex) include 

avoidance oflipid-solubility related toxicities and of differences in biodistribution. 

Moreover, the doxorubicin-potentiating effect of C8-GlcCer-enriched liposomes 

proved relatively insensitive to high serum concentrations, suggesting that in vivo 

application is feasible and might thus improve established doxorubicin formulation. 

Oiacylglycerol kinases (coUaboration with N Divecha) Diacylglycerol kinase (DGK) 

phosphorylates the second messenger diacylglycerol (DAG) to yield phosphatidic acid 

(PA), a putative second messenger in its own right. Since DAG activates protein 

kinase C (PKC), DGK may regulate PKC activation by extemal stimuli. We currently 

investigate the physiological functions of the DG K8 and -~ isotypes. 

We found that DGK8 translocates to the plasma membrane in A43! cells upon TPA 

or extracellular ATP treatrnent in a PKC-dependent fashion. In order to determine 

the role ofthe conserved domains in DGK8, we made truncations in its primary 

structure. Unexpectedly, any truncation introduced at either the C- or N-terminus 

inhibited the TPA-induced translocation as weU as the activity of DG K8. 

Translocation was associated with an over-all dephosphorylation of the kinase. 

Mutation analyses revealed three phosphorylation sites in the PH domain that are 

necessary for translocation, and three phosphorylation sites in the catalytic domain 

that seem to regulate translocation negatively. We are currently performing pulldown 

assays in unstimulated and TPA-treated A43! cells to unveal proteins that could 

interact with DG K8 in a PKC-dependent manner. 

The DG K~ isotype is localized in the nucleus, where it specifically binds the 

retinoblastoma protein, pRb. This 'pocket protein' is a key regulator of the 

E2F-mediated transcription and cell division. The interaction between pRb and 

DG K~ enhances DG K activity and is negatively regulated by PKCa, which 

phosphorylates a distinct Ser residue in the MARCKS-phosphorylation site 

domain ofDGK~. The function ofDGK~ and other pRb-binding lipid (PIP) 

kinases in the nucleus remains unknown. We are currently testing the hypothesis 

that DG K~ plays a role in PKC-mediated cell cycle regulation. 

Figure III+ Short-chain glucosylceramide 

(C 8 -GlcCer) coformulated with liposomal 

doxorubicin (Caelyx®) results in co-delivery of 

lipid and doxorubicin to ceUs, resulting in 

enhanced cellular uptake of the drug. 

Phosphatidylethanolamine-attached 

polyethylene-glycol (PEG 2ooo ) provides sterical 

shielding ofthe liposome.

SIGNALING THROUGH INOSITOL PHOSPHOLIPIDS 

Eukaryotic cells are constantly under the threat of oncogenesis induced by both 

cellular and environmental factors that lead to changes in the genetic makeup of the 

cello For example DNA damage in response to cellular stress (UV, X-radiation, 

reactive oxygen species, etc) can lead to tumorigenesis. Normal cells, however, have 

mechanisms (checkpoints), which sense the consequences of these changes and 

activate signalling pathways in order to counteract them. We are interested in the role 

that phosphoinositides, a group oflipid second messengers (Figure HLS), play in 

cellular stress pathways. Our recent work has concentrated on investigating three key 

questions: 

1. Are levels of phosphoinositides and the enzymes that regulate them targets for 

cellular stress? 

2. What are the potential downstream targets for phosphoinositides and how are 

they linked to stress pathways? 

3. What are the relationships between phosphoinositides and stress signalling in 

the model organism C. elegans? 

Croup leader Nullin Divecha 

Nullin Divecha PhD Group leader 

David Jones PhD Post-doe 

David Weinkove PhD Post-doe 

Jonathan Halstead PhD Post doe 

Jurgen Van Baal PhD Post-doe 

Alrik Los MSc Graduate student 

Yvette Bultsma Technicial staff 

Mireille Snel Technicial staff 

Tamara Chessa Undergraduate student 

Oxidative stress ~ 

pathway Cl) 

~2) 

Q'Y' ." ~~ 

34 ~ ~7~ 

Growth factor ~ • ' • ~ '"" U- 

pathway '\--fb T,pel PIP ' ~K ,(1) (5)~ (6) 

T;':::P4-K 

OB 

p 

(4) ~pellPI 

Figure III.S: Type I PIPS·k regulates the levels 0fPtdIns(4,s)P 2 (1) and also generates PtdIns(3,4,S)P J 

(3) 

in vivo in response to oxidative stress. The phosphorylation of PtdIns(4,s)P 2 

to PtdIns(J,4,s)P J 

is carried 

out by PIJ-kinase. The C. elegans homologue is called ACE-1. PtdIns(4,s)P 2 

can also be synthesised by 

phosphorylation of PtdIns(s)P (4) on the 4 position by a Type II PIP4-k. PtdIns(4,s)P 2 is also the 

substrate for a receptor-activated phospholipase C which generates two new second messengers, 

diacylglycerol (6) and inositol(l,4-s)trisphosphate (S). 

' 

Ptdlns(4,S)P 2 

: a role in regulating stress-induced apoptosis We demonstrated 

that exposure to oxidative stress regulates Ptdlns(3A,S)P 3 synthesised by PIPS-k 

(Figure HLS). However, prolonged exposure to oxidative stress eventually leads to a 

decrease in the phosphoinositides Ptdlns(3A,s)P 3 

and Ptdlns(4,S)P 2 

, an attenuation 

of PKB signaling and the onset of apoptosis. The changes in phosphoinositides occur 

in part via the catalytic inactivation of PIPS-k and its delocalisation away from the 

plasma membrane where its substrate, Ptdlns(4)P resides. Overexpression of PIPS-k 

attenuates the oxidative decrease in Ptdlns(4,S)P 2 levels and, surprisingly, inhibits 

oxidative stress induced apoptosis. Together with K Jalink (Division I, Cell Biology) 

we are using in vivo imaging techniques to study how Ptdlns(4,S)P 2 levels are 

modulated in response to different cellular stresses (Figure 111.6). Interestingly, 

many malignant tissues show enhanced PIPkinase activity, which may be a 

mechanism for tumour cells to avoid decreases in phosphoinositides thus preventing 

the onset of apoptosis in response to cellular stress. 

Cell migration The ability to migrate away from their natural environment and to 

invade other tissues is a hallmark of metastatic cancer cells and of ten defines a poor 

prognosis for the patient. A key player in migration is the small molecular weight G 

protein Rac, which regulates cytoskeletal dynamics. In collaboration with P. Hordijk 

(Central Lab Blood Transfusion Services, Amsterdam), we have defined a role for the 

cont 

uv 

Ptdlns( 4,5)P2 

Figure III.6: UV light rapidly decreases 

GFP-Type I PIPkinase 

PtdIns(4,s)P 2 levels. Cells were transfected with 

either CFP-PH domain (to monitor PtdIns(4,s)P 2 

levels; top panel) or CFP-Type I PIPkinase 

(bottom panel). In the top panel, only the cells 

within the circle were irradiated with UV.


Los AP, Van Baal J, De Widt J, Divecha N, 

Van Blitterswijk WJ . 5tructure-activity 

relationship of diacylglycerol kinase theta. 

Biochim Biophys Acta 2003 (in press). 

Gozani 0, Karuman P, Jones OR, 

Ivanov 0 , Cha J, Lugovskoy AA, Baird CL, 

Zhu H , Field SJ, Lessnick SL, Villasenor J, 

Mehrotra B, Chen J, Rao VR, Brugge JS , 

Ferguson CG , Payrastre B, Myszka DG, 

Cantley LC, Wagner G, Divecha N, 

Prestwich GD, Yuan J. The PHD finger of 

the chromatin-associated protein ING2 

fimctions as a nuclear phosphoinositide 

receptor. Ce1l 2003;11+99-111. 

Van Hennik PB , Ten Klooster JP, 

Halstead JR, Voermans C, Anthony EC, 

Divecha N , Hordijk Plo The C-terminal 

domain of RaC1 contains two motifs that 

control targeting and signaling specificity. 

] Biol Chem. 2003;278J9166-75. 

Age-l (hx-546) 

PIPS-k in the regulation of chemokine-induced migration. By using protein 

transduction technology, we have generated peptides that are able to cross the celi 

membrane and interfere with Rac/PIPs-k interaction. Moreover, we have now 

established mutant Type I PIPkinase that no longer interact with Rac. Using these 

tools, we have begun to define the role that PIPkinases play in Rac mediated 

migration. Furthermore, by using the interfering peptides, we hope to establish a 

proof of principle that small molecule inhibitors of the interaction between Rac and 

PIPS-k may be useful targets for therapy. 

Phosphoinositides and nuclear function Using a unique panel of sensitive mass 

assays for phosphoinositides developed in our laboratory, we showed that induction 

of cellular stress in response to various treatments (UV irradiation, DNA damaging 

agents and oxidative stress) leads to changes in nuclear monophosphoinositides, in 

particular Ptdlns(s)P. Furthermore, the levels ofPtdlns(s)P are controlled in part by 

cellular stress-mediated regulation of the Type II~-PIP 4-k, an enzyme that 

phosphorylates Ptdlns(s)P to generate Ptdlns(4,S)P 2 (Figure IILS). Overexpression of 

Type II~ PIP 4-k increases the resistance of cells to UV irradiation, while knockdown 

of the protein (using RNAi) sensitises cells to apoptosis induced by UV irradiation. 

Furthermore, we have now defined a mechanism by which cellular stress impinges 

on Type II~ activity. Ptdlns(s)P likely regulates nuclear processes through its 

interaction with modular protein domains. We showed that, similar to a FYVE 

domain, the plant homology domains (PHD), which consist of a double zinc finger, 

are able to interact with phosphoinositides. PHD domains are largely present in 

proteins involved in nuclear functions, including DNA repair, transcription and 

stress responses. One such protein is ING2, which modulates PS3 function. We 

found that the intranuclear localisation ofING2 and its ability to modulate PS3 

function is in part dependent on the levels ofnuclear Ptdlns(s)P. To extend these 

findings we have cloned, expressed and tested over sixty PHD domains from 

different proteins for their ability to interact with phosphoinositides. Our data 

demonstrate that PHD domains interact mainly with monophosphoinositides and 

that the subfamily of PHD domains that can interact with phosphoinositides regulate 

a variety of nuclear functions. Future studies will define the relationship between 

cellular stress, nuclear phosphoinositides and the function of PHD containing 

proteins. 

FEDLI 

Starved Ll 

Day2 

Starved Ll 

Day 1 

Starved Ll 

Day2 

Figure III. 7: Eggs were isolatedfrom control 

worms or worms carrying the age-1 mutation 

(hx-546) and allowed to hatch in the presence 

([ed) or absence offood (starved day 1 or day 2). 

The worms express GFP-DAF-16, visualized by 

jluorescence microscopy. One day starvation leads 

to nuclear translocation of DAF-16, which is 

reversed after prolonged starvation (day 2). This 

occurs in a phosphoinositide-dependent manner as 

reversal does not occur in the age-1 mutant. 

Phosphoinositides and stress signalling in C. elegans In C. elegans, there is a 

clear connection between life span, stress sensitivity and phosphoinositide signalling. 

Up on starvation or overcrowding, C. elegans induce genetic programmes to arrest 

development and increase stress resistance until conditions become more favourable 

(diapause). We found that Lr larvae arrest their development in response to oxidative 

stress and that they become more resistant to this treatrnent up on starvation. 

Increased resistance to oxidative stress upon starvation is dependent on daf16, 

which encodes a forkhead transcription factor that regulates genes involved in DNA 

rep air, apoptosis, stress resistance, and cell cycle arrest. MechanisticalIy, we showed 

that starvation (one day) induces nuclear translocation ofDAF-r6 (Figure IIL7). 

Upon prolonged starvation (day 2), however, DAF-r6 exits the nucleus in an age-l 

(Ptdlns 3-kinase)-dependent manner suggesting a complex feedback mechanism 

controlling DAF-r610calisation though phosphoinositide synthesis. In order to study 

the role of other phosphoinositides in the regulation of stress responses, we have 

isolated a deletion mutant in ppk-2, the only Type 11 PIP 4-k homologue that exists in 

C. elegans. The ppk-2 mutant exhibits higher levels of Ptdlns(s)P, is sensitive to 

oxidative stress and appears to regulate daf16 function independently of the age-r 

pathway. ppk-2 mutant worms are also more sensitive to UV irradiation. However, 

unlike oxidative stress, resistance to UV irradiation appears to be independent of 

daf16 function. This result suggests that other targets of Type 11 PIP 4-k exist that 

regulate responses to stress. The ease of genetic analysis in C. elegans should enable 

the elucidation of novel relationships between phosphoinositides and cellular stress, 

which can be translated into mammalian systems.

LYSOPHOSPHOLIPID SICNALlNC, CYTOSKELETAL 

RECULATION AND CELL-CELL COMMUNICATION 

Lysophosphatidic acid (LP A) is bioactive lysophospholipid that evokes a great variety 

of cellular responses, particularly as an inducer of cell migration, proliferation and 

survival. LP A signaling has been implicated in wound healing, embryonic 

development and tumor progression. LP A is produced extracellularly by a secreted 

lysophospholipase D, named autotaxin (ATX), previously identified as an 'autocrine 

motility factor' produced by melanoma cells. LP A signals through at least three 

distinct G protein-coupled receptors (GPCRs) , whose individual properties remain to 

be characterized. Since LP A drives tumor cell migration, invasion and proliferation, 

while LPA levels are elevated in cancer patients and its receptors as well as ATX are 

found overexpressed in certain cancers, LPA receptors and ATX qualify as potential 

targets for therapy. Our studies explore the mode of action of ATX and how LP A 

receptors signal cytoskeletal remodeling, cell migration and celI-celI communication. 

These studies should lead to novel ways of interfering with LP A receptor signaling in 

cancer cells and with inappropriate LP A production in their microenvironment. 

The ins and outs of LPA receptor signaling One ofthe long-standing challenges 

has been to understand how bioactive LP A is produced and its level regulated in the 

cellular microenvironment. Recent advances have shown that LP A is generated 

extracellularly from lysophosphatidylcholine (LPC) by a previously enigmatic 

ecto-phosphodiesterase, termed 'autotaxin' (ATX). ATX is implicated in tumor 

progression, since ATX overexpression enhances the metastatic capacity of 

Ras-transformed 3T3 cells and promotes tumor angiogenesis in nude mice. We are 

currently examining the biosynthesis, subcellular localization and secretion of ATX. 

To elucidate the importance of ATX in embryonic development and tumor 

progression, we set out to generate conditional ATX knock-out mice and cross them 

with cancer-prone mouse models (collaboration J Jonkers and A Berns). ES cell 

targeting has already been done, and we expect the ATX KO mice to become available 

in the beginning of 2004- We will evaluate the effects of ATX inactivation on tumor 

initiation and progression in a set of defined mouse models of cancer. 

Reminiscent of mammalian ATX is a secreted PLD from C. pseudotuberculosis, which 

hydrolyzes LPC (in addition to sphingomyelin) but not Pc. Bacterial PLD mimics 

LPA in provoking rapid internalization of LP A receptors (Figure 111.8). We found that 

the apparent Km value for LPC matches the normal LPC levels in blood. Thus, by 

hydrolyzing circulating albumin-bound LPC to LPA, toxic PLDs may exploit LPA 

receptors to stimulate host cell proliferation and survival. 

We have analyzed the effects of LP A receptor signaling on ceU behavior by expres sion 

of LPA 1 

,2 in receptor-negative cells. LPA receptors mediate activation ofthe Rac 

GTPase in a Gi-mediated and PI3-kinase-dependent manner. Rac activation is 

accompanied by myosin heavy chain phosphorylation and leads to lamellipodia 

formation, cell spreading and migration. We have established that the invasioninducing 

Tiaml guanine nucleotide exchange factor mediates LPA-induced Rac 

activation, with concomitant suppression of RhoA activation. Thus, LP A acts as a 

motility factor and chemoattractant by activation of a Gi-PI3kinase-TiamI-Rac 

signaling pathway. LPA also promotes GSK-3 protein kinase activity leading tot 

hyperphosphorylation of the microtubule-associated protein Tau in neuronal cells. 

Group leader Wouter Mooienaar 

Wouter Mooienaar PhD Group leader 

Ben Giepmans PhD Post-doe 

Laura Sayas PhD Post-doe 

Catelijne Stortelers PhD Post-doe 

Jacqueline Mulder MSc Graduate student 

Francis Van Horck MSc Graduate student 

Laurens Van Meeteren MSc Graduate student 

Agnes Van Rossum MSc Graduate student 

Leonie Van Zeijl MSc Graduate student 

Floor Frederiks Undergraduate student 

Dick Van den Boomen Undergraduate student 

Aafke Ariaens Technical staff 

Trudi Hengeveld Technical staff 

Paula Ruurs Technical staff 

LPA 

(1 !J.M) 

PLO 

+ 

LPC 

o 10 min 30 min 

.. 

Figure III.8: Bacterial PLD mimics LPA in 

provoking internalization of LP A receptors. 

Detection of LP Al receptors (fused to green 

fluorescent protein, GFP) expressed in HEK293 

ceUs. GFP fluoresence is depicted in grey-black. 

Note complete LPAI receptor internalization 

induced by LPA after 10 min. Bacterial PLD 

induces receptor internalization only in the 

presence of album in-LPC, and at a slower rate.

N 


Van Leeuwen FN, Olivo C, Grivell 5, 

Giepmans B, Collard JG, Mooienaar WH. 

Rac activation by lysophosphatidic acid 

LPAl receptors through the guanine 

nucleotide exchange factor Tiaml. J Biol 

Chem 2003; 278:400-6. 

Koop E, Lopes 5, Feiken E, Bluyssen H, 

Van der Valk M , Voest E, Mummery C, 

Mooienaar WH, Gebbink M . Analysis of 

receptor protein tyrosine phosphatase-mu 

gene expression using LacZ knock-in mice. 

Int J Dev Bio12003; 4T 345-54· 

Mulder J, Poland M, Gebbink M, 

Mooienaar WH, Kranenburg O. p116Rip 

is a novel F-actin-binding protein. J Biol 

Chem 2003; 278:27216-23 ' 

Mills GB, Mooienaar WH . The emerging 

role oflysophosphatidic acid in cancer. 

Nature Rev Cancer 2003; ]:582-91. 

Mooienaar WH, Van Meeteren L, 

Giepmans B. The ins and outs of 

lysophosphatidic acid signaling. BioEssays; 

(in press). 

TRPM7 

Van Rossum A, De Graaf J, 

Schuuring-Scholtes E, Kluin P, Fan Y, 

Zhan X, Mooienaar W, Schuuring E. 

Alternative splicing of the actin binding 

domain of human cortactin affects cell 

migration. J Biol Chem 2003; 278:45672-9. 

Van Meeteren L, Frederiks F, 

Giepmans B, Pedrosa M, Billington 5, 

Jost H , Tambourgi D, Mooienaar WH. 

Toxic sphingomyelinases D target 

cellular LP A receptors by hydrolyzing 

lysophosphatidylcholine. J Bial Chem 

(in press). 

HUHHHHUH 

lHHHHHlHH 

CBD 

CBD 

Calmodulin-binding 

domains (CBD) 

channel domain 

outside 

HUHnnnnn 

p HHHHHHHH 

2c 

kinase domain 

Figure III.9: Schematic structure ofTRPM7. 

Cation channel domain, kinase domain and 

calmodulin-binding domains (CBD) are 

indicated. 

GSK-3-mediated microtubule disassembly is thought to be a prerequisite for LPA to 

remodel the actin cytoskeleton. 

Cytoskeletal regulation by p116Rip and cortactin Rho family GTPases act in 

concert with actin-binding proteins to remodel the actin cytoskeleton. We have 

isolated and characterized pn6Rip, a putative scaffold protein containing two PH 

domains and a coiled-coil region. pn6Rip localizes to the actin cytoskeleton, whilst 

its N-terminal region binds directly to f-actin and shows actin-bundling activity in 

vitro. Overexpression of the actin-binding domain disrupts the actin cytoskeleton, 

indicating that pn6Rip is essential for maintaining cytoskeletal integrity. Through 

yeast two-hybrid assays, we have identified the regulatory subunit of myosin light 

chain phosphatase as a binding partner of pn6Rip. Knockdown of pn6Rip using 

RNA interference blocks cytoskeletal relaxation, consistent with pn6Rip serving to 

recruit an essential component of the RhoA-regulated contractile machinery to the 

actin cytoskeleton. 

Another actin-binding protein, cortactin, is found overexpressed in mammary 

carcinoma cells and serves an established role in actin polymerization and (tumor) 

cell migration. However, many questions remain about the precise physiological 

roles of cortactin in cytoskeletal regulation. We have successfully knocked down 

cortactin mRNA in both normal and transformed cells and are currently analyzing 

the phenotypic changes in cortactin-deficient cells. 

Cytoskeletal regulation by TRPM7, a cation channel and M HC kinase Little is 

known about how LPA induces cytoskeletal relaxation, which is a prerequisite for cell 

spreading and migration. LPA and other receptor ligands provoke a Ca 2 +-dependent 

phosphorylation of the myosin II heavy chain (MHC), concomitant with Rac 

activation and disassembly of the cortical cytoskeleton. We have identified a 

candidate human MHC kinase that shows siginificant similarity to Dictyostelium 

heavy chain kinases (Figure III.9). Intriguingly, the kinase domain is part of a 

transmembrane protein that belongs to the family of 'Trp-related' cation channels. 

This bifunctional protein is now known as TRPM7, but its function is not well 

understood. We find that ectopie expres sion ofTRPM7 promotes cell spreading and 

cell-matrix adhesion and, furthermore, TRPM7 enhances Ca2+-influx in response to 

LPA (collaboration K Jalink, Division I). TRPM7 associates with actomyosin and can 

phosphorylate myosin II heavy chain, which correlates with an increase in basal Ca 2 + 

levels. These results identify TRPM7 as a potential MHC kinase. Kinase-dead 

mutants ofTRPM7 are currently being tested to substantiate the link between Ca 2 + 

influx, regulation of the cortical actomyosin network and cell spreading (collaboration 

F Van Leeuwen, Nijmegen University). 

Cell-cell communication: connexin-43 Gap junctions are specialized cell-cell 

junctions that mediate intercellular communication. They are composed of connexin 

proteins, which form transmembrane channels for small molecules. The C-terminal 

tail of connexin-43 (CX43) , the most widely expressed connexin member, has been 

implicated in the regulation of CX43 channel gating by LPA and other mitogens. The 

CX43 tail contains various protein interaction sites, but little is known about binding 

partners. We have found that the CX43 tail binds directly the ZO-r protein, a 'tight 

junction' protein and to tubulin. Immunofluorescence and electron microscopy 

studies reveal that microtubules extend to CX43-based gap junctions in contacted 

cells. Since microtubules are dispensabIe for the regulation of gap junctional 

communication, these findings suggest that, in addition to its well-established role as 

a channel-forming protein, CX43 can anchor microtubule distal ends to gap junctions 

and thereby might influence the properties of microtubules in contacted cells. 

To establish how CX43-based gap junctions are regulated by receptor stimulation, we 

have determined how expres sion of (mutant) components of the phospholipase C 

pathwayaffects gap junctional communication. According to our working model, 

PIP2 breakdown liberates the positively charged C-terminal tail of CX43 from the 

plasma membrane, which allows active c-Src to bind to and phosphorylate the CX43 

tail on residue TYY26S leading to channel closure. Experiments using RNAi technology 

reveal that loss of CX43 function in fibroblasts inhibits wound healing in vitro. Our 

future studies will address how CX43 affects the migration behavior of contacted cells.

IV DIVISION OF IMMUNOLOGY 

LYMPHOCYTE ACTIVATION AND SURVIVAL 

We are interested in the molecular basis oflife/death decisions taking place in 

activated lymphocytes. Our work is inspired by the desire to improve immunotherapy 

of cancer by rational interventions directed at sustaining survival of lymphocytes 

upon their activation by tumor antigens. This is expected to improve anti-tumor 

immunity by enlarging the tumor-specific lymphocyte pool and by enhancing longterm 

maintenance of tumor-specific lymphocytes (immunological memory). The 

second aim of our work is to contribute to the design of novel therapies aimed at 

killing (lymphoid) tumors by activating apoptotic pathways. 

TN F receptor family members and the control of effector and memory 

immune responses From our work, the TNF receptor family member CD27 and its 

ligand CD70 have emerged as interesting targets to improve anti-tumor immunity, 

since this costimulatory system promotes generation and maintenance of CD4 + and 

CD8+ effector cells that have the capacity to migrate to tissue sites, as well as T-cell 

memory. Using CD2il- mice, we have established that CD27 acts complementary to 

the well-known costimulatory receptor CD28. CD27 makes an equal contribution to 

generation of the effector T-cell pool at the site of priming and is more important 

than CD28 for the establishment of CD4 + and CD8+ effector T cells at tissue sites. 

This was revealed in a model of intranasal infection with influenza virus. CD27 

makes its contribution to the size of effector cell pools by supporting the survival of 

antigen-primed T cells and has no effect on cell division. 

We have examined the relative contributions of CD27 and its close relatives 4-IBB 

and 0)40 to the memory T-cell response, using rnice lacking CD27, 4-IBB ligand 

(L), or 0)40L. We find that CD27, 4-IBB and 0)40 each made equal and nonredundant 

contributions to generation of the memory CD8+ T-cell pool. Adoptive 

transfer experiments showed that 4-IBB or 0)40 stimulation is not required during 

secondary expansion. Instead, 4-IBB and 0)40 determine the magnitude of 

secondary T-cell expansion by imprinting this potential into T cells during the 

primary response. The same may hold for CD27, but CD70-deficient mice are 

required to establish this definitively. Effects of CD27 and its relatives on activated 

T-cell survival and memory formation can be explained by transcriptional 

upregulation of anti-apoptotic Bcl-2 family members, which is emerging as a 

hallmark of these TRAF-linked receptors. Completely novel is our observation that a 

'licence to survive' during secondary expansion is imprinted by these type of 

receptors during the primary response. It suggests an instruction for increased 

transcriptional accessibility of anti-apoptotic genes. 

While CD27 is already expressed on naive T cells, on B cells it is acquired af ter 

antigenic stimulation. Our findings indicate that CD27 contributes to B-cell 

responses at the centroblast stage, i.e. during clonal expansion of activated B cells. 

CD27 accelerates the germinal center reaction, but has no effect on somatic 

hypermutation of Ig genes, class switching or serum Ig production. Lack of a severe 

B-cell phenotype suggested a redundancy between CD27 and its close relatives OX40 

and/or 4-IBB. However, in mi ce lacking the function ofboth CD27 and 0)40 or 

CD27 and 4-IBB, Ig production in response to influenza virus was normal. Even 

though survival ofCD4+ effector T cells in the lung ofinfluenza virus-infected mice 

is strongly impaired in CD27-deficient mice, in the spleen effector T-cell numbers 

are virtually not affected. This may explain why helper T-cell responses required for 

B-cell expansion and differentiation are apparently normal in CD2il- mice. 

Pro-apoptotic signaling in Iymphocytes Pro-apoptotic TNF receptor family 

members (death receptors) can recruit FADD and other adaptors, which allow them 

to in duce apoptosis. In lymphocytes, death receptors are thought to play a role in 

downregulation of the immune response and selection of memory cells. In our work 

on pro-apoptotic signaling, we focus on induction of mitochondrial permeability, 

which is critical for the life death/decision in response to most stimuli. 

Division head, Group leader jan/tie Borst 

Jannie Borst PhD Group leader 

Annemieke De Melker PhD Post-doe 

Stephen Tait PhD post-doe 

Yanling Xiao PhD Post-doe 

Jenny Hendriks MSc Graduate student 

Anna Keiler MSc Graduate student 

Arlette Werner MSc Graduate student 

Gerda Van der Horst Technical staff 

Evert De Vries Technical staff 

Lucas Maillette De Buy Wenniger 

Undergraduate student 

CD27 I OX40 I 4-1 BB 

r'o1+I',hiii1ii11"ty 

J..., .... J ................... ,J..,..;J..rJ 

TRAF 

- Effector function 

T cell survival 

- Memory 

Fig. IV.l: Closely related TNF receptor family 

members CD27, 4-lBB and 0)40 are critica I 

regulators ofT-cell survival,


Tesselaar K~', Xiao Y ;~, Arens R, 

Van Schijndel G, Schuurhuis DH, 

Mebius RE, Borst J, Van Lier RAW. 

Expression ofthe murine CD27ligand 

CD70 in vitro and in vivo. i Immunol 

2002; 169:]3 -4°. ;~ equal first author. 

Tesselaa r K, ~' Are ns R ;~ , 

Van Schijndel GMW, Baars PA, 

Van der Valk M, Borst J, Van Oers MHJ, 

Van Lier RAW. Lethal T-ceU 

immunodeficiency induced by chronic 

costimulation via CD27-CD70 interactions. 

Nature Immunol 2003; 4:49-54, 2003· 

;', equal first author. 

Hendriks J, Xiao Y, Borst J. CD27 promotes 

survival of activated T cells and 

complements CD28 in generation and 

establishment of the effector T-cell pool. 

i Exp Med 2003; 198:1369-80. 

Tait S, Werner AB , De Vries E, Borst J. 

Mechanism of action of Drosophila Reaper 

in human ceUs: Reaper glabally inhibits 

protein synthesis and induces apoptosis 

independent of mitochondrial permeability. 

Cell Death and Dilf (in press). 

Previous work has indicated that in the T leukemic cens under study, both death 

receptors and DNA damaging anti-cancer regimens require a mitochondrial 

contribution to activate the effector caspase program. The death receptors CD9S and 

Trail receptor signal to the mitochondria via the BH3 domain-only Bcl-2 family 

member Bid, which is converted into an active component by Caspase-8 or 10 that 

cleave it at aspartate residues. We have found that DNA damaging stimuli also 

require cleavage of Bid at aspartate residues to convey the apoptotic signal to the 

mitochondria. This cleavage is accomplished independent ofknown caspases and 

granzyme B. A key role for cleaved Bid in the DNA damage pathway explains the 

previously observed cross-resistance to death receptor-, gamma radiation- and 

etoposide-induced apoptosis in Jurkat variant clones. In these resistant cens, a 

cytosolic component is expressed that prevents cleaved Bid from activating the 

mitochondria. 

We have studied the function of Drosophila Reaper in mammalian cens, since it was 

suggested that this molecule could activate the mitochondrial pathway. Nevertheless, 

we find that Reaper efficiently induced apoptosis in the absence of mitochondrial 

permeabilisation and cytochrome c release. Moreover, its established capacity to 

downregulate Inhibitor of Apoptosis (lAP) proteins was not required for Reaper's 

pro-apoptotic activity in mammalian cens. Independent of lAP binding, Reaper could 

globany suppress protein synthesis. Twenty amine acids in its carboxy-terminus were 

required for inhibition of protein synthesis and apoptosis-induction. Our findings 

indicate that the newly identified capacity of Reaper to suppress protein translation 

can operate in mammalian cens and may be key to its pro-apoptotic activity. 

Regulation of Iymphocyte activation The threshold for lymphocyte activation is 

set by antigen dose and the amount of antigen receptors present at the cen surface. 

The Cbl family of ubiquitin ligase and scaffold proteins negatively regulates 

membrane expres sion of antigen receptors and a great variety of other tyrosine 

kinase-coupled growth factor receptor systems. Using the EGF receptor as a 

paradigm, we investigate the role of ubiquitination in receptor trafficking. Our 

finding that receptor ubiquitination occurs at the cen surf ace prior to receptor 

internalization begged the question whether this modification might regulate the 

endocytic process. We have now revealed that c-Cbl promotes the internalization of 

the EGF receptor from the plasma membrane into clathrin coated pits and vesicles. 

We have also elucidated the mechanism by which this is accomplished. By virtue of 

its ligase activity, c-Cbl appends ubiquitin moieties to the EG F receptor and to other 

components within the activated receptor complex. This anows the receptor complex 

to recruit EpSIS via its ubiquitin interacting motif (UIM) . Since EpSIS can recruit 

clathrin and other structural and regulatory components involved in clathrin coated 

pit formation, we postulate that via this mechanism, c-Cbl is instrumental in de nova 

formation of clatrin coated pits around activated tyrosine kinase receptors. In the 

absence of ubiquitin-UIM interactions, the EG F receptor is still internalized, but via a 

pathway that does not involve EpSIS. Such a ubiquitin-UIM driven mechanism for 

receptor internalization has been demonstrated in yeast, but not previously in 

mammalian cens. 

EGF receptor 

... 

... 

o ubiquitin 

__ -++-_____ clathrin-coated pit 

Fig. IV.2: Unique signaling role far ubiquitin in 

recruitment of activated growth factor receptors 

into clathrin-coated pits. 

o ubiquitin 

1!" . Ubc 

:lac-Cbl 

n'.UbC 

c-Cbl

DISSECTING VIRUS AND TUMOR-SPECIFIC T-CELL 

IMMUNITY 

The aim of our research is straightforward; I) to design novel technologies that can 

be used to examine and modify protein interactions that control T-cell immunity; 

2) to use these tools to unravel and manipulate the molecular processes underlying 

immune recognition by T lymphocytes. 

Tumor-specific cytotoxic T-cell immunity Tumors that arise in peripheral tissues 

can induce cytotoxic T-cell responses in mice and men. There has been a longstanding 

debate on the mechanism through which the immune system is alerted to 

such solid tumors that -at least initiaUy- grow outside of the lymphoid organs. It has 

been postulated that induction of cytotoxic T cells against tumor antigens occurs 

through direct interaction of CD8+ T cells and tumor ceUs that have migrated to the 

draining lymph nodes. Alternatively, evidence has been obtained for crosspresentation 

of tumor antigens as the relevant mechanism, in which host antigen 

presenting cells take up antigens and present these antigens to tumor-specific T cells 

in the lymphoid organs. To address the relative merits of these two processes, we 

have previously set up a murine tumor model in which tumor-specific T-cell 

responses can be directly visualized by Major Histocompatibility Complex (MHC) 

tetramer technology. Using a series of constructs that contain two T-cell epitopes, 

one in the signal peptide and one in the mature protein, we have now provided 

evidence that not all antigens that are presented weIl through the endogenous 

pathway are also presented weU through the exogenous pathway. Specifically, T-cell 

epitopes contained with signal peptides fail to induce detectable immunity through 

cross-presentation. One model to explain these data is that up on cleavage by signal 

peptidase, the signal peptide is degraded rapidly. As a consequence, the pool of 

antigen precursor available for processing by antigen pres enting cells may be 

limited. To test whether signal peptide cleavage is indeed a crucial factor in the 

observed bias, we are currently producing model proteins in which signal peptide 

cleavage is disrupted. Defining the rules that determine the efficacy of antigen 

cross-presentation should be of value for the design of improved vaccination 

strategies that rely on this process. 

While the physiological relevance of cross-presentation in the induction of 

cytotoxic T-cell immunity has become readily apparent, the cell-biological basis for 

cross-presentation remains poorly understood. To provide a better insight into the 

processing of exogenous antigens by dendritic cells, we have developed an in vitro 

assay system for cross-presentation. This assay system makes use of a model antigen 

complexed to immunoglobulins that can be taken up and processed by dendritic cells 

(DC). Introduction of a protease cleavage site adjacent to a known T-cell epitope 

within this antigen allows the liberation and hence quantification of DC-associated 

antigen and its processing intermediates. U sing this technology, we have begun to 

analyze the efficiency with which DC extract T-cell epitopes from internalized 

antigens for presentation by MHC class I. Preliminary experiments suggest that only 

a tiny fraction of the epitopes present in internalized antigens do eventually associate 

with MHC class I. These data suggest that while cross-priming may be highly 

efficient, the process of cross-presentation is not. 

Induction and maintenance ofT-cell immunity The abilityto visualize antigenspecific 

T-cell immunity and to determine the differentiation pathways and 

functional capacities of different subsets ofT cells is essential for our understanding 

of pathogen- and vaccine-induced immunity. In prior projects we have generated 

MHC class 1- and MHC class 11 tetramers for mi ce and men and have used (and are 

still using) these tools to visualize tumor and virus-specific CD4 + and CD8+ T-cell 

immunity. MHC tetramer technology makes it possible to follow the development of 

immunity at the T-cell population level. However, it doesn't allow the analysis of cell 

fate and cellular differentiation pathways. 

In a more recent project we have therefore begun to develop an approach in which 

individu al T cells are tagged with a genetic barcode. For this purpose, we have created 

a lentivirallibrary containing several thousand different barcodes. Infection of ceU 

populations by this library oflentiviral vectors and subsequent micro-array analysis 

Group leader Ton Schumacher 

Ton Schumacher PhD Group leader 

Ramon Arens PhD Post-Doe 

Anna Calogero phD post-doe 

Helmut Kessels PhD Post-Doe 

Monika Wolkers PhD Post-Doe 

Arne Bakker Graduate student 

Miriam Coccoris Graduate student 

Moniek De Witte Graduate student 

Koen Schepers Graduate student 

Jeanine Joling Technical staff 

Sonja Noback Technical staff 

Erwin Swart Technical staff 

Mireille Toebes Technical staff 

Jos Urbanus Technical staff 

Marly Van den Boom Technical staff 

Immune 

Complex 

Peptide extraction 

Processed peptides 

Lysis 

GFP 

NP 

À epitope 

y + 

Trypsin 

site 

Trypsin treatment 

+ 

Peptide extraction 

t 

Processed and 

unprocessed peptides 

Fig. IV.]: Detection of peptides and processing 

intermediates in cross-presentation.


De Visser KE, Schumacher TNM, 

Kruisbeek AM. CD8+ T cell tolerance and 

cancer immunotherapy. ] Immunother 

2003; 26:1-11. 

johnson BJ, Costelloe EO, Fitzpatrick OR, 

Haanen jBAG, SchumacherTNM, 

Brown LE, Kelso A. Single-cell peiforin and 

granzyme expression reveals the anatomical 

localization of effector C D8+ T cells in 

influenza virus-infected mice. Prac Natl 

Acad Sci USA 2003; 100:2657-62. 

Kessels HW, Wolkers MC, 

Schumacher TN. Adoptive transfer of 

MHC-restricted receptors. Methods Mol 

Med (in press). 

Kessels HWHG, De Visser KE, 

Coccoris M, Tirion FH, Kruisbeek AM, 

Schumacher TNM. The impact ofseiftolerance 

on the polyclonal CD8+ T cell 

repertoire. ] Immunol (in press). 

Olbrich ARM, Schimmer S, Heeg K, 

Schepers K, Schumacher TNM, 

Dittmer U. Effective post-exposure 

treatment of retrovirus-induced disease with 

immunomodulatory DNA containing CpG 

motifs· ] Viro12002; 76:11397-4°4. 

Ressing M, Van Leeuwen 0, Verreck F, 

Gomez R, Heemskerk B, Toebes M, 

Mullen M, jardetzky T, Longnecker R, 

OttenhoffT, Schilham M, Neefjes J, 

Schumacher TNM, Hutt-Fletcher L, 

Wiertz E. Inteiference with TCR-HLA-DR 

interactions by Epstein Barr virus gp42 

results in reduced T-helper cell recognition. 

Proc Natl Acad Sci USA 2003; 

100:11583-88. 

Schepers K, Schumacher TN. Tumors 

hijack fetal enzyme, escape killer T cells. 

Nat Med 2003; 9:1253-4 (2003). 

60 

40 

zo 

o 

o 

80 

4 1Z 

days post transfer 

....... Sx10E7 (n=4) 

-- mock (n=4) 

16 

can then be used to trace the progeny of individual cells. We expect that the 

development of this type of technology win prove valuable in the analysis of 

fundamental aspects ofT memory ceU maintenance and cell fate analysis within the 

T-celIlineage (for instance, how memory T cells are recruited from primary antigenspecific 

T-cell populations; are T-cell populations with distinct functional properties 

derived from a single or from distinct T -cell effector pools etc.?). 

TeR gene therapy There is increasing data indicating that adoptive T-cell therapy 

can result in - sometimes spectacular - tumor regres sion for tumors such as renal 

cell carcinoma and melanoma. Unfortunately, infusion oflarge numbers of cells 

expanded ex vivo remains a daunting and costly task and this approach is therefore 

unlikely to become widely accepted. In the past years, my group has pioneered the 

retroviral introduction of antigen-specific T-cell receptors into peripheral T cens as a 

means to induce virus- and tumor-specific immunity in vivo. In this strategy, 

autologous or donor-derived T-cell populations are equipped with a TCR of defined 

reactivity in short-term ex vivo procedures, and re-infusion of the redirected cens is 

used to supply T-cell reactivity against defined antigens. These experiments reveal 

that T cells that are redirected by TCR gene transfer expand dramatically upon 

antigen encounter in vivo. However, two issues that are essential for the application 

of this strategy in the human setting remained unaddressed: 

I. In a preferred setting, a collection ofT-cell receptors would be established that 

could be used for the treatrnent of patients that share the MHC restriction 

element used by a tumor-specific T-cell receptor, but that are mismatched at 

other MHC loci with the original T-cell antigen receptor (TCR) donor. However, 

T-cell receptors are normally selected against reactivity with all available self 

MHC molecules during thymic development, and it is unclear whether reactivity 

of the introduced TCR with recipient MHC products that were not encountered 

during thymic development could lead to the development of autoimmunity. 

To address this issue we have determined the feasibility ofTCR gene transfer in 

a large number of settings where the 'TCR recipient' is matched with the 

'TCR donor' for the relevant MHC allele (the restriction element), but is 

mismatched for other MHC alleles. The results of these experiments indicate 

that TCRs can be identified that are both safe and effective in a large number of 

partially MHC-matched recipients. 

2. The value ofTCR gene transfer seems most significant in cases where the 

endogenous T -cell repertoire is small or lacking, as is the case for many self 

antigens. However, it is unclear whether redirected T cells would function in 

settings of self-tolerance, where anergizing signals or suppressor T cells could 

potentially thwart their function. To test the feasibility of targeting self-antigens 

through TCR transfer, we have introduced the ovalbumin-specific OT-I TCR in 

T cells of RIP-Ova mice that transgenically express ovalbumin in the beta cells of 

the pancreas. Introduction of these modified cells in RIP-Ova mice in 

conjunction with vaccination leads to a rapid and dramatic hyperglycemia, 

indicative of an auto-immune attack. These data demonstrate that TCR transfer 

allows the induction of tissue-specific immunity in cases where the endogenous 

repertoire is lacking. In related studies we now aim to test the value of this 

technology in a mouse model of spontaneous pro state cancer. For this purpose, 

we have isolated T-cell receptor genes targeting a model prostate antigen. In the 

coming year we will establish whether administration on T cells that have been 

modified to express this receptor suffices to limit or halt tumor growth in vivo. 

Fig. IV+ Induction of diabetes mellitus through TCR gene transjèr. T cell from mice expressing chick en 

ovalbumin in beta cells of the pancreas were transduced with a retroviral vector encoding a T-cell receptor 

specific for an ovalbumin-derived peptide complexed to MHC class I. Upon reintroduction ofT cells and 

subsequent vaccination, all mice that received the ovalbumin-specific TCR developed rapid-onset type 1 

diabetes mellitus.

IMMUNOTHERAPY I 

The primary aim of this group is to develop cytokine-based immunotherapies and 

advanced technologies for characterization of immune responses in peripheral blood 

and at the tumor site. 

Effect of peri-operative immunotherapy in peripheral blood and at the tumor 

site in renal cell cancer Renal cell carcinoma (RCC) is an immunogenic tumor. 

Current treatment for metastatic RCC (mRCC) is immunotherapy with the cytokines 

IL-2 or IFNa. The combination oflow dose IL-2, IFNa and GM-CSF to expand T cells 

and activate DC was tested in mRCC and around nephrectomy in a phase I study. 

In mRCC a correlation was found between activated monocytes (CDr4/DR), CD4/DR 

pretreatment levels and CD3+, CD4+ and CD8+ T-cell post-treatment levels, but not of 

NK or B celIs, with patient survival. Durable complete remissions were found. 

Peri-operative immunotherapy around nephrectomy with the same combination of 

cytokines showed an increase of activated T cells and DC in the tumor. Pre-operative 

immunotherapy (day - 9 till-2) with the MTD of the phase I study showed an even 

stronger infiltration of CD8+ T cells. DC accumulation in the tumor was less af ter 

8 days compared to 3 days immunotherapy with the cytokine cocktail, possibly 

because DC had moved from the tumor to the regionallymph nodes (under 

investigation). In a patient with a complete remis sion of mediastinallymph nodes 

following pre-operative immunotherapy and nephrectomy, studies are ongoing to 

isolate and investigate the intratumoral and peripheral T cells for cytotoxicity against 

the tumor. 

Group leader Bert De Gast 

Bert De Gast MD PhD Group leader 

Florry Vyth-Dreese PhD Academic staff 

Natascha Verra MSc Graduate student 

Yeung-Hyen Kim MSc Graduate student 

Trees Dellemijn Technical staff 

Johan Sein Technical staff 

Willeke Van de Kasteele Technical staff 

In situ detection of activated De and effector T cells in tumor tissues from 

renal cell carcinoma patients treated with cytokine immunotherapy In the 

group of RCC patients treated with peri-operative immunotherapy around 

nephrectomy, a detailed immunohistological analysis was performed of tumor 

tissues obtained after 3 days immunotherapy or control patients undergoing 

nephrectomy only. In situ examination using 3-color immunofluorescence staining 

and confocallaser scanning microscopy (CLSM) revealed major treatment effects, 

particularly with higher dose GM-CSF, on DC and T-eell infiltration, activation and 

maturation. This was reflected in preferential invasion of tumor tissue and not 

adjacent normal renal tissues by CD8+ T cells and DC-SIGN (CD209)+ DC 

expressing CD8o, CD83, IL-r2 and the DC specific chemokine, DC-CK!. Using a 

novel assay for subtyping naïve, memory and effector T cells in tumor tissues in situ, 

naïve CD8+ T cells we re observed in control and lower GM-CSF dose group patients. 

In the higher GM-CSF dose group, also effector CD8+ T cells and TNFa positive 

T cells were detected. These findings stress the potential of peri-operative 

immunotherapy to in duce infiltration of activated, mature DC and effector CD8+ 

T cells in RCC tumors, which may lead to enhanced anti-tumor reactivity. 

In situ analysis of antigen presenting cell subsets DC activation, maturation 

and cytokine profil es were studied in inflammatory bowel disease lesions. 

Inflammatory lesions of patients suffering from inflammatory bowel disease were 

studied for expres sion of antigen presenting cells (in coUaboration with the groups of 

Drs S Van Deventer and A Te Velde, Academic Medical Centre, University of 

Amsterdam, Amsterdam and Y Van Kooyk, Department of Molecular Cell Biology, 

Free University Medical Center, Amsterdam, The Netherlands). The CLSM 

technique proved to be very useful to discrlminate subsets of antigen pres enting eells 

(DC and macrophages) in colon lesions from patients with Crohn's Disease as weU as 

in non-affected colon tissues from healthy donors or unrelated patients. Using 

multicolor analysis, insight was gained into the activation/maturation state and 

cytokine potential of these cell types; strong expres sion ofIL-r2, IL-r8 and TNFa 

pointed to the T helper-r character of this disease. These studies may have direct 

implications for future immunotherapy approaches and form the basis for analysis of 

tumor lesions. Expression of ubiquitinated proteins in aggresome-like structures 

within DC are probably important for MHC class I presentation and may reflect 

active antigen presentation in De. We investigated ubiquitinated proteins in a DC 

line used to present GFP/antigen aggregates to cytotoxic T cells (see group


De Gast GC, Batchelor D, Kersten MJ, 

Vyth-Dreese FA, Sein J, 

Van de Kasteele WF, Nooijen Wj, 

Nieweg OE, De Waal MA, Boogerd W. 

Temozolomide followed by combined 

immunotherapy with CM-CSF, low-dose 

JU and JFN alpha in patients with 

metastatic melanoma. Er ] Cancer 2003; 

88:175-80. 

Te Velde AA, Van Kooyk Y, Braat H, 

Hommes DW, Dellemijn TAM, Slors jF, 

Van Deventer Sj, Vyth-Dreese FA. 

Jncreased expression of DC-SJCW IL-12+ IL- 

18+ and CD8/IL-12'IL-18' dendritic cell 

populations in the colon ic mucosa of 

patients with Crohn's disease. EurJ 

Jmmunol2003; 3]:143-51. 

Verra NCV, jansen R, Groenewegen G, 

Mallo H, Kersten Mj, Bex A, 

Vyth-Dreese FA, Sein J, 

Van de Kasteele W, Nooijen W, 

De Waal M, Horenbias S, De Gast Ge. 

J mmunotherapy with concurrent 

subcutaneous CM-CSF, low dose IL-2 and 

JFN-a in patients with progressive 

metastatic renal cell carcinoma. Br ] 

Cancer 2003; 88:1346-51. 

Overwijk WW, Theoret MR, 

Finkelstein SE, Surman DR, De jong LA, 

Vyth-Dreese FA, Dellemijn TAM, 

Antony PA, Spiess PJ, Palmer DC, 

Heimann DM, Klebanoff CA, Yu Z, 

Hwang LN, Feigenbaum L, Kruisbeek AM, 

Rosenberg SA, Restifo NP. Tumor 

Regression and Autoimmunity after 

Reversal of a Functionally Tolerant State of 

SelJ reactive CDs+ T Cells. ] Exp Med 2003; 

198:569-80. 

Schumacher, this section of the SAR). Using a specific antibody and 3-color CLSM 

analysis, ubiquitinated proteins were detectable in stimulated DC, simultaneously 

with HLA-DR and the GFP complex that had been taken up. Further studies are 

underway to determine the kinetics of ubiquitinated protein expres sion in relation to 

antigen presentation activity. 

Detection of antigen specific T Iymphocytes in experimental and human 

tissues Immunohistological techniques have been developed to visualize tetrameric 

peptidejMHC complexes binding tumor and virus specific T lymphocytes in situ 

(Haanen et al. Nature Medicine 2000: 6:1056-60). In situ analysis of (subsets of) 

specific immune cells and accessory cells will increase our understanding of effector 

mechanisms operational in different models. 

Tumor destruction by specific T cells was analyzed in a TCR transgenic mouse 

melanoma model (in collaboration with Dr W Overwijk; see group Haanen, 

Immunotherapy 1I, this section of the SAR). In situ localization of specific effector 

T cells related to vitiligo effects as well as specific tumor attack. In this model, 

conditions were defined that lead to tumor regres sion by T cells specific for an 

MHC dass I-restricted epitope of the selfjtumor antigen gpIOO, viz. adoptive 

transfer of tumor specific T cells, vaccination with an altered peptide ligand and 

co-administration of IL-z. Histological analysis of tumors after treatrnent with T cells 

plus vaccination showed specific T-cell infIltration but no marked tumor cell death or 

tissue destruction. By contrast, when this therapy was followed by administration of 

IL-z, we observed extensive tumor cell death and loss of tissue integrity. These data 

show that strong vaccination by itself can induce proliferation and tumor localization 

of antigen specific T cells, but that these T cells require exogenous IL-z to effectively 

mediate tumor destruction. 

The in situ tetramer technique has been succesfully applied to detect minor 

Histocompatibility antigen (mHag)-specific T cells in a human skin explant model of 

Graft versus Host Reactivity. In ongoing studies with the group of Dr E Goulmy 

(Leiden University Medical Centre, Leiden), this technique allowed detection of 

antigen specific T cells, both in fresh and cryopreserved tissues and visualization of 

the functional status of cells in situ. Cryopreserved skin tissues, pre-labeled with 

tetrameric mHagjMHC complexes, could be used for multi-color analysis of 

membrane, intracytoplasmic as well as nudear proteins. Granzyme B cytotoxicityand 

Ki67 proliferation-associated proteins were only found in skin tissues invaded 

by specific, mHag directed T-cell dones and not by control dones. This model is 

now used to dissect pathways leading to HY- or HA-! mHag mediated Graft versus 

Host Reactivity. This immunohistochernical approach will provide a tooI for further 

detailed in situ studies ofhuman T-celljtumor cell interactions, the unravelling of the 

Graft versus Host Reaction in human skin and the monitoring of immunotherapy 

trials. 

Fig. IV.5: Cranzyme B expression pattern ofT lymphocytes activated through antigen specific (left) or non 

specific (right) stimulation.

IMMUNOTHERAPY 11 

The main objective of this line of research is the development of novel T-cell 

immunity-based strategies that can be translated to clinical application. The focus is 

mainly on treatment of patients with solid tumors, especially melanoma and renal 

cell carcinoma. 

Spontaneous T-cell immunity in advanced-stage melanoma patients In a 

group of 62 well-documented HLA-A~(020I-positive stage IV melanoma patients, we 

studied the presence of spontaneous CD8+ melanoma-specific T-cell immunity. 

This work was do ne in close collaboration with the groups of Bert De Gast and 

Ton Schumacher. Peripheral blood samples were taken before any treatment for 

stage IV disease was initiated. Analysis was performed using soluble HLA-A2 

tetramers complexed with peptides derived from melanoma-associated antigens. 

These tetramers stain TCR specific for melanoma-associated antigens MART-I, 

tyrosinase or gpIOO. In 33 patients (53%) MART-I-specific CD8+ T cells we re 

detectable and 17 patients (27%) displayed expansions of tyrosinase-specific CD8+ 

T cells directly ex vivo. Eight of these latter patients also had MART-I-specific T cells. 

Only I patient tested positive for gpIoo-specific CD8+ T cells; this patient also 

had high numbers of circulating MART-I-specific T eells (18% of total CD8+ T-eell 

pool). 

Patients were treated with ehemotherapy-based regimens. We tried to correlate 

the presenee of melanoma-speeifie T-eell immunity in these patients with their 

survival. 

At a median follow-up of 46 months 55 patients had died and the median survival of 

all patients was 7.8 months (95% eonfidence limits 6.0 - 9.7 months). This is 

comparable to median survival time found in other ehemotherapy-based treatment 

regimens. Kaplan-Meier curves for patients with and without melanoma-specific 

T-eell immunity were not signifieantly different. If anything, patients with 

melanoma-specifie T-eell immunity performed worse eompared to patients without 

tumor-specifie T eells. Based on these data, it seems that spontaneously occurring 

melanoeyte lineage-specifie immunity is largely a reflection of increasing tumor 

mass and spreading, and stage IV melanoma patients are unlikely to benefit from 

these immune responses other than in exeeptional cases. Efforts to enhanee these 

responses, in particular through adoptive therapy, are therefore worth pursuing and 

are meeting inereasing sueeess. 

In vivo requirements for T-cell-mediated regression of established tumors 

Tumors are notoriously poor indueers ofT-cell immunity. Vaccination ean give rise 

to specific immune responses, yet only results in durable tumor regression in a 

small fraction of ca neer patients. We have developed vaccination- and 

immunological adjuvant strategies in mi ce against a non-mutated 'self antigen, 

gproo, expressed on melanoma and normal melanoeytes. Upon vaccination with 

human gproo peptide (as altered peptide) alone we sawa small, poorly therapeutic 

increase in gpIoo-specific T eells in blood and spleen. To improve these responses 

we tested the adjuvant activity of the T-eell growth factor, (IL-2, and the antigen 

pres enting eell- and T-eell growth and differentiation factor IL-23. When 

administered af ter vaccination, both IL-2 and IL-23 greatly enhaneed T-eelilevels 

and anti-tumor effect (Figure IV.6). Immunohistoehemieal analysis revealed greatly 

enhaneed T-eell accumulation and tumor destruction in tumors from miee reeeiving 

IL-2 and IL-23, whereas flow cytometry showed that infJ.ltrating T eells from animals 

treated with IL-2 or IL-23 exhibited spontaneous IFN-y production direetly ex vivo. 

Together, these results show that IL-2 and IL-23 adjuvants ean inerease the level and 

aetivity of vaecine-indueed, tumor-specifie TeelIs that mediate tumor destruction. 

DNA vaccination for the treatment of melanoma patients Naked plasrnid 

DNA when injected into the skin or muscle is taken up and translated into protein. 

Although DNA uptake does not seem to be an efficient proeess, immune responses 

against antigens eneoded in the naked DNA plasmids ean be found, mostly upon 

in vitro antigen-speeific restimulation. Sinee DNA vaccines are relatively easy to 

produce, eheap and apparently safe, this mode of vaccination is explored for both 

Group leader John Haanen 

John Haanen MD PhD Group Leader 

Willem Overwijk PhD Post-doe 

Adriaan Bins MD Graduate student 

Annelies Jorritsma MSc Graduate student 

Raquel Gomez Technical staff 

Esmeralda De Jong Technical staff 

Felicia Tirion Technical staff 

Mieke Smits Technical staff 

200 -o-..-I-loIFA 

180 .,.....I-loYFA>


Johnson BJ, Costelloe EO, Fitzpatrick DR, 

Haanen JB, Schumacher TN, Brown LE, 

Kelso A. Single-cell perforin and granzyme 

expression reveals the anatomical 

localization of effector CD8+ T cells in 

influenza virus-inftcted mice. Proc Natl 

Acad Sci U S A. 2003; 100:2657-62. 

protection against infectious diseases and treatment of high-risk cancer patients. 

We have developed a novel strategy for DNA vaccination that results in a very rapid 

and strong antigen-specific T-cell response. This DNA vaccination strategy can be 

employed to treat tumor-bearing mice and protects animals from tumor challenge 

indicating that functional T-cell memory is generated. Further studies are needed to 

optimize this strategy for use in humans. To all ow production of clinical grade DNA 

vaccines, a GMP DNA plasmid production unit is being built at the NKI pharmacy 

department. 

Overwijk WW, Theoret MR, 

Finkelstein SE, Surman DR, De Jong LA, 

Vyth-Dreese FA, Dellemijn TA, Antony PA, 

Spiess PJ, Palmer DC, Heimann DM, 

KlebanoffCA, Yu Z, Hwang LN, 

Feigenbaum L, Kruisbeek AM, 

Rosenberg SA, Restifo NP. Tumor 

regression and autoimmunity after reversal 

of a functionally tolerant state of seifreactive 

CD8+ T ceUs. ] Exp Med. 2003; 

198:569-80. 

Gene therapy with melanoma-specific human T-cell receptor genes 

In collaboration with the group ofT Schumacher and several foreign groups, we 

have collected a number ofTCRa and ~ genes co ding for melanoma-specific TCR, 

including the MART-I26_35' tyrosinase 3 68-374' gpIO0209.217' and MAGE-A3168'I76 

receptors. These genes have been cloned into the Moloney-based retroviral vector 

using an IRES sequence to separate the TCRa and TCR~ genes. These retroviral 

vector systems enabled us to trans duce human peripheral blood lymphocytes. 

Although expres sion levels are still suboptimal, expres sion of introduced TCRs 

can be visualized with the aid ofTCR-specific monoclonal antibodies and MHC 

tetramers (Figure IV.7). 

We have developed a mouse model to validate the functional properties of these TCR 

re-directed human T-lymphocytes. RAGI/common y chain-/- mice lacking B-, T- and 

NK-cells, are injected with a human melonome cellline expressing both a mini gene 

encoding epitopes for MART-I-, tyrosinase-, gpIoo-, MAGE-A3- and influenza A 

virus matrix (MI)-specific cytotoxic T cells, and the fire fly derived luciferase. After 

intravenous injection of the tumor cells, these mi ce develop pulmonary tumors that 

can be followed in time using a CCD camera. Upon conversion of injected luciferin 

by luciferase in the tumor cells, light will be emitted that can be detected by the 

camera. The amount of emitted light is a reflection of the tumor mass present in the 

lungs of these mice. When mi ce are injected with cloned influenza-specific T cells 3 

days after tumor cell administration, no tumors grow out, showing the efficacy of the 

specific T-cell population to reject tumor: cells. By comparing different melanomaspecific 

TCR-redirected T-cell population in this model we hope to be able to select 

TCR gene pairs for use in a human phase I study. 

TeR gene transfer to human PBL 

Tyro i oas 3~3n - P itic TCRaf3 Mek 

1,54 ro . 0,35% 

- \.~.' _ .• t' . 

~'t.~~~""' . ~. 

f:::' :~Çl/· .. I:'. ·; 

D8 

Fig. IV.7: TCRaf3 genes from an HLA-A2 restricted tyrosinase]69-J77-specijic human T-ceU clone were 

cloned into a Maloney-based retroviral vector. Human PBL were transduced with th is vector or a mock 

vector. Percentage transduced CD8+ T ceUs are depicted in right upper quadrant.

NORMAL AND MALIGNANT DEVELOPMENT OF 

LYM PHOCYTES 

The research of our group aims at the dissection of molecular pathways controlling 

normal and malignant development oflymphocytes. We focus on I) the mechanism 

underlying secondary immunoglobulin (Ig) gene diversification and its impact on 

genetic stability and; 2) the identification and functional characterization of genes 

differentially expressed during lymphocyte development and malignant 

transformation. Our approaches involve basic as weIl as advanced molecular genetics, 

biochemistry, immunology and recombinant mouse genetics. 

Linking secondary immunoglobulin diversification to genetic instability and 

Iymphomagenesis Secondary diversification ofIg genes is confined to activated 

B cells and comprises somatic hyperrnutation (SHM) and class switch recombination 

(CSR). SHM efficiently introduces point mutations into the Ig variabIe region genes 

and CSR mediates Ig isotype switching. Both SHM and CSR are transcriptioncontrolled, 

'region-specific' processes that require the catalytic activity of the 

Activation Induced Cytidine Deaminase (AID). AID deaminates cytidines within 

transcribed SHM- and CSR-regions. The resulting uracils are removed by a uracil 

glycosylase that generates abasic sites. Error-prone polymerases can resume DNA 

synthesis across these lesions (translesion synthesis, TLS) and establish somatic 

mutations. In fact, several TLS polymerases have recently been associated with SHM. 

At present, we are making use of novel recombinant mouse models to deterrnine the 

exact contribution of individual TLS polymerases and their functional domains to the 

mutation process. In addition, we are developing inducible mouse models to address 

the impact of AID and TLS polymerases on genome integrity and their potential role 

in the generation of follicular and post-follicular lymphomas. 

Group leader Heinz Jacobs 

Heinz Jacobs PhO Group leader 

Silke Schnell Graduate student 

Petra Langerak Graduate student 

Paul Van den Berk Technical staff 


Bross L, Wesoly J, Buerstedde J M, 

Kanaar R, Jacobs H. Somatic 

hypermutation does not require RadS4 

and RadS4B-mediated homologous 

recombination. Eur J Immunol2oo3; 

33J5 2 -7· 

Genes controlling Iymphocyte development and malignant transformation 

We are interested in the developmental programs controlling norrnal and aberrant 

development oflymphocytes. Precursor lymphocytes depend on the signaling of 

antigen receptor precursors, the pre-BCR or pre-TCR, which resembIe critical check 

points in early B- and T lymphocytes, respectively. By means of differential mRNA 

display from sorted thymocyte subsets we have identified two novel differentially 

expressed signaling molecules. At. present, we define their role in vitro as weIl as 

in vivo with re gard to lymphocyte activation, differentiation, selection, and survival. 

In addition, we use proviral tagging of Moloney Murine Leukemia Virus induced 

RAG-deficient lymphomas to identify 'proto-oncogenes' capable of controlling the 

differentiation and expansion oflymphocytes. Immuno-phenotyping and screening 

of the tumor panel for known common insertions indicated their absence in the 

most immature lymphomas, suggesting that other proto-oncogenes have been 

activated and need to be identified in this subset.

V DIVISION OF MOLECULAR 

BIOLOGY 

MOUSE MODELS FOR HEREDITARY CANCER 

Division head, Group leader Hein Te Riele 

Hein Te Riele PhD Group leader 

Jacob Hansen PhD Post-doe 

Marije Scholte PhD Post-doe 

Camiel Wielders PhD Post-doe 

Nanna Claij MSc Graduate student 

Floris Foijer MSc Graduate student 

Marjolein Sonneveld MSc Graduate student 

Tinke Vormer MSc Graduate student 

Kristel Kemper Undergraduate student 

Karishma Palande Undergraduate student 

Reina Paulis Undergraduate student 

Jaco Van der Torre Undergraduate student 

Conny Brouwers Technical staff 

Marleen Dekker Technical staff 

Elly Delzenne-Goette Technical staff 

Sandra De Vries MSc Technical staff 

Valerie Doodeman Technical staff 

Anja Van der Wal Technical staff 

Genetic instability and deregulated eell cyele control are hallmarks ofhuman cancer. 

Research in our group involves both aspects focusing on I) cancer predisposition by 

loss of DNA mismatch repair functions and 2) the role ofthe retinoblastoma gene 

family in cell cyele control and tumor suppression. The principle tools inelude gene 

inactivation in murine embryonic stem (ES) eells and analyses of the phenotypic 

consequences in homozygous, heterozygous and chimeric knockout mice and in cell 

lines derived thereof. 

DNA mismatch repair Genetic defects in DNA mis match rep air (MMR) underly 

the cancer predisposition syndrome hereditary non-polyposis eolorectal cancer 

(HNPCC). While the primary function MMR, the correction of DNA replication 

errors, is well-established, its role in two other mutation-avoidance mechanisms is 

less well-studied. These are suppression ofhomologous recombination between nonidentical 

DNA sequences and suppression of DNA damage induced mutagenesis. 

To study these MMR functions, we have generated ES celllines carrying disruptions 

in the MMR genes Msh2, Msh6 and Mshj. These genes encode two heterodimeric 

protein complexes MSHz/MSH6 and MSH2/MSH3, whieh have overlapping but 

also specific mismatch recognition capacities. As a readout for MMR-dependent antirecombination, 

we have introduced in wild-type and MMR-defective ES cells a 

recombination reporter consisting of a neo gene that was rende red inactive by either a 

two base-pair insertion in the open reading frame or a base-pair substitution in the 

start codon. We found that restoration of neo activity by deletion, insertion or 

substitution of one or a few base pairs can be achieved by introducing into cells 

single-stranded oligonueleotides consisting of zo-60 deoxyribonueleotides. The 

oligonueleotides are largely identical to the target sequences except for the one or few 

nueleotides to be modified. However, 'oligo targeting' was only effective in Msh2- 

deficient cells; in wild-type cells, the presence of MSH2 reduced the efficiency up to 

300 fold. The level of suppression depended on the type of alteration. Therefore, 

oligo targeting provides a unique readout for the specificity of mismatch recognition 

ofMSH2/6 and MSHz/3 in a living cello Furthermore, we showed that oligo 

targeting can be used to modify endogenous genes by using speciflc PCR protocols to 

identify modified eells. However, its general application may be hampered by the 

accumulation of inadvertent genetic alterations eaused by MSHz deficiency. 

Therefore, we are developing procedures to transiently inactivate MSH2, 

allowing oligo targeting to oceur effeetively while the accumulation of spontaneous 

mutations remains low. Alternatively, we are exploring the possibility that specific 

alterations escape detection by MMR and can be effectively introduced into wild-type 

cells. 

Figure V.I: Intestinal MSH2 expression in 

Msh2+/+, Msh2 Iow /+, Msh2 1ow /. and Msh2 I mice. 

In addition to editing DNA replication and recombination, MMR mediates the 

toxicity and suppresses the mutagenicity of methylating agents, an activity that has 

been attributed to MMR-dependent inhibition of replication of 0 6-methylguanine 

lesions in DNA. We have found that mismatch correction and DNA-damage 

response functions ofMMR can be uncoupled: a ten-fold reduced level ofMSH2 

protein still allowed effeetive correction of mismatches but failed to mediate the 

toxieity of alkylating agents and to suppress alkylation-damage-indueed mutagenesis. 

We have now generated miee expressing a low level ofMSHz. While lymphomas and 

intestinal tumors rapidly developed in Msh2 -/- mice, development of these tumors 

was fully suppressed in Msh2 low /- mice, even though MSHz expres sion was 

undeteetable in the thymus and the intestinal epithelium (Figure V.I). This result 

indicates that loss of post-replicative mis match correction is largely responsible for 

the strong eancer predisposition phenotype in MSH2-deficient mice. However, low 

MSHzlevel sensitized mice to tumor induction by the alkylating agent ENU. Thus, 

low MSH2 protein level causes a moderate cancer predisposition phenotype by 

potentiating the carcinogenicity of a DNA-damaging agent. A similar condition may

exist in humans but would not be readily detectable as it is not manifested by 

microsatellite instability_ 


Wheeler VC, Lebel LA, Vrbanac V, Teed A, 

Te Riele H, MacDonald ME. Mismatch 

The retinoblastoma gene family The retinoblastoma gene family consists ofthree 

repair gene Msh2 modifies the timing of 

genes Rb, P107 and PI30 encoding the so-called pocket proteins, which have partially 

early disease in Hdh(Q111) stnatum. Hum 

overlapping roles in regulation of the cell cycle. We have analyzed the response of 

Mol Genet 2003; 12:273-81. 

pocket-protein-deficient primary mouse embryonic fibroblasts (MEFs) to a number 

of growth-inhibiting conditions. Ablation of all three pocket proteins fullyalleviated 

Savouret C, Brisson E, Essers j, Kanaar R, 

the G 1 

cell cycle arrest imposed by serum withdrawal, cell-cell contact and DNAdamage. 

However, unrestrained proliferation of pocket protein deficient cells under 

Gourdon G. CTG repeat instability and 

Pastink A, Te Riele H, junien C, 

serum-deprived conditions was prevented by extensive apoptosis and a cell cycle 

size variation timing in DNA repairde.ficient 

mÎGe. EMBO J 2003; 22:2264-73. 

checkpoint operating in G 2 

• By studying MEFs expressing a single pocket protein, we 

found that each of them can mediate a GI arrest upon cell-cell contact. However, pRb 

is the critical mediator of a G 1 

arrest up on growth factor deprivation and irradiation. 

Dekker M, Brouwers C, Te Riele H. 

Pl07 can partially compensate for the absence of pRb under both conditions. PI30 

Targeted gene modijication in mismatchrepair-de.ficient 

embryonic stem eeUs by 

can mediate a modest response upon serum withdrawal which is additive to that of 

Pl07, but does not play a role in the response of cells to ionizing radiation. 

single-stranded DNA oliogonucleotides. 

The (partially) compensating role of Pl07 plus PI30 for the absence of pRB observed 

Nucleic Acids Res 2003; 31: e27. 

in vitro, mayalso suppress tumorigenesis as a consequence of Rb loss in vivo. To 

address this possibility, we have studied cancer predisposition caused by combined 

Claij N, Van der Wal A, Dekker M, 

ablation of pocket proteins in rnice. However, combinations ofloss of function alleles 

jansen L, Te Riele H. DNA mis match 

of the Rb gene family often leads to embryonic lethality or reduced viability. We 

repair de.ficiency stimulates N-ethyl-Nnitrosourea-induced 

mutagenesis and 

could circumvent this problem by generating cohorts of chimeric mice from ES cells 

carrying single and compound loss-of-function mutations in the Rb gene farnily. We 

lymphomagenesis. Cancer Res 2003; 

indeed found that the mild cancer predisposition phenotype associated with 

63:2062-66. 

hemizygosity for Rb was stron 1 aggravated b concomitant inactivation of PI07 

~~~~~~~~~~~----~--~~-=~~~~~~ 

(Figure V.2). Furthermore, we found that complete inactivation of Rb and PIo7 or Rb 

Claij N, Te Riele H. Msh2 deficiency does 

and P130 resulted in development of retinoblastoma. In addition, Rb-l-pI30-/- chimeras 

not contribute to cisplatin resistance in 

developed bilateral adrenal medullary tumors and small celllung hyperplasia. LacZ- 

mouse embryonic stem eeUs. Oncogene 

tagged ES cells are used to follow the fate of mutant cells and to identify the earliest 2003; 23:260-266. 

tumorigenic lesions. Our results demonstrate that in the absence of pRB, its 

homologs PI07 and PI30, either alone or together, act as potent suppressors of 

Claij N. MSH2, more than mismatch 

tumorigenesis in a variety of tissues. Our current work is focusing on the 

repair? {dissertation] University Utrecht 

identification of additional genetic events that collaborate with loss of the Rb gene 

2003. 

farnily in oncogenic transformation in vitro and in vivo. 

Tumor 

pituitary gland tumor 

adenocarcinoma coecum 

osteosarcoma 

lymphosarcoma 

leiomyosarcoma 

thymoma 

ovarytumor 

thyroid tumor 

adrenal gland tumor 

intestinal tumor 

lungtumor 

testis tumor 

total 

number 

22 

8 

8 

8 

4 

3 

2 

2 

2 

2 

loss of Rb+ allele# 

20/20 

4/6 

3/5 

4/5 

I/I 

0/3 

2/2 

I/I 

I/I 

I/I 

I/I 

I/I 

20/28 

Figure V.2: Tumor incidenee in 53 Rb+/-p1oi/-chimeric miee and frequency of Rb LOH. 

# Number of tumors showing /,oss ofthe Rb wild-type aUelejnumber of tumors tested.

ANTIGEN IC VARIATION IN TRYPANOSOMES 

Group leader Piet Borst 

Piet Borst MD PhD Group leader 

Henri Van Luenen PhD Academic staff 

Jan Wijnholds PhD Academic staff (honorary) 

Henk De Vries PhD Post-doc 

Nobuhito Ono MD PhD Post-doc 

Glen Reid PhD Post-doc 

Koen Van de Wetering PhD Post-doc 

Peter Wielinga PhD Post-doc 

Paul-André Genest MSc Graduate student 

Cristiane Bentin Toaldo MSc Graduate student 

Sebastian Ulbert MSc Graduate student 

Zhong Vu MSc Graduate student 

Noam Zelcer MSc Graduate student 

Sietske Bakker Undergraduate student 

Stefanie Hundscheid Undergraduate student 

Chris Streuper Undergraduate student 

Daniël Warmerdam Undergraduate student 

Marcel De Haas Technical staff 

Rudo Kieft Technical staff 

Annemieke Kuil Technical staff 

Liesbeth Van Deemter Technical staff 

Ingrid Van der Heijden Technical staff 

The African trypanosome Trypanosoma brueei can freely multiply in the bloodstream 

of its mammalian host. It escapes complete destruction by the host immune system 

by antigenic variation of its surface coat. We are studying the molecular mechanisms 

of antigenic variation in strains of T. brueei that are not infectious in humans and 

that can readily be grown in mice and in culture. Our recent work has focussed on 

two issues: the biosynthesis and function of the unusual DNA base J and antigenic 

variation of the transferrin receptor of T. brueei. 

Base J ~-glucosyl-hydroxymethyluracil (base J) , which we discovered in 

trypanosomes in 1993 (Gommers-Ampt et al., Cell 1993; 75:II29-II36), is a base 

present in all kinetoplastid flagellates and in Euglena. It replaces 1% of thymine in 

DNA and is predominantly located in repetitive sequences, such as telomeric repeats. 

Base J is synthesized by modification ofthymine in DNA. We have not yet found the 

enzymes involved, but are looking at candidate genes by siRNA knock-down, which 

is efficient in trypanosomes. We have characterized a J-binding protein (JBP1) that 

binds with high specificity to J-containing duplex DNA (Cross et al. EMBO J 1999; 

18:6573-6581). A JBPl gene KO in T. brueei has the remarkable effect of reducing the J 

content of trypanosome DNA to 5% of wild type, without any detectable physiological 

consequences. In the related kinetoplastid Leishmania, a JBP1 KO is lethal and we are 

studying why. 

The biosynthesis of J involves hydroxymethyluracil (HMU) as an interrnediate. This 

is also a product of oxidative DNA damage and many organisms contain one or more 

DNA glycosylases that take out HMU. Such enzymes are lacking in trypanosome 

extracts, and introduction of an inducible gene for an HMU glycosylase (SMUG1) 

kills the trypanosome. We have been unable to show, however, that the absence of 

this class of DNA repair enzymes makes trypanosomes hypersensitive to some DNA 

damaging agents. In a wide survey of glycosylases from pro- and eukaryotes, we have 

not found a single enzyme able to excise J from DNA. Probably, the bulky glucose 

group prevents access to the HMU. 

Transferrin receptors To cover its need for iron, the trypanosome takes up 

transferrin (Tf) from its mammalian host by means of a heterodimeric transferrin 

receptor (Tf-R). We have shown that T. brueei can make about 20 different Tf-Rs 

differing slightly in amine acid sequence. This diversity in Tf-Rs allows the 

trypanosome to cope with the diversity in Tfs in the large range of mammals that it 

can colonize (Bitter et al., Nature 1998; 391: 499-502). When trypanosomes are 

starved for Tf, they increase the level ofTf-R. The increase starts within hours, long 

before cellular iron stores are depleted. We have shown that saturation of the Tf-R 

with apo-Tf (no Fe) does not prevent upregulation and we infer that the signal for 

upregulation is cytostolic iron. Tf-R levels appear to be regulated by changes in RNA 

processing. We are investigating how. 

MULTIDRUG RESISTANCE OF CANCER CELLS 

We are interested in mechanisms of drug resistance in cancer cells and focus on 

resistance caused by increased ATP-dependent transport of drug out of the celI. We 

have isolated genes for these transporters and are characterizing their substrate 

specificity and sensitivity to inhibitors in transfected cells. We make use of the 

baculovirus system to produce insect cell membrane vesicles containing high 

amounts of transporter protein and suitable for vesicular transport studies. We also 

use transfected polarized kidney cell monolayers in which the transporters either 

route to the apical or the basolateral membrane, allowing the study of vectorial 

transport through the celllayer. Drug transporters causing chemotherapy resistance 

in tumors are present in (some) normal tissues, so we are also interested in their 

physiological function in metabolism and defence of the body against drugs and 

xenotoxins. We have therefore inactivated several genes for drug transporters by 

targeted gene disruption in mice. Initially we looked at P-glycoproteins; most recently

we have studied the Multidrug Resistance Protein family members MRP3, 4 and 5, 

and have begun to study MRP8 (collaboration with Ira Pastan, Bethesda, USA) and 

MRP9 (collaboration with Toshi Ishikawa, Tokyo, Japan). 

M RP3 We have shown that MRP3 is an organic anion transporter, like other MRPs, 

but only marginally associated with drug resistance. Some of the major bile salts are 

low-affinity substrates for MRP3 (collaboration with R. Oude Elferink, AMC, 

Amsterdam), but glucuronidated bile salts, steroids and drugs appear to be the 

preferred substrates. The Mrp3 KO mouse, generated by our group, has no obvious 

alterations in bile salt metabolism, but is affected in the handling of morphineglucuronides 

(collaboration with J. Beijnen, NKI/AvL) and acetaminophenglucuronide 

(collaboration with R. Oude Elferink). We are setting up transfected cell 

systems, containing MRP3 and glucuronosyl transferases to study the cellular export 

of glucuronides by MRP3 in detail. 

M RP4 and M RPS We have shown that MRPS is an organic anion transporter with 

the unusual ability to transport nucleotide analogs. Similar results were obtained 

with MRP 4, a close relative of MRPS (collaboration with J. Schuetz, Memphis). Other 

groups have found that MRP4 and MRPS can transport the cyclic nucleotides cGMP 

and cAMP. U sing intact transfected cells and vesicular transport assays, we have 

found that the affinity ofMRP4 and MRPS for nucleotide analogs and cyclic 

nucleotides is low, making a major role in drug resistance or cyclic nucleotide 

disposition doubtful. In search for other functions, we found that MRP 4 is able to 

transport some conjugated bile acids and steroids with high affinity. Interestingly, we 

found that MRP 4 (but not MRPI, 2, 3) can transport prostaglandins EI and E2, and 

that transport is inhibited by some non-steroidal anti-inflammatory drugs, such as 

indomethacin, which are also known to block synthesis of these prostaglandins. We 

are studying whether MRP 4 is a limiting factor in the cellular excretion of 

endogenously made prostaglandin (Figure V.3). Still we are searching for a highaffinity 

substrate for MRPS. Our Mrps KO mouse has no phenotype thus faro In view 

ofthe overlapping substrate specificities ofMRP4 and MRPS, we have generated a 

Mrp4/S double KO (collaboration with J. Schuetz, St. Jude Children's Research 

Hospita!, Memphis) and are studying the disposition of MRP 4/5 substrates in these 

mice. 


Martin W, Borst P. Secondary loss of 

chloroplasts in trypanosomes. Proc Natl 

Acad Sci USA 2003; 100:765-7. 

Mussmann R, Janssen H, Calafat J, 

Engstier M, Ansorge I, Clayton C, Borst P. 

The expression level determines the surface 

distribution of the transferrin receptor in 

Trypanosoma brucei. Mol Microbiol2003; 

47:23-35. 

Reid G, Wielinga P, Zelcer N, De Haas M, 

Van Deemter L, Wijnholds J, Balzarini J, 

Borst P. Characterization ofthe transport 

of nucleoside analog drugs by the human 

mu!tidrug resistance proteins MRP4 and 

MRP5. Mol Pharmaco!2oo3a; 63:1°94-1°3. 

Reid G, Wielinga P, Zelcer N, 

Van der Heijden I, Kuil A, De Haas M, 

Wijnholds J, Borst P. The human 

mu!tidrug resistance protein MRP4 

functions as a prostag!andin elflux 

transporter and is inhibited by nonsteroida! 

antiinjlammatory drugs. Proc Natl Acad 

Sci USA 2oo3b; 100:9244-9. 

Wielinga PR, Van der Heijden I, Reid G, 

Beijnen JH, Wijnholds J, Borst P. 

Characterization ofthe MRP4- and MRP5- 

mediated transport of cyclic nucleotides 

from intact ceUs. ] Bio! Chem 20°3: 

278:17664-71. 

Zelcer N, Huisman MT, Reid G, 

Wielinga PR, Breedveld P, Kuil A, 

PGE . 

-.... , -- 

~ 

U 

I 

PGE 

PGE 

Knipscheer P, Schellens JH, Schinkel AH, 

Borst P. Evidence for two interacting !igand 

binding sites in human MRP2 (ATP 

binding cassette C2). ] Biol Chem 20°3: 

278:23538-44 . 

Zelcer N, Reid G, Wielinga P, Kuil A, 

Van der Heijden I, Schuetz JD, Borst P. 

Steroid and bile acid conjugates are 

substrates ofhuman mu!tidrug-resistance 

protein (MRP)4 (ATP-binding cassette 

C4). BiochemJ 20°3: 371:]61-7. 

Figure v.J: Schema tic representation ofprostaglandin E (PGE) transport processes in transfected ceUs. 

CeUs are transfected with DNA constructs encoding the prostaglandin transporter (PGT) , which mediates 

rapid uptake ofPGE, and MRP4, which mediates elflux. There is elflux in the absence ofMRP4 and it is 

not known wh ether this is due to dijfUsion or an unknown carrier (see Reid et al., 2003b). 

Zelcer N, Saeki T, Bot I, Kuil A, Borst P. 

Transport ofbile acids in multidrug 

resistance protein 3 overexpressing ceUs cotransfected 

with the ilea! sodium-dependent 

bile acid transporter. BiochemJ 20°3: 

369: 2 3-3°.

CEll CYClE CHECKPOINTS 

Group leader René Medema 

René Medema PhD Group leader 

Alexandra Bras Post-doe 

Jamila Laoukili Post-doe 

Susanne Lens Post-doe 

Marc Schmidt Post-doe 

Rob Wolthuis Post-doe 

Marie Stahl Graduate student 

Gerben Vader Graduate student 

Barbara Van de Weerdt Graduate student 

Renske Van Leuken Graduate student 

Marcel Van Vugt Graduate student 

Matthijs Kooistra Undergraduate student 

Yemima Budirahardja Undergraduate student 

Marvin Tannenbaum Undergraduate student 

Jos Kauw Technical staff 

Rob Klompmaker Technical staff 

Figure V+ FOXO-mediated protection from 

oxidative stress. In the presence of active PKB/Akt, 

ceUs are stimulated to proliferate, at least in part 

due to the stabilising effect of P KB / Akt on cyclin D 

protein levels, and the nuclear exclusion of p2t ip1 . 

PKB/Akt promotes the association ofhexokinase 

to the vo!tage-dependent anion channel (VDAC), 

in a glucose-dependent fashion . This is required to 

prevent mitochondrial hyperpolarization and 

cytochrome c release, and therefore promotes ceU 

survival under conditions ofhigh glucose turnover. 

On the contrary, activation of FOXO factors, 

when P KB / Akt is silenced, causes the 

transcriptional upregulation of p2tip1, repression 

of cyclin D and a consequent exit from the cel! 

cycle. In addition, the anti-oxidant enzymes 

catalase and MnSOD are induced. MnSOD 

functions to protect ceUs from undergoing apoptosis 

as the glucose-dependent maintenance of 

mitochondria! integrity ceases to function. 

Upregulation of catalase may accomodate a shift 

from a glucose-based to a fatty-acid based 

metabolism, and enhance protection against 

peroxisomal H 2 

0 2 

production due to betaoxidation. 

Moreover, expression ofGADD45 is 

stimu!ated by FOXO factors, possibly to function 

as a safeguard in the event that DNA damage may 

still occur. 

Cell division of eukaryotic cells is a highly regulated process. cens can respond to a 

variety of extracellular signaIs, which together dictate ceUular behaviour, including 

the decision to proliferate, differentiate, or undergo apoptosis. CeU proliferation is 

controUed by multiple growth-regulatory pathways that act together to ensure proper 

cen division. At the late Gr restriction point (R point) the cen weighs the activity of 

positive and negative regulatory signals. Part of our research is focused on the 

mechanisms by which growth factors , through their cognate receptors, modulate the 

activity of ceU cycle regulatory proteins that control passage through Gr- After passage 

through the R point, mitogenic growth factors are no longer required for cens to 

complete division and cens become refractory to growth inhibitory signaIs. Instead, 

ceUs come to rely upon the intrinsic regulators of the cell cyele machinery for orderly 

progression through the remainder of the ceU cyele. Several checkpoints have been 

defined that monitor progression through the various stages and can block the 

initiation of the next cen cycle phase if errors are detected. For example, DNA 

damage checkpoints arrest ceU cyele progression at different stages so that the 

damage can be repaired before celI division. In addition, the spindIe checkpoint 

prevents separation of sister chromatids before all chromosomes have attained 

bipolar attachment to the spindle. This checkpoint ensures accurate separation of the 

sister chromatids to the daughter cells. As such these checkpoints are essential for 

genomic stability. 

Forkhead transcription factors and cell cycle regulation We have studied the 

function of the FoxO and FoxM subfamilies of forkhead transcription factors in 

regulating eell cycle progression. We have shown that FoxO factors positively regulate 

transcription of the CDK-inhibitor P27kipr, but in addition cause the repression of 

cyelin Dr/D2 expres sion. As such, activation of FoxO factors is sufficient to force 

cells into a state of quiescence, as we have shown in a variety of cell types. We have 

been able to demonstrate that FoxO-induced quiescence is tightly coupled to an 

altered metabolic state of the quiescent cells. FoxO directly regulates the expres sion 

of the anti-oxidant enzyme MnSOD, and this induction is responsible for protection 

against oxidative stress under conditions oflow glucose availability. Finany, in sharp 

contrast to our observations in fibroblasts, we have shown that the activation of FoxO 

in lymphocytes results in programmed celI death and the activation of apoptosispromoting 

Bc12-related proteins. Future experiments win be directed at a better 

understanding how the same pathway can cause such very distinct cellular responses 

in different cell types. 

In parallel, we have shown that FoxMr plays an important role in the G 2 

transcriptional program. We have identified a collection of G 2 

-specific genes that are 

Mitochondria 

Quiescence 

Mitochondria 

Proliferation

specifically induced by FoxMI. In the absence of FoxMl, cells fail to align their 

chromosomes and of ten undergo mitotic failure. As a consequence, FoxMl-deficient 

celIs become polyploid at a high frequency and display a elear defect in chromosome 

stability. Thus, these results demonstrate that FoxMl plays a critical role in the 

expression of G 2 

-specific genes and is essential to maintain chromosome stability. 

We are currently investigating which FoxMl target genes are involved in the 

maintenance of chromosome stability. 

Cell cycle checkpoints in G 2 

and mitosis During the last years we have 

concentrated on checkpoint functions in G 2 

and mitosis. Firstly we have studied the 

role of Polo-like kinase-l (PIk!) during these phases of the celI cyele. Plk-l performs 

multiple essential functions during the celI cyele. By RNAi-mediated depletion of Plkl 

we could show that Plk-l deficient cells are unable to separate their centrosomes, 

fail to form a bipolar spindle and undergo a Mad2jBubRl-dependent prometaphase 

arrest. Interestingly, depletion of PIk-l does not interfere with mitotic exit in spindle 

checkpoint-deficient cells as determined by degradation of cyelin Band chromosome 

decondensation. AIso, eleavage furrow ingression and cytokinesis does take place, but 

nuelear division is highly asymmetrie. Similar division defects are observed in 

spindie checkpoint-deficient cells treated with monastrol, indicative that the aberrant 

cell division may be an indirect consequence of a failure to form a bipolar spindie. In 

addition, we have shown that PIkl is inhibited in an ATMjATR-dependent fashion by 

DNA damage in G 2 

and in mitosis, which constitutes a novel mechanism by which 

DNA damage can prevent activation of CdC25e. We are currently investigating the 

effect of Plk-l depletion in the response of cells to genotoxic stress. 

In addition, we have studied the role of Survivin, a protein highly expressed in many 

types of cancer, in cell cyele progression. We have shown that Survivin is essential for 

chromosome alignment and cytokinesis. Moreover, Survivin is required for spindie 

checkpoint function. Cells lacking Survivin are unable to align their chromosomes, 

fail to recruit the Aurora B kinase to kinetochores and become polyploid at a very 

high frequency. Survivin-depleted cells exit mitosis prior to completion of 

chromosome congression and without sister chromatid segregation, indicating that 

the spindie assembly checkpoint is affected. Indeed, we could show that spindle 

checkpoint proteins BubRl and Mad2 are prematurely displaced from kinetochores 

in the absence of Survivin. Furthermore, our data demonstrate that Survivin plays a 

crucial role in the branch of the checkpoint that monitors tension on the 

kinetochores, but is not essential for the branch that signals microtubule-attachment. 

These findings provide important new insights into the wiring of the spindie 

checkpoint. This insight can help us predict the response of a tumor cell to different 

spindie poisons such as paclitaxel or vinblastin, used in treatment of cancer. 


Lens SM, Medema RH. The 

survivinjAurora B complex: coordinating 

tension and attachment. Cell Cycle 2003; 

6:5°7-10. 

Lens SM, Wolthuis RM, Klompmaker R, 

Kauw J, Agami R, Brummelkamp T, 

Kops C, Medema RH. Survivin is required 

for a sustained spin die checkpoint arrest in 

response to lack oftension. EMBOJ 2003; 

22:2934-47· 

Sunters A, Fernandez De Mattos S, 

Stahl M, Brosens JJ, Zoumpoulidou C, 

Saunders CA, Coffer PJ, Medema RH , 

Coombes RC, Lam EW. Fox03a 

transcriptional regulation of Bim controls 

apoptosis in paclitaxel treated breast cancer 

celilines. ] Biol Chem 2003 (in press). 

Van Vugt MATM, Medema RH. Polo-like 

kinase-1: Activity measurements and RNAimediated 

knockdown. Methods in 

Molecular Biology 2003; In: Cell Cycle 

Protocols: Methods and Techniques, edited 

by Drs. Gavin Brooks and Timothy C. 

Humphrey. The Humana Press Inc, 

Totowa , USA (in press). 

Baas AF, Boudeau J, Sapkota CP, Smit L, 

Medema R, Morrice NA, Aless i OR, 

Clevers He. Activation ofthe tumour 

suppressor kinase LKB1 by the STE20-like 

pseudo kinase STRAD. EMBOJ 2003; 

22:3°62-72. 

Burgering BMT, Medema RH . Decisions 

on life and death: FOXO proteins are in 

control when PKB jAkt is oJf duty. J. Leuk. 

Biol. 2003; 73:689-7°1. 

Figure V.5: Model explaining the role ofthe survivinjAurora B complex in spindie 

checkpoint signaling. Survivin and Aurora Bare localised together with INCENP at 

the inner centromere (circle) which is surrounded by kinetochores (half moons). In an 

unattached situation the kinetochore is in close contact with the inner centromere and 

signal (most likely phosphorylation events) eminatingfrom the survivinjAuroro. B 

complex can be picked up by kinetochore associated proteins. Mad2 and BubR1 are 

localised at the unattached kinetochores and the spindie checkpoint is active. When 

microtubules attach in a synthelic or merotelic (not shown) fashion, Mad2 is displaced 

from the kinetochores. However, these type of attachments do not create tension and 

the centromere remains in close proximity to the kinetochores. SurvivinjAurora B 

dependent phosphorylations most likely destablilize these misattached microtubules 

and enable BubR1 to remain at the kinetochore. When microtubules attach in a 

bipolar fashion, Mad2 is displaced from the kinetochores. These types of attachments 

create tension between the sister-kinetochores and this wil! pull the kinetochores away 

from the survivinjAurora B complex. The absence of Aurora B mediated 

phosphorylation events wal result in stabilization of the microtubule-kinetochore 

attachments, displacement of BubR1 and silencing of the spindie checkpoint. 

normal cell 

no 

survivin!Aurora B 

complex 

No attachment 

No tension 

MaaZ high 

BubRl high 

Attachmeltt 

No tens/on 

MaaZ low 

BubRI high 

G

MOUSE MODELS OF BREAST CANCER 

Group leader Jos Jonkers 

Jos Jonkers PhD Group leader 

Patriek Derksen PhD Post-doe 

Xiaoling Liu PhD Post-doe 

Bastiaan Evers Msc Graduate student 

Henne Holstege Msc Graduate student 

Arne Waalkens Undergraduate student 

Charlotte Ijsveld Undergraduate student 

Daphne Van Leeuwen Undergraduate student 

Nurcan Yagci Undergraduate student 

Marcelle Treur-Mulder Technical staff 

Hanneke Van der Gulden Technical staff 

Matthew Smalley PhD Guest 

Breast carcinoma is the most common type of cancer affecting women in Western 

countries. However, the multiplicity of genetic lesions found in human breast 

tumors has complicated the identification of mutations that are responsible for 

tumor formation. The main goal of our laboratory is to establish such causal relations 

by using advanced mouse mammary tumor modeis, and to identify novel cancer 

genes that cooperate with the initiating mutations in mammary carcinogenesis. 

Conditional mouse models of BRCA1- and BRCA2-associated breast cancer 

To establish mouse models ofhereditary breast cancer, we have used the Crej LoxP 

system for conditional mutagenesis of the Brcal and Brca2 tumor suppressor genes. 

In addition to conditional Brcal and Brca2 mouse mutants, we have generated P53 

conditional knockouts to investigate possible collaboration between BRCA loss and 

PS3 inactivation in tumorigenesis. We have crossed mice with different combinations 

of these conditional alleles with KI4cre transgenic mice, which express the Cre 

recombinase in basal cells of stratified epithelia. U sing the various compound 

mutant strains, we were able to show that BRCAIj2 and PS3 loss-of-function 

effectively collaborate in skin- and mammary tumorigenesis, indicating a pivotal role 

of the PS3 pathway in BRCAI- or BRCA2-associated breast cancer. We have also 

shown that the presence of a single conditional Brcal or Brca2 allele has no effect on 

tumor latency in our conditional P53 mammary tumor model, indicating that BRCAl 

and BRCA2 are not haploinsufficient for suppression of mammary tumorigenesis. 

o 

~ 1 

Cl:: 

;:;;' 0 

~ 

....1 -1 

-2 

Genome-wide Profile 

5 6 l ,,, 11 12 13 '" IS 1617 1819 X 

I 

, 

500 1000 1500 2000 2500 

Chromosome 6 

-3 ~ ____ ~ ____ -T ____ ~~ __ ~ ____ ~~ 

2 • (l 

.,r- ..,.···.... r-:'''' ..-:.a,..·~... ~~ ....."..._.. ~--#-:,' ..,.~. 

Molecular profiling of BRCAl and BRCA2 tumors We have thus far isolated 

approximately ISO mammary carcinomas from the conditional Brcal;P53 and 

Brca2;P53 tumor modeis, as well as a series of control tumors from the conditional 

P 53 mammary tumor model. We have subsequently determined the gene expres sion 

profiles of these tumors using ISK mouse cDNA arrays produced by the NKI microarray 

facility. In addition, we have determined DNA copy number changes in the 

same set of tumors using array-based CGH analysis (Figure V.6). For this purpose 

we have developed a 3K mouse BAC array in collaboration with the laboratory of 

A Bradley (Sanger Institute, Hinxton, UK). To identify specific differences between 

the BRCAI and BRCA2 tumors, we have performed pair-wise comparisons of the 

molecular profiles from the BRCAI and BRCA2 groups. We have also compared the 

molecular profiles of the different BRCAI and BRCA2 tumors with the profiles of 

control tumors to identify alterations that are specific for BRCAI- or BRCA2- 

associated breast cancer. We are currently using these data sets to identify novel 

oncogenic mutations that cooperate with BRCAI- andjor BRCA2-loss in mammary 

tumorigenesis 

-2 

-3 

5Ó 1ÓO 150 

Figure V.6: Array-CGH profile ofa mammary 

tumor from a K14cre;Brca2LOx/Lox;pslox/LOX 

fomal e, showing KraS2 amplification and regional 

DNA copy number changes. Confidence levels were 

calculated according to the Rosetta Error Model. 

Grey dots indicate significant gain or loss in DNA 

copy number; black dots indicate no significant 

change. 

Establishment of mouse models for metastatic breast ca neer None of the 

mammary tumors from our conditional PS3 model show local invasion or metastatic 

capacity, and this model is therefore suitable for investigating phenotypic 

consequences of additional (conditional) mutations in genes involved in tumor 

progression and metastasis. A strong candidate is the cell adhesion molecule 

E-cadherin, of which decreased expres sion is associated with poor prognosis in breast 

cancer. We have introduced conditional E-cadherin alleles into our existing P53 

mammary tumor model via crossbreeding. Our data indicate an effective 

collaboration between E-cadherin loss and PS3 inactivation in skin and mammary 

tumorigenesis. Histopathologic analysis of the resulting tumors indicates that loss of 

E-cadherin results in highly metastatic malignancies, and in a switch from ductal to 

lobular mammary carcinomas, reminiscent of the human condition (Figure V.7). 

Conditional gene mutation in a mammary gland fat-pad transplantation 

system To measure both early and late consequences of tumor suppressor gene 

inactivation in mammary gland epithelium, we have applied CrejloxP-based 

conditional mutagenesis to a mammary gland tissue reconstitution system involving 

isolation, culture and transplantation of primary mouse mammary epithelial cells 

(MMECs). Prior to transplantation, short-term cultures of primary MMECs from 

conditional mutant mi ce are subjected to expression of a tamoxifen-inducible

Cre-recombinase to activate the conditional mutations. Using this system we were 

able to effectively recapitulate BRCA2-associated tumorigenesis in a relatively short 

time period of 10 weeks (Figure V.8). 

The MMEC culture and transplantation model also offers the opportunity to search 

for complementing mutations in BRCA-deficient MMECs by introducing mutations 

in candidate genes that are identified by the aforementioned comparative molecular 

profiling of BRCAI and BRCA2 tumors. Furthermore, the system may be used to 

assess the effects of (candidate) tumor progression genes on tumor development, 

local invasion and metastasis formation. Finally, the use of non-invasive in vivo 

imaging technology may permit us to quickly assess whether these additional 

mutations will provide selective advantages to the cells and confer increased survival, 

proliferation and/or metastatic capacity. 

~ 

Isolate / _ 4-HT .. 


Chung Yj. Jonkers j. Kitson H, Fiegler H, 

Humphray S, Scott C, Hunt S, Yu Y, 

Nishijima I, Velds A, Holstege H, 

Carter NP, Bradley A. A whole-genome 

mouse BAC microarray with 1 Mb 

resolution for analysis of D NA copy number 

changes by array CG H. Genome Research 

(in press). 

Jonkers J, Berns A. Animal models of 

cancer. In: Knowies M, Selby P (Editors). 

Introduction to the Cellular and Molecular 

Biology of Cancer 4th ed. Oxford University 

Press (in press). 

BrcaLox/LOX ; p53Lox/Lox 

R26R ; R26creERT 

MMECS ' 

ti 

+ 4-HT 

transplant into 

cleared fat-pad of 

immunodeficient females 

- 4-HT 

+ 4-HT 

Carmine staining 

LacZ staining 

Figure V.8: A mammary gland fat-pad tranplantation model for BRCA2-associated tumorigenesis. Shortterm 

cultures of primary mammary epithelial cells (M ECs) from conditional mutant jemales carrying a 

tamoxifen (4·HT) inducible cre allele (R26creER T ) are incubated with or without 4-HT prior to 

transplantation into fat pads that have been cleared from endogenous epithelium. The presence of the 

R26R reporter allele permits detection of Cre-mediated gene switching by LacZ staining. Whereas nontreated 

MECsfrom R26creERT;Brca2Lox/Lox;P5/ox/Lox;R26Rjemales produce normal ductal structures 

upon transplantation (top panels), tamoxiftn-treated MECs develop into hyperplasias and tumors due to 

Cre-mediated inactivation of Brca2 and P53 (bottom panels) . 

Figure V. T A conditional mouse model for human 

infiltrating lobular carcinoma (ILC). Top panel: 

Human ILC showing regular neoplastic noncohesive 

ceUs in an 'Indian file ' arrangement 

(arrow) . Bottom panel: mouse mammary tumor 

induced by tissue-specific inactivation of E 

cadherin and P53, resembling human ILC 

(Haematoxylin and eosin stain).

VI DIVISION OF TUMOR BIOLOGY 

ANTIG EN PRESENTATION SY M HC CLASS I AN 0 

MHC CLASS 11 MOLECULES 

Division head, Group leader Jacques NeeJjes 

Jacques Neefjes PhD Group leader 

Eric Reits PhD Post-doctoral fellow 

Carla Herberts PhD Post-doctoral fellow 

Alexander Criekspoor MSc Graduate student 

Ingrid Jordens MSc Graduate student 

Marije Marsman MSc Graduate student 

Coenraad Kuyl MSc Graduate student 

Joost Neijsen MSc Graduate student 

lennert Janssen BSc Technicial staff 

Nico Dantuma PhD Guest 

Wilbert Zwart Undergraduate student 

Martijn Van der Velde Undergraduate student 

Rachid EI Betar Undergraduate student 


Reits E, Griekspoor A, Neijssen J, Groothuis T, 

Jalink K, Van Veelen P, janssen H, Calafat J, 

Drijfhout jW, Neefjes J. Peptide diffusion, 

protection, and degradation in nuclear and 

cytoplasmic compartments before antigen 

presentation by MHC class J. Jmmunity 2003; 

18:97-108. 

Herberts C, Reits E, Neefjes J. Proteases, 

proteases and proteases for presentation. Nat. 

Jmmunol. 2003; 4:3 06-308. 

Yewdell jW, Reits E, Neefjes j. Making sense of 

mass destruction: quantitating the MHC class J 

pathway. Nat. Rev. Jmmunol. 2003, in press. 

MHC class land 11 molecules present degradation products of self and antigenic 

proteins to the immune system. The immune system can subsequently respond by 

killing such cells, which is one of the concepts in immunotherapy. The aim of our 

studies is to characterize the various steps in these processes and to develop 

techniques to visualize them in living cells. Thus, to define methods for improving 

the immune response to tumor cells. 

M HC class I molecules The vast majority of degradation fragments (in the form of 

peptides) is produced by a large protease called the proteasome. The proteasome is 

located in the cytoplasm and in the nucleus and recognizes substrates modified by 

so-called polyubiquitin trees. We have visualized the polyubiquitin trees in living cells 

and are currently studying their interaction with the proteasome. The consequence of 

proteasomal degradation is that proteins are degraded into fragments. These 

fragments are either trimmed to MHC class I binding peptides (of usually 9 amine 

acids) or are fully degraded. We have followed peptide degradation in living cells by 

internally quenched peptides. Peptides are degraded within 6 seconds by cytosolic 

peptidases. We show that the implication of this is that most peptides are degraded 

before even being considered by MHC class I molecules. Peptides that are protected 

against degradation should then have a better chance ofbeing presented by MHC 

class I molecules. However, peptides can be protected by interacting with chromatin, 

a situation that could be important with necrotic cells. In addition, we have varied the 

peptide sequence to scan for sequences affecting the peptide half-life. No such 

sequences were found with one exception. This is currently studied in more detail. 

Major effects were observed with elongated peptides. Using various combinations of 

drugs, RNAi and peptides, we show that the peptidase TPPI is exclusively involved in 

the degradation of peptides longer than IS amine acids. TPPII is a protease critical 

for trimming most proteasomal degradation products before MHC class I molecules 

can handle them. 

We have shown that peptides are exclusively degraded by intracellular 

aminopeptidases. Consequently, we can follow peptides in living cells when 

stabilized by N-terminal protection. Surprisingly, such peptides can move from 

one cell to the next through GAP junctions. This is onlya small portion of total 

peptides because the majority is degraded by peptidases. This peptidase activity 

prevents spreading of peptides through monolayers (important to have an 

immune response only to the cancer or infected cells). Still peptide transfer 

into professional antigen pres enting cells could be important for a process called 

cross-presentation. 

M HC class 11 molecules MHC class 11 molecules sample protein fragments in 

another compartment, the Mlle. MIIC is a late-endosomal structure containing 

MHC class 11 molecules in transit and HLA-DM. HLA-DM is a specialized 

chaperone required for porter MHC class 11 loading. We have reconstituted the 

MHC class 11 loading system in living cells after introduction of CFP-Iabelled 

HLA-DR3 (a particular MHC class 11 type) and YFP-Iabelled HLA-DM (and the 

invariant chain). We have subsequently shown that these modifications did not affect 

peptide loading of HLA-DR3 and that the various proteins were properly transported 

in the cells. FRET imaging showed that the two proteins interacted within Mlle. 

Further experiments showed that the interaction occurred on the intemal vesicles 

exclusively and not on the limiting membrane. The spatial separation ofMHC 

Figure VJ.1: Jdentification ofRabGAP7. RabGAP7 was downregulated by RNAi in GFP-Rab7 expressing 

MeljuSo ce lis. GFP-Rab7 containing late endosomes swell dramatically and GFP-Rab7 is maintained in 

the activated state on vesicular membrane.

elass 1I loading with peptide adds a novel aspect to the process of antigen 

presentation by MHC elass 1I molecules. 

MHC elass 1I molecules are transported for the MlIC to the plasma membrane. This 

transport is controlled by various factors ineluding ILIO. We have identified some of 

these factors. MlIC is transported along microtubules by the kinesin and dynein 

motor proteins. The small GTPase Rab7, through its effector RILP, controls the 

dynein motor. This also controls the replicative cyele of Salmonella. We have 

now identified proteins controlling the Rab7 cyele ineluding a GAP and a GEF 

(Figure Vl.r). How these proteins are subsequently controlled is currently being 

studied in further detail. 


Neefjes J, Dantuma N . Fluorescent probes 

for proteolysis: tools for drug discovery. Nat. 

Rev. Drug Disc., in press. 

Koppers-Lalic D, Rychlowski M , 

Van Leeuwen D, Rijsewijk FA, 

Ressing ME, Neefjes JJ , 

Bienkowska-5zewczyk K, Wiertz EJ. 

Bovine herpesvirus 1 interferes with 

T AP-dependent peptide transport and 

intracellular trafficking of MHC class I 

molecules in human cells. Arch Virol. 2003; 

148:2023-37. 

Ressing ME, Van Leeuwen D, Verreek FA, 

Gomez R, Heemskerk B, Toebes M , 

Mullen MM, Jardetzky T5, Longnecker R, 

5chilham MW, OttenhoffTH, Neefjes J, 

5chumacher TN, Hutt-Fletcher LM, 

Wiertz EJ. Interference with T cell receptor 

HLA-DR interactions by Epstein-Barr virus 

gp42 results in reduced T helper cell 

recognition. Proc Natl Acad Sci U SA. 

2003; 100:11583-11588. 

Jordens I, Marsman M , Kuijl C, Janssen L, 

Neefjes J. The smal! GTPase Rab7 and its 

effector protein RILP regulate lysosomal 

transport. Pigment Cell Res. 2°°3;16:583. 

Reits E, Neefjes J. The immunological 

synapse: an exclusive zone for directed cell 

death after specific recognition. Ned 

Tijdschr Geneeskd. 2oo3;14T1102-6. 

Van Lith M , Van Ham M, Neefjes J. 

Stabie expression ofMHC class I heavy 

chainjHLA-DO complexes at the 

plasma membrane. EurJ Immunol. 2003; 

3Y1145-51. 

Marsman M, Jordens I, Kuijl C, Janssen L, 

Neefjes J. Dynein-mediated vesicle 

transport controls intracellular Salmonella 

replication. Mol. Biol. CeU, in press.

DNA DAMAGE CHECKPOINTS AND TUMORIGENESIS 

Group leader Reuven Agami 

Reuven Agami PhD Group leader 

Ingrid Kolfschoten PhD Post-doe 

Mathijs Voorhoeve PhD Post-doe 

Anja Duursma MSc Graduate student 

Martijn Kedde MSc Graduate student 

Carlos Le Sage MSc Graduate student 

Bart Van Leeuwen BSc Technical staff 

Tumor suppressors are genes found inactivated by mutations or deletions in a subset 

ofhuman cancers. However, each individual tumor contains many genetic 

alterations, complicating the study of the contribution of each variation to 

carcinogenesis. We have developed a vector-based system that mediates suppression 

of gene expression through RNA interference. With this system we demonstrate 

persistent inactivation of a dominant RAS oncogene that results in loss of 

tumorigenicity. We also studied the tumor suppressive functions of the human 

INI

cancers and also may possibly serve as genetic tools for cancer therapy. 

In another approach, we looked at the tumor suppressive functions of the human 

1N1

NUCLEOCYTOPLASMIC TRANSPORT 

In our research group we study macromolecular transport between the nucleus and 

the cytoplasm, and investigate how this fundamental process is used in the 

regulatory circuits of the ceU. 

Group leader Maarten Fornerod 

Maarten Fornerod PhD Grou p leader 

Helen Pickersgill PhD Post-docteral fellow 

Rafael Bernad-Fernandez MSc Graduate student 

Dieuwke Engelsma MSc Graduate student 

Jolita Hendriksen MSc Graduate student 

Heila Van der Velde BSc Technical staff 

Adriana Mejita Undergraduate student 

Figure VI+ Nuclear pore complexes in Xenopus 

laevis oocyte nuclear envelopes visualised by 

scanning electron microscopy from the cytoplasmic 

(upper) or nuclear (lower) side. Arrows point to 

cargoes caught in transit (Images courtesy of 

Terry Allen, Patterson Institute, Manchester, UK). 

Eukaryotes are characterised by a separation of their transcription and translation 

machineries in different subceUular compartrnents, the nucleus and the cytoplasm. 

This topology requires a large amount of traffic between the nucleus and the 

cytoplasm. Nucleocytoplasmic transport allows regulation of nuclear and cytoplasmic 

activities that is rapid and easily reversible. Indeed, it has become clear in the last 

years that many essential ceUular processes, such as signal transduction, ceU cycle 

progression and apoptosis are co-regulated on the level of nuclear transport. 

Nuclear transport The ceU has several mechanisms to maintain the nuclear and 

cytoplasmic identities in interphase. First, passage to or from the nucleus is 

selectively aUowed or blocked by nuclear pore complexes (NPCs, Figure VI.4), the 

channel forming units between the nucleus and the cytoplasm. These 125 MDa 

complexes restrict free diffusion of molecules or particles with a diameter larger than 

9 nm, corresponding to the size of a globular protein of 40-60 kD. However, 

complexes of up to 25 nm in diameter (e.g. ribosomal subunits) are efficiently 

transported, provided they carry a nuclear import or export signal. 

Nucleocytoplasmic transport is accomplished by distinct classes of soluble transport 

receptors that interact with transport signals and nuclear pore complex. One such 

class consists of importins and exportins, a superfamily of transport receptors that 

are responsible for the majority of nuclear transport pathways identified to date. 

Importin and exportin mediated transport is dependent on a signal within the 

transport substrate. Examples of transport signals are the basic nuclear localization 

signal (NLS) that is found in many nuclear proteins, and the nuclear export signal 

(NES) that mediates rapid nuclear export. Leucine-rich-type NESs have recently been 

identified in a diverse range of ceUular proteins that include eeU cycle regulators, 

stress response signal transducers and mediators of apoptosis. It consists of a short 

amine acid peptide with regularly spaced hydrophobic residues. The consensus NES 

sequence is highly degenerate. 

A critical mechanism to maintain nuclear and cytoplasrnic identities is the existence 

of the nuclear marker protein RanGTP. Nuclear RanGTP binds to and dissociates 

importin-cargo complexes and thereby provides directionality to nuclear import. The 

import substrate is released into the nucleoplasm, and the RanGTP-bound importin 

recycles back to the cytoplasm (Figure VI.5). RanGTP imposes directionality to 

nuclear export as weU, since RanGTP jexportinjcargo heterotrirneric complexes are 

several orders of magnitude more stable than exportinjcargo heterodimers. Af ter 

translocation through the nuclear pore complex, trimeric export complexes are 

Ron 

Q P 

Figure VI.S: Nuclear RanGTP regulates the direction ofimportin and exportin mediated 

nucleocytoplasmic transport (adapted from Fornerod et al., Cell90:10S1) .

destabilized by RanBPI or RanBPI-like domains in RanBP2/NuP3S8. The export 

reaction is made irreversible by RanGAPI-stimulated RanGTPase activity 

Synthetic nuclear export signals as tools to identify nuclear transport 

reaction intermediates in vivo The essential role of RanGTP for stabie NES/CRMI 

binding and the relative weakness of these interactions provoked us to look for 

artificial sequences that either bound to CRMI in a RanGTP independent way or 

bound very strongly. A phage library displaying Is-mer random peptides was used to 

select for signals binding to immobilised CRMr. In the presence of RanGTP, we 

strongly enriched a peptide consistent with the leucine rich NES consensus 

sequence. Surprisingly, also in the absence of RanGTP, a peptide was enriched that 

resembied a leucine-rich NES. 

To assess the export capacity in vivo of these synthetic NESs, we fused the selected 

peptide sequences, and several permutantions thereof to G FP and a nuclear 

localisation signal (NLS). Transfection of M CF7 cells using the majority of peptide 

sequences resulted in predominantly cytoplasmic signais, indicating that these 

sequences mediate efficient nuclear export in vivo. However, one of the permutations 

showed a very strong CRMI-dependent nuclear rim staining indicative oflocalisation 

at the nuclear envelope (Figure VI.6). Immunofluorescence and electron microscopy 

showed that this NES colocalised with CRMI at the cytoplasmic side of the NPC. A 

knockdown of the cytoplasmic filament protein NUP3S8 using RNAi resulted in a 

clear reduction of this NES-GFP-NLS at the nuclear rim. In vitro, binding of this NES 

was RanGTP independent. In vitro, it was the strongest NES currently known. 

These findings support the idea that NUP3S8 is a docking site for CRMI where it 

releases its cargo to the cytoplasm. Our findings also indicate that naturalleucinerich 

NESs are suboptimal in CRMI binding affinity to all ow efficient release from the 

nuclear pore complex, and, more in general, artificial transport signals can be used to 

reveal nuclear export reaction intermediates in vivo. 

The role of specific nucleoporins in nucleocytoplasmic communication With 

the advent of RNAi technology, it has become feasible to study the function of 

vertebrate NPC by in vivo depletion. U sing this approach we have this year targeted 

Nup3S8, NUp2I4 and Nup88, the three peripheral nucleoporin localised at the 

cytoplasmic face of the NPC. We have shown that NUP3S8 associates with the NPC 

via a novel interaction with Nup88 and NUp2I4. Knockdown of Nup88 or NUp2I4 

using RNAi in human cells caused a strong decrease of NUP3s8/RanBP2 at the NE, 

whereas total protein levels remained constant. Conversely, knockdown of 

NUP3s8/RanBP2 showed no reduction of Nup88 or NUp2I4/CAN at the NE. This 

implied a role for both Nup88 and NUp2I4/CAN in anchoring the cytoplasmic 

filaments to the NPC. Depletion of Nup88 caused a reduction of NUp2I4/CAN from 

the nuclear envelope, and depletion of NUp2I4/CAN a reduction ofNup88, 

suggesting an interdependence of these two nucleoporins at the NPC. Coir.:.hmnoprecipitation 

studies revealed an interaction between NUP3s8/RanBP2 and 

the export receptor CRMr. Knockdown of NUP3s8/RanBP2 caused an associated 

decrease in CRMI-dependent export of a GFP reporter construct and, consistently, 

CRMI was mislocalized from the nuclear envelope. These results suggest that the 

cytoplasmic filaments of the NPC play a supporting role in CRMI mediated nuclear 

protein export. 


Roth P, Xylourgidis N, Sabri N, Uv A, 

Fornerod M, Samakovlis C. The 

Drosophila nucleoporin DNup8810calizes 

DNup214 and CRM1 on the nuclear 

envelope and attenuates NES-mediated 

nuclear export. J Cel! Biol. 16]:701-6. 

Bernad R, Van der Velde H, Fornerod M, 

Pickersgill H. Nup358/RanBP2 attaches to 

the NPC via association with Nup88 and 

NUp214/CAN, and plays a supporting role 

in CRM1-mediated nuclear protein export. 

Mol. Cel! Biol., in press. 

Greber U, Fornerod M. Nuclear 

trafficking in viral infections. Curr. Top. 

Microbiol. Immunol. , in press. 

• • 

• 

Rev 

p 

G 

+ LMB 

Figure VI. 6: A synthetic nuclear export signal 

arrests at the nuclear periphery. GFP-NLS reporter 

proteins were Jused to a natural (Rev) or two 

synthetic NESs (P and G) and expressed in HeLa 

ceUs. The Rev and P NESs are exported and 

localise to the cytoplasm, whereas the G NES 

arrests the nuclear envelope. Leptomycin B (LM B ) 

blocks the export receptor CRM1, and all three 

proteins accumulate in the nucleus.

GENES INVOLVED IN BREAST CANCER PROGRESSION AND 

METASTASIS 

Group leader John Hilkens 

The main aim of our research is to identify novel genes involved in breast cancer 

progression. In particular, we intend to identify genes involved in breast cancer 

metastasis and unravel the coliaborating genetic pathways leading to breast cancer. In 

addition, we want to understand the biological function of celi surface associated mucins, 

notably episialin/MUC 1, which have been implicated in breast cancer progression. 

John Hilkens PhD Group leader 

Hubert De Leeuw phD Post-doe 

Melanie Kimm MSc Graduate student 

Vassiliki Theodorou MSc Graduate student 

Mandy Boer BSc Teehnieian 

Catheleyne Puts Undergraduate student 

Petra Niesten Undergraduate student 

Seleeted publications 

Thingstad T, Hilkens J. Tumor-Host 

Interactions Regulate Glucocorticoid 

Mediated Epiglycanin Expression in 

TA3Ha Murine Mammary Carcinoma 

Cells. Tumor Biol2oo3; 24: 116-129. 

Identification of novel genes involved in mammary tumor progression and 

metastasis by M MTV tagging Mouse mammary tumor virus (MMTV) proviral 

insertion in the genomie DNA of murine mammary epithelial cells can activate 

flanking proto-oncogenes leading to mammary tumor induction by a process called 

insertional mutagenesis. The availability of the almost complete mouse genome 

sequence in data bases and semi-high throughput PCR and sequencing teehnology 

has recently faeilitated the identification of retrovirally activated oneogenes. By using 

this new teehnology, we have searched for common MMTV integration sites (CIS) in 

primary tumors ofMMTV infected BALBjc mice. A CIS indicates the presence of 

nearby oneogenes. 

We have found several CIS, whieh may aetivate genes of the Wnt and Fgf families: 

Wntl, Wnt3, Wnt3a, Fg{], Fg{4, Fgf8 and Fgfio and two CIS which may aetivate yet 

unidentified genes. Fgf-l0 and Wnt3a have not previously been reported as MMTV 

targets. Further analysis of the Fgfio CIS revealed that MMTV had inserted in the 

Fgfio promoter region in the opposite transeriptional orientation, suggesting that the 

MMTV LTR enhancer activated the Fgfio gene in these tumors. Indeed, RT-PCR 

studies showed that Fgfio is expressed only in those tumors with a MMTV proviral 

insertion in the Fgfio locus. Subcutaneous injection into BALB/e rnice ofHCll 

mammary epithelial eelis retrovirally transdueed with Fgfio gave rise to highly 

vaseularized invasive subcutaneous tumors proving that Fgfio is an oncogene. A 

survey of randomly selected human primary breast tumors for Fgfio and Wnt3a 

expression revealed elevated transcription of Fgfio in 20% and Wnt3a in 15% of the 

specimens tested. We also found expression of both genes in human breast 

carcinoma eelilines, suggesting that Fgfio, normally only expressed in stromal eells, 

is ectopiealiy expressed in human breast carcinoma eelis. Sinee the receptor for 

FG FIO is present on breast epithelial celis, co-expres sion of Fgfio wiU trigger an 

autoerine loop, which may contribute to human and mouse mammary 

carcinogenesis. Our data show that Wnt3a and Fgfio ean induee murine mammary 

tumorigenesis and may play a role in a subset ofhuman breast earcinomas. 

Membrane-associated mucins in breast cancer Membrane associated mucins 

are frequently upregulated in various types of eaneers. Severallines of evidenee 

indicate that these mucins promote metastasis and are of prognostic significanee; 

however their exact biologieal function is unknown. Episialin (also designated EMA, 

CA 15-3 antigen, CD 227), encoded by the MUCI gene, is a paradigm for this class of 

mucins. We have shown that episialin reduces eeU-eeU and eell-matrix adhesion and 

promotes metastasis due to shielding of the adhesion reeeptors by its extremely 

elongated extra cellular domain. The function of episialin in normal eelis has not yet 

been established. We have found that episialin is localized in the trailing edge of 

migrating epithelial eelis and fibroblasts, of ten in uropod-like structures, whereas it 

is mainly eonfined to mierovilli in non-migrating eeUs. Moreover, episialin is present 

in cholesterol-sphingolipid rich lipid rafts. Our recent experiments show that the 

molecule is present in the same lipid rafts and colocalizes with adhesion molecules, 

like the u3 integrins, a proportion of ~1 integrins, and CD44- The accumulation of 

episialin/MUC 1 at the trailing edge wili increase the loeal density of the molecule to 

a level that will be sufficient to exert its anti-adhesive properties even in eells with low 

overall amounts of episialin/MUCl. An indication that episialin/MUCI might also 

be funetional in vivo during celi migration eomes from our observation that 

episialin/MUC 1 is upregulated in skin fibroblasts during wound healing and shows a 

polarized expres sion in these eelis. We propose that the anti-adhesion aetivity of 

MUC 1jepisialin at the trailing edge of a migrating eell may prevent inadvertent 

adhesion at the rear of the celI and thus facilitating eeli migration.

ESTROGEN RECEPTOR AND BREAST CANCER 

Aim of the project: elucidation of the mechanism of anti-estrogen resistance 

in breast cancer Estrogen receptor a_(ERa)-positive breast cancer patients are 

commonly treated with tamoxifen, a potent and widely used anti-estrogen_ Only half 

of the recurrences in ER+ breast tumors respond to tamoxifen while the other half is 

resistant The mechanism of tamoxifen-resistance has remained elusive thusfar. 

The conformation of a helix-l2 domain in the ligand binding domain of ER 

determines the ability to recruit and interact with the components of the 

transcriptional machinery and thus to elicit an estrogen-sensitive response by ER. 

We, therefore, developed a novel way of measuring conformational changes in the 

ER by fluorescence resonance energy transfer (FRET)_ Using this assay, we studied 

the efficacy of antiestrogens to inactivate ERa under different experimental 

conditions, and found that tamoxifen resistance is caused by phosphorylation of 

serine-30S in the hinge region of ERa by Protein Kinase A (PKA) _ Tamoxifen binds 

but then fails to induce the inactive conformation, invoking dependent 

transactivation instead_ PKA activity thus induces a switch from antagonistic to 

agonistic effects of tamoxifen on ERa. Microarray analysis ofbreast tumor samples 

(performed in collaboration with dr Laura van 't Veer and colleagues, dept of 

Experimental Pathology) reveals significant downregulation of the inhibitory subunit 

ofPKA, PKA-RIa, in tamoxifen-resistant tumors prior to treatrnent. In tissue culture, 

enforced downregulation ofPKA-RIa using RNAi keeps ERa in its active 

conformation and imp airs tamoxifen action, resulting in tamoxifen resistant 

proliferation (see Figure VL7). These cells are, however, still sensitive to ICI r82780. 

Activation of PKA (via downregulation of the inhibitory subunit PKA-RIa) prevents 

inactivation of ERa controlled gene transcription by tamoxifen and creates resistance 

to one of the most successful anti-cancer drugs, tamoxifen. 

This FRET approach is also used to characterize other anti-estrogens. Anti-estrogen 

ICI r82780 (Fulvestrant) induces a stringent FRET change that is overcome by the 

combined action of PKA activation and increased levels of cofactors cyclin Dr and 

SRC-r. This indicates that different mechanisms are responsible for resistance to 

anti-estrogens. Other anti-estrogens, including raloxifen, EM-S62, toremifen and 

chlomifen, are characterized in a similar way, yielding a biophysical taxonomy of 

anti-estrogens. The FRET change induced by Raloxifen, EM-S62, and Chlomifen is 

released by PKA, as is the case with Tamoxifen. These fmdings compare with 

ER transactivation studies using ERE-reporter assays. They show that 

activation/inactivation of the ER is directly to be measured in single cells and 

provides a direct way of evaluating SERMs (selective estrogen receptor modulators) 

and putative inhibitors of these. 

Group leader Rob Michalides 

Rob Michalides PhD Group leader 

Wilbert Zwart MSc Graduate student 

Astrid Balkenende BSc Technical staff 

Desiree Verwoerd BSc Technical staff 


Bindels E, Lallemand F, Balkenende A, 

Verwoerd D, Michalides R. 

Overexpression of GljS cyclins affects 

oestrogen independent growth by 

sequestration of inhibitors. Oncogene 21, 

8158-8165,2002. 

Van Diest P, De Jong J, Baak J, 

Michalides R, Van der Veld E. Prognostic 

value of proliferation and apoptosis in 

breast cancer. In: Prognosis and predictive 

factors in breast cancer. Ed Walker ed, 

PP57-8J,200J. 

Van Diest P, Michalides R. Cel! cycle 

regulators: interactions and their role in 

diagnosis, prognosis and treatment of 

cancer. In: Cel! cycle inhibitors in cancer 

therapy: Current strategies. Giordano A, 

Soprano KJ eds. Humana Press, Totowa, 

New Yersey USA, 207-252, 200J. 

pSuper 

pSuper 

PKARI

ULTRASTRUCTURAL STUDIES ON ENDOCYTOSIS AND 

SIG NALI NG 

Group leader Peter Peters 

Peter J Peters PhD Group leader 

Sue Godsave PhD Post-doe 

Bea Krenn PhD Post-doe 

Pekka Kujala PhD Post-doe 

Pieter Res PhD Post-doe 

Nicole Van der Wel PhD Post-doe 

Tom Groothuis MSc Graduate student 

Erik Bos MSc Technical staff 

Martijn Romeijn BSc Technical staff 

Donna Broekhuis Fluitsma BSc 

Technical staff (Free University) 

Figure vl.8: Lower leJt: Slice from an electron 

tomogram constructed using a series of tilted 

images from -7°. to 7°. at tilt intervals of Oj· 

with the boxed region (230 nm wide) 

corresponding to a slice in the region that has 

been segmented. 

Model: Representation of a small region from a 

cellular tomogram. Aside from the interaction 

between the cytoplasmic domains in apposing 

bilayers, a complementary interaction between the 

periplasmic domains also appears likely. 

The focus of our work is on understanding the machinery and organization of 

molecular sorting within the endomembrane system of a cello We concentrate our 

work on vesicular transport mechanisms within the endocytic pathway and the link 

to pathogenesis. Cancer cells depend on signaling via growth factors; its termination 

is determined primarily by endocytosis of growth factor receptor complexes. Cryo 

immunogold-electron microscopical methods and 30 cryo electron tomography are 

our main techniques to evaluate these membrane trafficking events. 

Three-dimensional imaging of large maeromoleeular protein assem blies in 

eells Technique development. Prokaryotic and eukaryotic cells are highly efficient 

'factories' that house a large number of miniaturized molecular machines. Our next 

great challenge will be to understand the three-dimensional architecture of the cell in 

terms of the spatial arrangement of organelle-bound protein complexes, and to 

describe quantitatively how this arrangement alters as the cells respond to stimuli. 

We are working on new technologies to provide 30 maps of cellular components at 

molecular resolution. 

Helium cooled-electron tomography of unfIxed, ultra-thin cryo-sections from high 

pressure frozen cells is the method of choice for determining the three-dimensional 

structures of large macromolecular assemblies (like growth factor receptor 

complexes) in cell organelles where crystallographic methods are generally not 

applicable. The essence of this approach is to obtain a series of projection images in 

an electron microscope by tilting the specimen relative to the electron beam and 

combining the images computationally to re construct the three-dimensional 

structure. We are currently determining the molecular architecture of receptor 

networks. Tomograms constructed from cryo-sectioned cells reveal remarkable 

membrane features at high resolution. X-ray crystallographic studies provide 

constraints on the possible modes of receptor packing in the membrane. By placing 

receptor molecules into the density map derived from tomography, we are beginning 

to construct a three-dimensional molecular model for the membrane protein 

network. There is currently great excitement in this fIeld since ongoing advances in 

cryo-sectioning of native material at ultra low temperature as weIl as cryo- EM 

instrumentation and computational methods may ultimately make it possible to 

obtain resolutions in the range of 20 A to 40 A, i.e. potentially high enough to locate 

individual proteins in a cello We will focus on receptor-mediated signaling and 

endocytosis; a process, when in a state ofkinetic imbalance may result in cancer. 

Collaborators: Sriram Subramaniam and Jacqueline Milne (National Cancer 

I nstitute , NIH, USA) 

Antigen-presentation CD1, cancer and anti myco-bacterial immunity. It has recently 

been shown in mice that COr-restricted T cells can potently promote tumor rejection. 

Evidence for the importance of COr in human tumor immunity comes from a recent 

analysis of the genes expressed by chronic lymphocytic leukemic cells. 

COr molecules can present (microbial) lipids to T cells and thereby induce the 

production of cytokines and perforin. In addition, COr-restricted T cells can kill 

immature dendritic cells that are infected with Mycobacteria. COr molecules are 

composed of transmembrane MHC class I-like heavy chains and a non-covalently 

associated beta 2 

-microglobulin. MHC molecules present peptide antigens whereas, 

the non-polymorphic CDr family present (glyco)lipid antigens. 

Besides differences in antigen pres enting capacity and T cell activation, the CDr 

isoforms follow discrete intracellular trafflcking routes that correlate with their ability 

to intersect with the lipid antigens that each presents. The CDrb, CDrc and CDrd 

isoforms contain a cytoplasmic tyrosine-based sorting motif, YXXZ (Y =tyrosine, 

X=any amino acid, Z=bulky hydrophobic amino acid) that recognize adaptor protein 

complexes (AP) to mediate trafflcking towards various intracellular compartrnents. 

Interaction with AP-2 is known to be important in their internalization from the 

plasma membrane. We and others showed that the tyrosine-based sorting motif of 

CDrb inter acts with AP-3, the adaptor protein complex is critical for the localization 

of CDrb to late endosomes and lysosomes. We also showed that AP-3 directly

interacts with the single murine CDrd isoform and controls its targeting to 

lysosomes. AP-3 deficiency was found to prevent CDrd localization to lysosomes and 

increase expres sion at the cell surface of dendritic cells. The altered trafficking of 

CDrd in AP-3 deficient mice results in a significant reduction ofNK T positive cells, 

consistent with a defect in CDrd self-antigen presentation and thyrnocyte positive 

selection. Lysosomal sampling appears to be of great importance in antigen 

presentation. 

Dendritic cells derived from monocytes are a good model system to study trafficking 

of antigen presenting molecules. We have shown that up on activation mature 

dendritic cens do not lose their ability to endocytose CDrb molecules via clathrin 

coated vesicles. Thus, the constitutive endocytosis of CDrb molecules and the 

differential sorting ofMHC class II from lysosomal MHC class II compartments 

(MIIC) separates peptide and lipid antigen presenting molecules during dendritic 

cell maturation. 

Collaborators: Michael Brenner, Harvard Medical School and Juan Bonifacino, 

Cell Biology and Metabolism Branch, NIH, USA. 

Neuro degenerative diseases - Immunotherapy cen biological events are thought 

to be at the heart of the alterations in kinetic imbalance of protein folding and 

degradation found in several neuro degenerative diseases such as Alzheimer, 

Huntington and prion diseases. Recently, several in vitro and in vivo studies have 

shown that antibodies that recognize an oligomeric state eommon to different 

amyloidogenic proteins can reduee the levels of the pathogenic, abnormally folded 

form of the protein in eells. In a EU consortium with the labs of Prusiner in San 

Francisco, Kristensson in Stockholm, Taraboulos in Jerusalem and Zurzolo in 

N apoli, we are perforrning experiments to assess the effects of passive immunization 

against the abnormally folded form of the prion protein (PrP Se ) in miee and to 

investigate how antibodies reduee the prion load. PrPSe distribution and 

ultrastructural changes are currently investigated in cultured eells and in mice 

treated with prornising combinations of anti-PrP antibodies and ehemo-therapeutic 

agents. 

- Prion in white blood eells and dendritie eells. Reeently it has been shown that prions 

can be transrnitted by blood transfusion from an infected individual in the pre clinical 

phase of the illness. This is a strong argument that white blood cells are most likely 

involved in the pathogenesis of infectious forms of prion disease, either solely as 

carriers of PrPSe or perhaps also as sites where conversion of the normal prion 

protein (Prp c ) to PrPSe takes plaee. To study the role of blood eells in the etiology of 

prion disease, we are first evaluating the normal expres sion ofprpc in blood cells of 

healthy individuals. Once it is known which normal blood cells express Prp c , we will 

try to unravel the preeise subcenular location of PrPSe in infected mutine white blood 

cells and to deterrnine where inside the cell the conversion of Prpc to PrPSe takes 

place. 

- Uptake of prions via the gastro-intestinal tract. It has become increasingly important 

to establish the subcellular trafficking routes of Prpc in peripheral tissues since our 

understanding of the pathway of prion transrnission from periphery to central 

nervous system is poor. Recent work suggests that migratory dendritic cens present 

in the gut epithelium have the potential to transport PrPSe. However, the 

contribution of these cells to prion disease is not known. We are currently in a EU 

consortium with MacPherson in Oxford, Mabbott in Edinburgh, Aucouturier in Paris 

and Rayrnond in Liege investigating the role of epithelial cells, M-cells, and dendritic 

cells in PrPSe invasion. We are determining the cellular and subcellular distribution 

of Prpc and PrPSe in different tissues, especially focusing on the digestive tract, 

principally by using the technique of cryo-immunogold electron microscopy. 


Mironov A Jr., Latawiec D, Wille H, 

Bouzamondo E, Legname C, 

Williamson RA, DeArmond Sj, 

Prusiner SB, Peters PJ. Cytosolic prion 

protein in neurons, journal ofNeuroscience 

(2003; 162:703-17). 

Van der Wel N, Sugita M, Fluitsma DM, 

Cao X, Schreibelt C, Brenner MB, 

Peters PJ. CD1 and MHC dass 1I 

molecules foUow a different course during 

dendritic ceU maturation. Mol Biol CeU 

(2003; 14=3378-88). 

Cao X, Sugita M, Van der Wel N, Lai j, 

Rogers RA, Peters PJ, Brenner MB. 

CD1 molecules efficiently present antigen in 

immature dendritic ceUs and traffic 

independently ofMHC dass 11 during 

dendritic ceU maturation. j Immunol. 2002 

Nov 1;169(9):4770-7. 

Peters PJ, Mironov A Jr., Vey M, Peretz D, 

Van Donselaar E, Leclerc E, Erpel S, 

DeArmond Sj, Burton DR, 

Williamson RA, Prusiner SB. Trafficking of 

prion proteins through a caveolae-mediated 

endosomal pathway, j CeU Biol (2003 Aug 

18;162(4)7°3-17). 

Weis RM, Hirai T, Chalah A, Kessel M, 

Peters PJ, Subramaniam S. Electron 

Microscopic Analysis ofMembrane 

Assemblies Formed by the Bactenal 

Chemotaxis Receptor Tsr. j Bactenot. 2003 

jun 1P85(12):3636-3643. 

Puertollano R, Van der Wel NN, 

Creene LE, Eisenberg E, Peters PJ, 

Bonifacino JS. Morphology and dynamics 

of dathrinjGGA1-coated camers budding 

from the trans-Golgi network. Mol Biol CeU. 

2003 Apr;14( 4):1545-57· 

Cernadas M, Sugita M, Van der Wel N, 

Cumperz JE, Behar SM, Besra CS, 

Peters PJ, Brenner MB. NK T cetl 

development is criticaUy dependent on 

murine CD1d lysosomal localization and 

interaction with the AP-3 complex. joumal 

ofImmunol (2003 Oct 15;171(8):4149-55). 

Savellano D, Bos E, Blondet C, Sato F, 

Abe T, Josephson L, Weissleder R, 

Caudet J, Sgroi D, Peters PJ, Basilion JP. 

The TransJemn Receptor: A Potential 

Molecular Imaging Marker for Human 

Cancer, Neoplasia. (2003 Dec. Vot. 5, 

No. 6,495-506).

VII DIVISION OF MOLECULAR 

GENETICS 

ROlE OF MOUSE POlYCOMB-GROUP GENES IN 

TRANSCRIPTIONAl REPRESSION AND TUMORGENESIS 

Division Head, Group leader Maarten Van Lohuizen 

Maarten Van Lohuizen PhO Group leader 

Koen Braat PhO Post-doe 

Maria Hernandez pho Post-doe 

Anders Lund PhO Post-doe 

Inhua Muyrers-Chen PhO Post-doe 

Bas Tolhuis PhO Post-doe 

Erwin Boutsma MSc Graduate student 

Sophia Bruggeman MSc Graduate student 

Merel Lingbeek MSc Graduate student 

Konstantin Matentzoglu Msc Graduate student 

Panthea Taghavi MSc Graduate student 

Petra Van der Stoop MSc Graduate student 

Florence Van Tienen Undergraduate student 

Oanielle Huisman Technical staff 

Jan Paul Lambooij Technical staff 

Sonja Noback Technical staff 

Ellen Tanger Technical staff 

Els Verhoeven Technical staff 

b 

d 

We are studying the mechanism of stabie inherited transcriptional repression by 

Polycomb-group (Pc-G) protein complexes, and the effects of deregulation ofPc-G 

genes on Homeobox (Hox) gene expression, development, Cell cycle control and 

cancer formation. Furthermore, we study the critical role of Pc-G complexes in the 

maintenance of stem ceU fate. In addition, we are performing large-scale genetic 

screens in primary cells and in cancer-predisposed mice to identify cancer-relevant 

combinations of collaborating oncogenes and tumor-suppressor genes. 

Functional characterization of Pc-G protein complexes Repressive Pc-G 

proteins and counteracting Trithorax-group (Trx-G) of nucleosome remodeling 

factors are involved in maintenance of proper gene expres sion patlerns during 

development at the level of chromatin structure. As such, these factors constitute a 

crucial 'ceUular memory' mechanism of transcriptional states. Important targets 

include the Hox gene clusters but also critical ceU cycle regulatory genes, such as the 

INK4ajARF tumor suppressor locus. At least two biochemical distinct evolutionary 

highly conserved Pc-G protein complexes can be distinguished. The first contains 

EnxljEnx2 (SET domain proteins acting as Histone H3 methylases), SU(Z)I2, Eed and 

histone deacetylases. The second large complex encompasses BmiIjMeh8, 

M33jMPC2, MphljMph2 and RinglbjRingla together with other proteins yet to be 

characterized and is required throughout development. To study Pc-G function we 

focus on representative members of these groups: Bmil, Me118, M33, Ringlb and Eed, 

respectively in gain- and loss-of-function studies in mice. New insights have come 

from our recent analysis of Ringlb-deficient rnice. Unlike other single Pc-G gene 

knockout mice (which display various defects but are viabie, see below), Ringlb null 

mutant mice die shortly after gastrulation. This may relate to the central Ringlb 

positioning of the Ringlb protein in the Pc-G complex, and in addition suggests that 

the closely related Ringla gene cannot compensate for loss of Ringlb. This severe 

phenotype resembles that of the Eed null mutant mice, and suggests that loss of 

Ringlb results in complete loss of Pc-G maintenance function. In addition, we have 

generated Ringlb-conditional knockout ES celllines and mice, which are being used 

to study Pc-G function in relation to differentiation and development in a timed and 

cell type specific manner. To study the molecular mechanism of Pc-G repression we 

are characterizing local changes in chromatin structure as a function of changes in 

the expression levels of Pc-G proteins, using Chromatin Immunoprecipitation 

(CHIP) and chromatin profiling using E.coli dam methylase (DAMid, in close 

collaboration with Dr. B. Van Steense!, this Division) Main focus of these studies is 

on the currently best-characterized in vivo relevant cell cycle target gene: Cdkn2A 

(p16INK4ajp19ARF) and on Hox cluster genes. 

e Primary neurospheres 

f 

Secondary spheres 

i~lLL 

** 

o 

WT HET KO 

Figure VII.l: Bmil controls neuronal stem cell 

renewal. Bmil deficient pnmary neurospheres 

(b) are reduced in size when compared to wild 

type (a) and upon replating, Bmil deficient 

secondary neurospheres (d) fail to form in contrast 

to wild type controls (c). This indicates severely 

reduced self-renewal of neural stem cells in Bmil 

deficient mice. (e and f) quantification of pnmary 

and secondary sphere formation. 

Connections between Pc-G gene repression, cell cycle regulation and Cancer 

formation We originally identified Bmil as an oncogene that cooperates with the 

cMyc oncogene in the induction of Band T cellieukemia in mice, underscoring the 

connection between deregulation of Pc-G repression and cancer. In line with this, 

Bmil-overexpressing transgenic mice have a high incidence of Band T cell 

lymphomas. In contrast, Bmil knockout mice show severe progressive proliferation 

defects and increased apoptosis oflymphoid and myeloid cells, resulting in severe 

lymphopenia. In addition, also primary embryo fibroblasts (MEFs) and neurons in 

the cerebellum of Bmil knockouts show such defects. We demonstrated that these 

defects are due to increased levels of the INK4ajARF-encoded tumor suppressors 

p16INK4a and P19ARF, that in turn are critical regulators of the Rb and the P53 tumor 

suppressor pathways. As such, the INK4ajARF locus acts as an important tumorprevention 

mechanism in normal cells. Our recent results indicate that P19ARF is 

the most critical Bmil target in mice, whereas p16INK4a is more important in 

primary human cells.

Bmi1/Pc-G repression is required to maintain stem cell fate A key 

characteristic of cancer cells is their unlimited self-renewal. In this respect, cancer 

eells resembie stem eells, and aeeumulating evidenee suggests that many forms of 

eancer may indeed eontain eells earrying stem eell markers. In studying the 

proliferation defeets and progressive loss of cells in Bmi! deficient mice we reeently 

discovered that Bmi! is required for proliferation and self-renewal of neural stem 

eells (Figure VIL!, collaboration with D. Zencak and Y. Arsenijevic, Lausanne 

University). Furthermore, we demonstrated a critical role for Bmi! in controlling 

proliferation of cerebellar granule precursor eells (CGCs) and showed that Bmi! 

expression is controlled by a major developmental and stem cell proliferation 

signaling cascade, the Sonie Hedgehog pathway (Figure VIL2). In addition, we 

showed that BmiI is overexpressed in a majority of primary human 

medullobastomas, a major childhood brain tumor thought to originate from 

inappropriate maintenanee of proliferation of CGC eells (collaboration with C. Leung 

and S. Marino, University ofZfuich). These results, together with the reeently 

established role of BmiI in hemapoeitic stem cells and leukaemie stem eells, suggest 

a eommon conserved role for BmiI-eontaining Polycomb complexes in maintenanee 

and expansion of stem eells or committed progenitors and in the pathogenesis of 

tumors originating from the neoplastic transformation of these cells. The possible 

broader relevance of these findings for human ca neer is further underscored by the 

amplification of BMI1 in Mantle celllymphomas and the overexpression of BMI! in 

various tumor types induding non-small eelllung caneer. 

In vivo and in vitro genetic screens to identify new groups of collaborating 

oncogenes or tumor suppressors We have reeently applied MuLV-insertion 

mutagenesis on a genome-wide seale in INK4ajARF deficient leukemia-predisposed 

mice. In dose collaboration with A. Berns (this Division), J. Jonkers (Division V) and 

A. BradleyjThe Sanger Center, Hinxton, U.K., we are now extending and optimizing 

these types of screens to other eaneer relevant models such as breast eaneer. We have 

also developed in vitro high-eopy suppressor screens, to screen for collaborating sets 

of oneogenes and tumor suppressor genes. One sueh a screen is aimed at identifying 

genes eontributing to tumor-progression. Hereto, combinations of predisposing 

oncogenes such as TBX2, Myc or Ras Vl2 are introduced in MEFs or eonditionally 

immortalized human primary epithelial eells prior to transduction with tumorderived 

retroviral eDNA libraries or RNAi-hairpin pRETROSUPER libraries. 

Transformed dones are subsequently seleeted in semi-solid medium, and the 

relevant genes are isolated using efficient PCR strategies. So far we have isolated 

several known and novel eandidate oncogenes that eooperate with Myc, one novel 

transcription factor that transforms in combination with Ras V12 and two RNAi's 

direeted at chromatin-regulating genes. The role and mechanism of aetion of these 

new putative oneogenes or tumor suppressors, and their interferenee with adhesion 

and invasion of primary mouse and human cells is under investigation. 


Itahana K, Zou Y, Itahana Y, Martinez J-L, 

Beausejour C, Jacobs JJL, 

Van Lohuizen M, Band V, Campisi J, 

Dimri GP. Control ofreplicative lift span of 

human fibroblasts by the polycomb protein 

Bmi1 and p16. Mol Cel! Bio12003; 

23:402413. 

Voncken JW, Roeien BA). Roefs M , 

De Vries S, Verhoeven M, Marino S, 

Deschamps). Van Lohuizen M. Rnf2 

(Ring1b) deficiency causes gastrulation 

arrest and cel! cyele Inhibition. Proc Natl 

Acad Sci USA 2003; 100:2468-73. 

Smith KS, Chanda SK*, Lingbeek M*, 

Ross DT, Botstein D, Van Lohuizen M, 

Cleary ML. Bmi-1 regulation ofINJG0. 

ARF is a downstream requirement for 

transformation ofhematopoietic progenitors 

by E2a-Pbx1. Mol Ce1l2003; 12:393-400. 

(*These authors contributed equally) . 

Kranc KR, Bamforth SD, Braganc J, 

Chris Norbury C, Van Lohuizen M, 

Bhattacharya S. Transcnptional coactivator 

cited2 induces Bmi1 and Me118 and controls 

fibroblast proliferation via Ink4ajARF. Mol 

Cel! Bio12003; 237658-66. 

A. CGC's CGC's +Shh 

Dl D2 D3 D4 Dl D2 D3 D4 

Bmil 

Cyelin D2 

Sufu --- 

Actin 

~..--------------------- 

B. CGC RK3E 

- - 

- 

6 

f.:! 

~ 

;:> (3 (3 

Bmil 

- - Tubulin 

Figure VII.2: Bmi1 is regulated by the Shh 

pathway. (A) : Western blot illustrating Bmil 

induction upon Shh stimulation of iso!ated 

Cerebellar precusror cells (CGes) concomitant 

with the proliftration marker CyclinD2 and 

(B): induction of Bmi1 by Shh pathway 

transcription fa ctors (Gli) in RK3 E ceUs and 

CGCs.

MOUSE MODELS FOR CANCER 

Group leader Ton Berns 

Anton Berns PhD Group leader 

Paul Krimpenfort PhD Academic staff 

Margriet Snoek PhD Academic staff 

Eric Bindels PhD Post-doc 

Joaquim Calbo PhD Post-doc 

Tamás Csikós PhD Post-doc 

Johan Jongsma PhD Post-doc 

Jaap Kool PhD Post-doc 

Ralph Meuwissen PhD Post-doc 

Martijn Nawijn PhD Post-doc 

Anthony Uren phD Post-doc 

Andrej Alendar MSc Graduate student 

Ate Loonstra MSc Graduate student 

Carla Martins MSc Graduate student 

Haraid Mikkers MSc Graduate student 

Renée Van Amerongen MSc Graduate student 

Erwin Van Montfort MSc Graduate student 

Rahmen Bin Ali technical Staff 

Joost De Moes Technical Staff 

Jan Paul Lambooij Technical staff 

Loes Rijswijk Technical staff 

Fina Van de Ahé Technical staff 

Vedrana Tabor Technical Staff 

John Zevenhoven Technical staff 

The mouse is used as a model organism for establishing the role of oncogenes and 

tumor suppressor genes in tumor development. Using Cre/Lox mediated switching, 

multiple oncogenes and tumor suppressor genes can be mutated in a loco temporal 

manner. This permits more accurate mimicking of tumorigenesis as it occurs in 

man. Specific genotype-phenotype correlations can be established in this way. These 

models are also suited to test prevention and intervention strategies when combined 

with sensitive in vivo imaging techniques. Furthermore, the models permit us to 

identify new oncogenes and tumor suppressor genes involved in tumor progression 

using a variety of techniques, such as array CG H, expres sion profIling and proviral 

insertional mutagenesis. 

Functional analysis of oncogenes and tumor suppressor genes We utilize 

mice carrying combinations of different oncogene and conditional tumor suppressor 

gene alleles to model a range of tumors. Current focus is on lung cancers and 

mesotheliomas. To achieve sporadic induction of these tumors at a defined point in 

time, we use Adeno-Cre virus to (in)activate the relevant genes in a subset of the 

cells. Infection with Adeno-Cre virus will result in a temporarily high level of Cre 

recombinase causing the switching of most if not all of the conditional alleles. 

Subsequently, tumor growth can monitored by noninvasive imaging using a 

conditionalluciferase reporter that is switched together with the conditional 

oncogenes and tumor suppressor genes. 

Oncogenes 

Tumor 

suppressor 

genes 

Blood 

! 1 ~,---------,. 

~;;:al~ ____ • '$ 

:çrT \~~ ~ 

Immune Stroma Angiogenesis WTcelis 

Figure VII.y Why animal models are needed: Only in the intact organism all the relevant factors are 

present that impact on tumor initiation, progression, and metastasis. 

Lung tumors When a conditional Ki-Ras allele is switched on in lung by Adeno-Cre 

administration, rapid onset of tumorigenesis ensues, yielding pulmonary 

adenocarcinomas with 100% incidence after a short latency. When concomitantly P53 

is inactivated tumors arise even faster. They appear highly vascularized and more 

aggressive. However, they retain the adenocarcinoma phenotype. In contrast, when 

Rb and P53 we re inactivated by AdenoCre exposure, primarily small-cell-lung-cancer 

(SCLC) is observed. Both the loss of Rb and P53 are required for the development of 

SCLC. The marker profile of these tumors is indistinguishable from human SCLC. 

The tumors also metastasize to the same sites. We have established a series of cell 

lines from these SCLC. These will serve as a basis to further pin down the sequence 

of events in SCLC development. We want to identify the cell type that gives rise to 

SCLC. In addition, we want to find other lesions occurring in SCLC. Clearly, 

additional mutations are required for SCLC development as the early observed 

hyperplasia that is evident shortly af ter AdenoCre administration can persist for a 

long time. These celllines will be subjected to CG H analysis and expres sion 

profIling. Furthermore, the capacity of the celllines to give rise to local or 

metastasizing tumors will be assessed. Finally, we will determine their response to 

cytotoxic drugs to which SCLC usually become refractory after an initial response and 

determine the genes that play a critical role in this resistance.

Mesotheliomas Very little is known about the genetic lesions contributing to the 

development of mesotheliomas. Inactivation of NF2 and INK4a has been reported in 

a subset ofhuman mesotheliomas. We have used these observations to design a 

mesothelioma mouse model and have inactivated Nf2 in combination with either Rb, 

Ink4ajArf, P53, or P53 + Ink4a by Adeno-Cre infection of the mesotheliallining of the 

thoracic and intraperitoneal cavity. Mesotheliomas were found at low incidence upon 

loss of Nf2. Concomitant loss of Ink4ajAif or P53 but not Rb strongly accelerated the 

development of mesotheliomas. Interestingly, tumors occurring in conditional 

Nf2;Ink4ajAifmice were more invasive than tumors arising in mutant Nf2;P53 mice 

although tumors in Nft;P53 mice occurred after a shorter latency. Likely Ink4a is 

responsible for this feature as tumors arising in mi ce that carry conditional Nf2;P 53 

alleles on an Ink4a null background showed invasive growth af ter an even shorter 

latency than observed in conditional Nf2;P53 mice. We have established celIlines 

from these mesotheliomas and are following similar approaches as described above 

for our lung cancer modeis. 

Identification and characterization of new oncogenic pathways Proviral 

insertional mutagenesis in transgenic and compound mutant mice can mark genes 

acting in speciflc signal transduction pathways. Emphasis in this program that is 

conducted in close collaboration with the group of Maarten Van Lohuizen, is on 

saturation proviral insertional mutagenesis to identify new oncogenes in sensitized 

backgrounds. We now have established conditions to amplify insertions from single 

cells for high throughput analysis of insertion sites (collaboration with The Sanger 

Instiute). In this way we can prove which oncogenic insertions resided in the same 

cell and, therefore, collaborated in tumorigenesis. We hope that this will allow us to 

assign these oncogenes to speciflc complementation groups in transformation. 

We are studying the pathway of two protein families that we re found earlier by 

insertional mutagenesis, Piml and Frat1. The Pim' s are potent oncogenes 

collaborating with Myc in lymphomagenesis. The Pim family of proto-oncogenes 

encodes a distinct class of serinejthreonine kinases consisting of PIMr, PIM2 and 

PIM3- Although the Pim genes are evolutionary highly conserved, the contribution of 

PIM proteins to mammalian development is unclear. PIMr-deficient mice were 

previously described, but showed only minor phenotypic aberrations. To assess the 

role of PIM proteins in mammalian physiology, compound Pim knockout mice were 

generated. Mice lacking expression of Piml, Pim2 and Pim3 are viabie and fertile. The 

phenotype of Pim mutant mice indicates that the PIM kinases have redundant and 

essential roles in cytokine and growth factor signaling. PIM -deficient mice show a 

profound reduction in body size. In addition, the response of distinct hematopoietic 

cell populations to growth factors is severely impaired. In particular, PIM proteins 

are required for the synergistic activation of peripheral T lymphocytes by T cell 

receptor (TCR) and 112 signaling. Under these conditions, absence of PIM proteins 

affects cell cycle entry rather than survival. These results indicate that PIM acts as a 

gain-setter of growth factor signaling by lowering the threshold for cells to enter the 

cell cycle in response to distinct receptor-mediated signais. 

Pratr-3 is the second gene family being studied. Pratr has been cloned as a tumor 

progression gene. The FRAT proteins bind to and inhibit the kinase activity of GSK3 

and are therefore intimately connected with the B-catenin pathway activation. The 

three members of this protein show substantial overlap in expression. Inactivation of 

the complete family in mice does not lead to any obvious developmental aberrations 

indicating that FRAT proteins are not essential for embryonic development. Possibly, 

FRAT proteins fulml a role in responding to specific signals arising from stress 

conditions. FRAT proteins likely fulfill a role in brain, as FRATr and 2 are specifically 

highly expressed in subsets of neuronal cells. In order to gain insight into 

components acting upstream of FRAT proteins we have begun to characterize in 

more detail the FRAT proteins. Two-hybrid screens will be performed to search for 

proteins that interact with FRATr and 2, in order to gain access to the signaling 

cascade upstream ofFRAT. We will also perform expression array analysis on 

carefully matched celllines to find a signature of endogenous FRAT proteins under 

various growth conditions. 


Mikkers H, Berns A. Retroviral insertional 

mutagenesis: Tagging cancer pathways. Adv 

Cancer Res 2003; 88:53-99. 

Marino S, Hoogervoorst D, Brandner S, 

Bern s A. Rb and P107 are required for 

normal cerebellar development and granule 

cell survival but not for Purkinje cell 

persistence. Development 2003; 130,3359-68. 

Berns A. Tumour suppressors: timing will 

teil. Nature 2003; 42+140-1. 

Meuwissen R, Linn SC, Linnoila RI, 

Zeven hoven J, Mooi WJ, Berns A. 

Induction of small ceillung cancer by 

somatic inactivation ofboth Trp53 and Rb1 

in a conditional mouse model. Cancer Cell 

2003; 4:181-9. 

Lyons SK, Meuwissen R, Krimpenfort P, 

Berns A. The generation of a conditional 

reporter that enables bioluminescence 

imaging of CrejLox-dependent 

tumorigenesis in mice. Cancer Res (in 

press). 

Jonkers J, Berns A. Animal models 

of cancer. Cell Mol Biol Cancer 2003; 

(in press). 

Yu BD, Becker-Hapak M, Snyder EL, 

Vooijs M, Denicourt C, Dowdy SF. 

Distinct and non-overlapping roles for pRB 

and cyclin D:Cdk4j6 activity in melanocyte 

survival. Proc Natl Acad Sci USA 2003; 

(in press). 

70 tumor free 

100 • 

90 

80 

70 

60 

50 

40 

30 

20 

10 

.. . , 

• ti 

. ~ 

: , 

• I 

All : 

invasive : 

. 

Nf2 ;p53;lnk4a"{" 

• Nf2;p53 

•• Fraction 

.. 

invasive 

. .. 

o ------------------------------------ 

o 50 100 150 

Time (days) 

Figure VII+ Mesothelioma development in 

NF2;P53 and Nf2;P53;Ink4a mutant mice. 

200

CHROMATIN CENOMICS 

Group leader Bas Van Steense I 

Bas Van Steensel PhD group leader 

Romeo lascaris PhD post-doe 

Martin lodén PhD post-doe 

Celine Moorman PhD post-doe 

Elzo De Wit MSc graduate student 

Marike Feenstra MSc graduate student 

Frauke Creil MSc graduate student 

Maart je Vogel MSc graduate student 

Ben Abbas Technical staff 

The main goal of our laboratory is to understand how gene expres sion programs in 

higher eukaryotes are regulated. In particular, we focus on mechanisms of 

chromatin-mediated gene regulation. Using Drosophila and mammalian cells as 

model systems, we develop and apply new whole-genome approaches to study the 

mechanisms of gene regulation. 

Chromatin profiling in Drosophila In 200r we published a novel technique 

(named 'DamID chromatin profiling') for the genome-wide mapping of in vivo target 

loci of chromatin proteins [van Steensel et al (200r) Nature Genet. 2T304-308]. This 

microarray-based method allows us to test thousands of genomic sequences for in 

vivo binding of achromatin protein or transcription factor of interest. In a series of 

collaborations with the laboratories ofJeffrey Delrow, Steven Henikoff, and Robert 

Eisenman (all at the Fred Hutchinson Cancer Research Center, Seattle, USA) , we 

have now completed and published 6,000-gene binding profiles of 7 transcription 

factors and chromatin proteins in Drosophila. 

We identified several hundreds of target genes for each of the functionally related 

transcription factors Myc, Max, and Mad/Mnt in Drosophila cells. These datasets 

revealed that each of these factors associates with hundreds of genes covering a broad 

spectrum of cellular pathways. In addition, the results have provided new insights 

into the molecular 'code' underlying target specificity of the transcription factors. 

Likewise, a genomic map of targets of G A GA factor was constructed. U sing 

computational methods, we uncovered some of the sequence features that determine 

in vivo binding of GAGA factor (see below). 

We have also constructed global binding maps of the chromatin proteins HPr, HPrc, 

and Su(var)3-9. Comparative analysis of these binding patterns has revealed that 

these proteins can form distinct complexes. HPr and Su(var)3-9 can bind together or 

independently of each other, and the different complexes show distinct chromosomal 

distributions. Moreover, we found that some of these chromatin complexes are 

preferentially associated with specific classes of developmentally co-regulated genes. 

For example, HPr and Su(var)3-9 bind together to a set of genes that are primarily 

expressed during embyogenesis; strikingly, these genes are concentrated in the 

pericentric chromosomal regions. 

The above-mentioned experiments were performed with cDNA arrays, which only 

all ow detection of protein binding to regulatory regions close to transcription units. 

In collaboration with the laboratory of Kevin White (Yale University) we have started 

to employ genomic 'tiling' arrays, which consist of -rkb genomic fragments covering 

large contiguous genomic regions. Using these tiling arrays, we have obtained a 

highly detailed view of the binding patterns of GAGA factor and HPr in a 2.9Mb 

region of the fly genome (Figure VII.7). One of the surprising results of this work 

was that HPr binds to the gene ck in the coding region rather than at the promoter, 

possibly indicating a role in transcription elongation. 

0.5 1.5 

position (Mb) 

2.5 

Figure VII.7: Binding ofGAGAfactor as detected by genomic 'ti/ing' arrays covering 2.9 Mb ofgenomic 

sequence divided into overlapping segments of -0.9 kb. Each verticalline represents one probe. A value of 

-1 indicates background signal; higher va lues indicate binding ofGAGAfactor. 

In addition to the generation of genomic maps of protein binding, we are also 

developing methods for global mapping of chromatin structure. When E. coli Dam 

methyltransferase is expressed in eukarytic cells, the resulting adenine methylation

pattern in gen om ic DNA is known to reflect local chromatin accessibility. Based on 

this, we have developed a microarray-based mapping assay of chromatin accessibility. 

Preliminary studies in a Drosophila cellline suggest that this method can indeed be 

used to map the degree of 'packaging' of genes at a genomic scale. This new 

technique will allow us to perform detailed studies of the molecular mechanisms that 

control chromatin accessibility. 

Chromatin profiling in human cells We have adapted the chromatin profiling 

technology for use in human cells. Most steps of the procedure have been carefully 

tested and optimized, although we are still seeking ways to further enhance the 

sensitivity of the assay. Using r8k human cDNA arrays, we have obtained 

reproducible genomic binding profiles of members of the HPr protein family in the 

breast cancer cellline MCF7. The resulting list of>roo putative target genes is 

currently being validated and will serve as a basis for functional studies of HPrcontrolled 

gene networks in human cells. We have also initiated experiments with 

r2k human CpG island arrays (University Health Network Microarray Centre, 

Toronto, Canada), which should enable us to detect protein binding in many 

regulatory non-coding regions. 

Bioinformatics Targeting of transcription factors and chromatin complexes to 

specific sets of genes must be guided at least in part by cis-regulatory elements. To 

find sequence elements that may mediate the recruitment of a protein, we collaborate 

with bioinformaticist Or. Harmen Bussemaker (Columbia University, New York, 

NY). The REDUCE algorithm enables us to identify the preferred sequence context 

for in vivo binding of a given protein. For many of our protein binding maps 

REDUCE identified specific sequence motifs that are correlated with protein binding. 

For example, we found that binding of Drosophila Myc is not only correlated with the 

canonical E-box CACTGT, but also with the palindromic sequence TATCGATA, 

suggesting the existence of a factor that binds to this motif and creates a preferred 

environment for Myc binding. 

For most of our chromatin profiling experiments, we used cDNA arrays to detect the 

protein binding patterns. Because the resolution of chromatin profiling is - 2kb, 

binding to transcribed regions as well as to nearby upstream and downstream 

regions can be detected with cDNA arrays, but the precise region of binding within 

this range cannot be pinpointed. We showed that RE DUCE can be used to 

circumvent this limitation and reveal wh ether a protein binds preferentially to 

specific gene features such as upstream regions, downstream regions, introns, exons, 

etc. Using this approach, we found that GAGA factor binds abundantly to GAGAG 

motifs in introns, but not to the same motifs in exons. Thus, by combining cDNAarray 

based chromatin profiling and REDUCE analysis, the effective mapping 

resolution can be increased, and new insights can be obtained into the targeting 

specificity of DNA-binding proteins. 

We have initiated a detailed analysis of the genomic features that may be involved in 

recruitrnent of HPr in Drosophila. The results indicate that HPr binding may be 

partially guided by transposable elements. In addition, we found that HPr displays a 

preference for large genes. 

We have established a computational pipeline (named 'ChromaPilot') for automated 

analysis of our chromatin profiles. Datasets are now automatically processed and 

subjected to quality control and statistical analysis. Moreover, ChromaPilot includes a 

series of standardized algorithms to extract biologically relevant information, such as 

detailed chromosomal maps, statistical analysis of chromosomal distribution 

patterns, REDUCE analysis (see above) , searches for biological pathways, and 

comparison with all other datasets previously generated in the laboratory. 

ChromaPilot greatly speeds up the analysis of the complex chromatin profiling data. 

We are in the process of developing a similar set of algorithms for the analysis of 

mammalian data. 


Van Steensel B, Delrow J, Bussemaker HJ . 

Genomewide analysis of DrosophilaGAGA 

fa ctor target genes reveals context-dependent 

DNA binding. Proc Natl Acad Sci USA 

20°3: 100:2580-85. 

Orian A, Van Steensel B, Delrow J, 

Bussemaker HJ, Li L, Sawado T, 

Williams E, Loo LW, Cowley SM, Yost C, 

Pierce S, Edgar BA, Parkhurst SM, 

Eisenman RN . Genomic binding by the 

Drosophila Myc, Max, MadjMnt 

transcription factor network. Genes Dev 

20°3: 17: 1101-14. 

Sun LV, Chen L, Greil F, Negre N, Li TR, 

Cavalli G, Zhao H, Van Steensel B, 

White KP. Protein-DNA interaction 

mapping using genomic tiling path 

microarrays in Drosophila. Proc Natl Acad 

Sci USA 20°3: 100:9428-33. 

Greil F, Van der Kraan I, Delrow J, 

Smothers JF, De Wit E, Bussemaker HJ, 

Van Driel R, HenikoffS, Van Steensel B. 

Distinct HP1 and Su(var)3-9 complexes 

bind to sets of developmentally co-expressed 

genes dependi ng on chromosomallocation. 

Genes Dev 20°3: (in press ).

VIII DIVISION OF 

EXPERIMENTAL THERAPY 

MECHANISMS AND PREDICTION OF TUMOR RESPONSE 

TO RADIATION 

Division Head, Group leader Adrian Begg 

Adrian Begg PhD Group leader 

Frank Hoebers MD Radiation Oncologist 

Conchita Vens Post-doe 

Hilde )anssen MD Graduate student 

Gaby Rumping MSc Graduate student 

Christie Vermeulen MSc Graduate student 

Ben Floot Technical staff 

Ingrid Hofland Technical staff 

Debbie Sprong Technical staff 

Manon Verwijs Technical staff 

Iris Groeneveld Undergraduate student 

We have had a long-standing interest in developing ways to predict response to 

radiotherapy, alone or in combination with chemotherapy. In recent years our 

research has moved towards obtaining a better understanding of fundamental 

processes involved in radiation damage, in order to more rapidly attain this goal and 

provide better leads for intervention. 

Mechanisms and modulation of radiosensitivity Polymerase beta is the key 

enzyme in Base Excision Repair (BER), a DNA damage rep air pathway which 

removes bases damaged spontaneously or by genotoxic agents. Proteins involved in 

BER are also thought to be responsible for the rep air of single strand DNA breaks 

(SSB). We previously showed that expres sion of a dominant negative to DNA 

polymerase beta (polbetaDN) resulted in radiosensitization ofhuman tumor celi 

lines, demonstrating a role for BER in determining radiosensitivity. We hypothesized 

that inhibition of BER willlead to increased numbers of double strand breaks from 

convers ion of unrepaired SSBs during replication. To test this, we measured rates of 

chromosome aberrations and sister chromatid exchange. PolDN expressing cells 

showed increased chromosome aberrations (Figure VIILr), demonstrating an 

increased load of DSBs in these cells. We also found increased spontaneous sister 

chromatid exchanges, reflecting increased homologous recombination events due to 

BER inhibition. Results to date are therefore consistent with the hypothesis that the 

polbetaDN leads to increased SSBs which are converted to DSBs during replication, 

which in turn are subject to repair by homologous recombination. In apparent 

contradiction to these results, polymerase beta knock-out cells exhibit wildtype 

radiosensitivity, indicating either BER is unimportant, or a role for redundant 

polymerases. We found that expression of polbetaDN also radiosensitized polbeta 

knock-out cells. Since we previously showed that polbetaDN inhibited BER, this 

leaves the possibility that redundant polymerases are indeed involved in dealing with 

radiation damage. Current experiments are directed at the relationship between 

different parts of the radiation induced DNA damage repair and tolerance network. 

A project has started this year to study the role of apoptosis and senescence in a 

sporadic lung tumor model in mice (collaboration with Meeuwissen, Berns, Verheij, 

J. Borst in this institute). Adenovirus containing the cre enzyme is instilled into 

lungs of genetically manipulated FVB/n mice. Removal of floxed blocking DNA by 

cre activates oncogenic K-ras and luciferase, producing sporadic tumors which can be 

followed before and af ter treatment by light emission on luciferin administration. 

4 

Figure VIIJ.1: Expression of a 'dominant negative' 

to DNA polymerase beta in A549 human lung 

tumor cells increases the production of 

chromosome aberrations, as detected by FISH 

with 'painting' probes to chromosomes 1,2, ad 4. 

Strand breaks are apparently converted from single 

to double by inhibiting base excision reapir. 

(j) 

u 

polbeta ON 

~ 

Q) 3 

c.. 

(/) 

c 

0 

:iJ 

cu 3 

~ 

~ 

Q) 

..0 

cu 

Q) 2 

E 

0 vector control 

(/) 

0 

E 2 

0 

~ 

..c 

Ü 

0 2 3 4 5 6 7 

Oase (Gy) 

T

Genetic determinants of radiosensitivity in this clinically relevant model will be 

determined by crossing with mi ce containing alterations in apoptosis and senescence 

genes. To date, the sensitivity of FVB jn mice to locallung irradiation has been 

determined. 

Hypoxia At a basic level, we are pursuing the interesting finding that irradiation 

under hypoxia appears to produce greater numbers of DNA-protein cross links than 

under oxic conditions. A collaboration has been set up with Dr David Murray 

(Edmonton), who found increased sensitivity ofXPF and ERCCr mutant rodent cells 

only when irradiated under hypoxia. The XPF-ERCCr dimer has been shown to be 

important in repairing DNA interstrand cross links, and by implication DNA-protein 

cross-links. This implies that relative radioresistance under hypoxia (3x more 

resistant than oxic cells) has a biochemical as weIl as a radiochemical basis, with 

implications for possible targets. We are now investigating this in human cells 

(fibroblasts and tumor lines) , which are known to exhibit significant differences in 

cross-link rep air and nucleotide excision repair (NER) compared with rodents. We 

have constructed putative dominant negatives to both XPF and ERCCr, targeting the 

known binding regions of these proteins to their dimerization partner. The aim is to 

compete out dimer formation by the wildtype proteins, thus inhibiting cross-link 

repair. Constructs have been introduced into target cells using a retroviral vector, also 

expressing GFP for cell selection. Oftwo ERCCr constructs made so far (XPF 

binding domain), one showed increased sensitivity to both cisplatin and UV in 

several stabie subclones, indicating compromised cross-link rep air andjor NER. Oxic 

and hypoxic radiation sensitivity tests are underway. The putative XPF dominant 

negative construct (ERCCr binding domain) showed no altered drug phenotype. 

Whether these truncated proteins bind their respective wildtype partners is under 


To assess the role ofhypoxia in he ad and neck tumors, we have continued input into 

the clinical study involving patients treated by surgery, with or without post-operative 

radiotherapy. The goal is assess the importance of two forms ofhypoxia, chronic and 

acute, in determining outcome. Patients receive both pimonidazole to assess 

primarily chronic hypoxia, and IUdR (iododeoxyuridine) to assess the proportion of 

non-perfused blood vessels, providing an estimate of acute hypoxia. In the latter, the 

proportion ofCD3rj34 stained blood vessels which have no surrounding IUdRlabeled 

tumor cells are scored. In addition, endogenous markers for hypoxia are 

being evaluated, particularly HIF-ru. Immunostained sections are evaluated by both 

image analysis and serni-quantitative visual scoring. To date, we have accrued 

roo patients and have evaluated 60 for pimonidazole and IUdR-negative vessels. 

A wide range of values has been found for all parameters. The trial aims to accrue 

r50 patients (UZ Leuven and UCL Brussels), at which time outcome correlations will 

be made. 


Begg AC. Is HIF-lalpha a good marker for 

tumor hypoxia? Int] Radiat Oneol Biol 

Phys 2003; 56:917-9. 

janssen HL, Haustermans KM, Sprong D, 

Blommestijn G, Hofland I, Hoebers Fj. 

Blijweert E, Raleigh JA, Semenza GL, 

Varia MA, Balm Aj, Van Velthuysen M L, 

Delaere P, Sciot R, Begg AC. HIF-IA, 

pimonidazole, and iododeoxyuridine to 

estimate hypoxia and perfusion in human 

head-and-neek tumors. Int] Radiat Oneol 

Biol Phys 2002; 54:1537-49. 

janssen HL, Hoebers Fj. Sprong D, 

Goethals L, Williams Kj, Stratford Ij, 

Haustermans KM, Balm Aj, Begg AC. 

Differentiation-assoeiated staining with 

anti-pimonidazole antibodies in head and 

neek tumors. Radiotherapy Cf( Oneology 

(in press). 

Cisplatin A trial in which the Netherlands Cancer Institute played an integral role 

showed that daily or weekly cisplatin, concurrently with radiotherapy, improved 

outcome in NSCLC. In a subsequent smaller study, we further showed that cisplatin 

adducts in buccal cens taken early during therapy significantly correlated with 

outcome. The two main advantages of the assay are that buccal cens are easily 

accessible, and it utilizes a unique antiserum capable of detecting cisplatin-DNA 

adducts immunocytochemically. Previous studies employed immunperoxidase 

staining and a now outdated scanning spot cytometer. We have now optimized 

staining and analysis using two-color fluorescence, DAPI DNA staining to select cens 

for measurement (artifact exclusion), a high resolution CCD camera for image 

capture, and have automated analysis with ImageProPlus software. Human tumor 

cells, or buccal cells from volunteers, treated in vitro with a range of cisplatin 

concentrations provide reproducible calibration curves for use with each staining of 

patient material. This is now being applied within a North American trial (GOG: 

Gynecology Oncology Group) of cervix cancer patients treated with combined 

cisplatin and radiation. To date, more than IOO patients have been submitted to us 

for analysis. This will provide an excellent test of the predictive potential ofbuccal cen 

adducts. Assays of several other potential predictors are concurrently being carried 

out in other centers.

VASCULAR MEDIATED TISSUE DAMAGE AND THERAPY 

The focus of our lab is on vascular damage related to cancer therapy. 

Group leader Fiona Stewart 

Fiona Stewart PhD Group leader 

Paul Baas MD PhD Academie staff 

Nicola RusselI MD PhD Academie staff 

Maurice Aalders PhD Post-doe 

Jacqueline Kruse PhD Post-doe 

Martijn Triesscheijn MSc Graduate student 

Marjan Ruevekamp Teehnieial staff 

Johannes Te Poele Teehnicial staff 

Radiation induced vascular damage Cancer survivors treated with radiotherapy 

are at risk for late radiation-induced damage to normal tissues, much of which is 

related to vascular injury. In large vessels this causes atherosclerosis, resulting in 

severe thrombo-embolic events like stroke. In smaller vessels it presents as 

telangiectasia, causing cosmetic and functional damage in the skin and more severe 

problems, e.g. excessive bleeding, in internal organs. Both types of vascular injury can 

become progressively more serious over periods of many years af ter radiotherapy. 

To design intervention strategies for minimizing the risk oflate vascular damage, it 

is necessary to understand the molecular mechanisms responsible for these changes. 

The purpose of our work is to use in vitro and animal models to investigate 

mechanisms of radiation induced vascular damage and to examine the extent to 

which there are similarities with hereditary, or age related vascular damage in the 

absence of ra dia tion. We are focusing on the functional roles of TG F ~ and 

Notch signaling and the initiation of thrombotic and inflammatory cascades, with 

respect to vascular damage. 

It is known that an imbalance between signals for endothelial cell (EC) sprouting and 

vessel maturation leads to telangiectasia and defects in vascular development. TGF~ 

signaling plays a crucial role in these processes and mutations in either ALKlor 

Endoglin (TGF~ type land accessory receptors expressed in ECs) can cause 

hereditary hemorrhagic telangiectasia (HHT) in both man and mouse. Sirnilarly, 

mutations in N otch leading to defective signaling are associated with hereditary 

vascular abnormalities. 

We have now demonstrated that irradiation of mouse kidneys causes a significant 

increase in expres sion levels ofboth TGF~I and endoglin mRNA during periods of 

developing telangiectasia (evident in both the renal cortex and glomeruli). 

Immunohistochemistry also showed a marked increase in endoglin protein level in 

the glomerular capillaries of irradiated kidneys. Whether endoglin plays any 

causative role in radiation-induced vascular changes, or whether it represents a 

negative feedback mechanism to block further sclerotic and fibrotic changes, needs 

further investigation. Experiments are underway to investigate whether irradiation 

alters the relative expres sion levels of ALKljALK5, the TGF~ receptors that control 

the balance between EC sprouting and maturation, and their downstream effectors. 

The N otch ligand, J agged I, which is expressed at very low levels in normal 

vasculature, was also consistently upregulated in irradiated samples ofboth mouse 

kidney and rectum during periods of developing telangiectasia. The functional 

consequences of these changes will be explored in future studies. 

Mice are normally very resistant to the development of atherosclerosis and they have 

low levels of plasma cholesterol. They are therefore not good models of human 

atherosclerosis. ApoE-j- mice, however, have a five-fold increase in plasma 

cholesterol and develop atherosclerosis spontaneously with age. We have recently set 

up studies with localized irradiation of the carotid arteries of these rnice to study the 

influence of irradiation on the development and phenotype of atherosclerotic plaque 

(in collaboration with Mat Daemen, Cardiovascular Research Institute, Maastricht). 

Preliminary results indicate that plaque with a high inflammatory component 

(unstable plaque) develops in the irradiated carotid of irradiated ApoE-j- rnice from 

about 22 weeks. These studies will be continued to investigate whether the acute 

inflammatory response that occurs after irradiation triggers early development of 

atherosclerosis in young mice and wh ether irradiation of older mice increases the 

risk of developing unstable plaque, prone to rupture. 

In parallel with the mouse studies, we are analyzing human plaque from irradiated 

cancer patients for comparison with atherosclerotic plaque from unirradiated 

subjects. These studies should help to identify suitable prophylactic or treatrnent 

strategies for patients at risk for radiation induced atherosclerosis. 

Photodynamic Therapy (PDT) is an effective treatrnent for smalilocalized cancers 

and it is routinely used in the AvLjNKI for oral cavity tumors and multiple basal celi 

carcinomas (BCC). Treatrnent involves systemic delivery of the photosensitizer

Foscan (mTHPC) followed by local tumor illumination with laser light of 652 nm. 

This generates free radicals and singlet oxygen, which leads to local tissue damage. 

Clinical protocols for PDT are based on the assumption that optimum intervals 

between photosensitizer administration and illumination are times of maximum 

differential between drug uptake in tumor and surrounding normal tissue; i.e. 4 days 

for Foscan. However, there is increasing evidence that vascular mediated damage is 

an essential component of curative PDT in vivo and that endothelial cells (ECs) in 

vessels feeding the tumor may be more important determinants of PDT response 

than the tumor cells themselves. This could have a major influence on the design of 

optimal clinical protocols. 

In vitro experiments were carried out to compare Foscan uptake and toxicity of PDT 

in human tumor cells (HMESO-I and HNXOE), ECs and fibroblasts. These 

experiments demonstrated that PDT toxicity was directly proportional to drug 

concentration for all cell types. The drug was distributed throughout the cytoplasm 

within 3 hours but was excluded from the nucleus even af ter 24 hours incubation. 

Co-staining with a mitochondrial probe showed increased drug uptake in these 

organelles. ECs were not intrinsically more sensitive than fibroblasts or tumor cens 

for a given intra-cellular drug concentration, but they did accumulate higher levels of 

drug. Maximum drug loading was achieved within 24 hours in tumor cenlines and 

fibroblasts but ECs continued to accumulate drug during at least 48 hours 

incubation. Phase contrast time-lapse microscopy showed that ECs entered a rapid 

apoptotic death within 2 hours oflow dose PDT, whereas tumor cens showed a 

delayed cen death (16 hours). 

In vivo studies demonstrated a major discrepancy between times of maximum 

photosensitizer levels in HMESO-I or HNXOE tumors grown in nude mice and 

optimum drug-light intervals for PDT response (tumor cure and growth delay). There 

was a much closer correlation between plasma drug levels and PDT response; with 

maximum effect seen for drug-light intervals of I-3h. This supports oU! hypothesis of 

a major influence of vascular mediated PDT response and suggests that specific 

targeting of the ECs, by using shorter drug-light intervals, should be considered for 

clinical protocols. 

We are specificalIy investigating the relationship between PDT response and 

photosensitizer uptake and distribution in patients with multiple basal celI 

carcinomas. Skin lesions are illuminated at intervals of 12 hours to 4 days after 

Foscan. Serial blood samples are taken from each patient, as weil as a single biopsy of 

an untreated tumor at the time of illumination. These samples are being analyzed for 

drug content and results win be correlated with tumor response to determine 

whether plasma levels give a better prediction of PDT effect than tumor drug levels, 

as was found in animal studies. 

To further study PDT-mediated vascular effects in vivo, optical coherence tomography 

(OCT) was used to make high resolution, cross-sectional images of superficial tumor 

(HNXOE in nude mice) and overlying skin before, during and af ter PDT. Structural 

imaging was combined with cross-sectional imaging of blood flow in superficial 

vessels to obtain information about the development of damage in normal and tumor 

tissue. The algorithms that were developed to extract flow data were first validated 

with a control group of animals that received either nicotinamide or hydralazine to 

induce vasodilatation. The depth resolved perfusion measurements showed the 

expected transient increase in skin perfusion from IS to 45 minutes after hydralazine. 

Several PDT experiments were then performed with varying drug doses and time 

intervals between Foscan and illumination. These experiments showed the value of 

OCT for non-invasive assessment of the development of phototoxic damage. These 

studies will be extended to determine whether vascular mediated changes in tumors 

and normal tissues can be quantified in real time. 


Copper MP, Tan IB, Oppelaar H, 

Ruevekamp MC, Stewart FA. Foscan® 

mediated photodynamic therapy in early 

stage squamous cel! carcinoma of the head 

and neck. Arch Otolaryngol Head Neck Sur 

2003; 1297°9-11. 

Cramers P, Ruevekamp M, Oppelaar H, 

Dalesio 0, Baas P, Stewart FA. Foscan® 

uptake and distribution in relation to 

photodynamic therapy. BrJ Ca neer 2003; 

88:283-29°. 

Kuin A, Kruse JJ, Stewart FA. Proteinurea 

and vascu/ar changes after renal 

irradiation: the role of reactive oxygen 

species (ROS) and vascular endothelial 

growthfactor (VEGF). Radiat Res 2003; 

159:174-181. 

Kruse JJCM, Te Poele JAM, Russeli NS, 

Boersma Lj, Stewart FA. Microarray 

analysis to identiJY mo/ecular mechanisms 

of radiation induced microvascular damage 

in normal tissues, Int] Rad Onco/ Biol 

Phys (in press). 

Kruse JJCM, Te Poele JAM, Velds A, 

Kerkhoven RM, Boersma LJ, Russeli NS, 

Stewart FA. Identification of differentialLy 

expressed genes in mouse kidney after 

irradiation using microarray analysis. Rad 

Res (in press). 

Schouwink H, Oppelaar H, 

Ruevekamp M, Van der Valk M, Hart G, 

Rijken P, Baas P, Stewart FA. Oxygen 

depletion during and after mTHPC 

mediated photodynamic therapy in RIF1 

and H-MESO 1 tumors. Radiat Res 2003; 

159:19°-8. 

Zeamari S, Floot B, Van der Vange N, 

Stewart FA. Pharmacokinetics and 

pharmacodynamics of cisplatin after intraoperative 

hyperthermie intraperitoneal 

chemopeljUsion (HIPEC). Anticancer Res 

2003; 23:1643-8. 

Zeamari S, Rumping G, Floot B, Lyons S, 

Stewart FA. In vivo bioluminescence 

imaging oflocalLy disseminated coloncarcinoma 

in rats. BrJ Cancer (in press).

GENES AND PROTEINS INVOLVED IN ANTICANCER DRUG 

RESISTANCE AND PHARMACOKINETICS 

Group leader 

Alfred Schinkel PhD Group leader 

Johan Jonker PhD Post-doe 

Gracia Merino Pelaez PhD Post-doe 

Maarten Huisman MSc Graduate student 

Antonius Van Herwaarden MSc Graduate student 

Marijn Vlaming MSc Graduate student 

Barbara Karnekamp Undergraduate student 

Ellen Boischer Technical staff 

Corina Van der Kruijssen Technical staff 

Els Wagenaar Technical staff 


Huisman MT, Smit JW, Wiltshire HR, 

Beijnen JH , Schinkel AH. Assessing safety 

and efficacy of directed P-gp inhibition to 

improve the pharmacokinetic properties of 

saquinavir used in combination with 

ritonavir. J Pharmacol Exp Ther 20°3: 

3°4:596-602. 

Schinkel AH , Jonker Jw. Mammalian drug 

elflux transporters ofthe ATP binding 

cassette (ABC) family - an overview. Adv 

Drug Deliv Rev 20°3: 55:3-29. 

Allen JO, Van Dort SC, Buitelaar M, 

Van Tellingen 0, Schinkel AH . Mouse 

breast cancer resistance protein 

(BCrpl/ Abcg2) mediates etoposide resistance 

and transport, but etoposide oral 

availability is limited primarily by P 

glycoprotein. Cancer Res 20°3: 6]:1339-44. 

out 

in 

Figure VIII.2: Structure of a prototpic ABC drug 

transporter. 

Our research focuses on genes and proteins that cause drug resistance in tumors, 

and/or influence the pharmacological and toxicological behavior of anticancer and 

many other drugs and toxins, including carcinogens. Insight into these systems may 

improve chemotherapy approaches for cancer and HIV/AIDS, as weIl as 

pharmacotherapy in a broader sense, and increase insights into factors determining 

susceptibility to carcinogens. To study the physiological, pharmacological and 

toxicological roles of the proteins involved, and their interactions, we generate and 

analyze knockout or transgenic mi ce lacking or overexpressing relevant genes. Cell 

lines obtained from these mice are further used as tools to characterize drug 

resistance genes. 

Impact of active drug transporters We have a long-standing interest in plasma 

membrane proteins of the ATP binding cassette (ABC) transporter family, including 

P-glycoprotein (P-gp) , MRP2 and BCRP (Figure VIII.2). These proteins actively 

export a wide range of anticancer, anti-HIV/AIDS, and many other drugs from cells. 

This ATP-dependent drug extrusion can cause multidrug resistance (MDR) in tumor 

cells. P-gp, MRP2 and BCRP alIlocalize to the apical membrane of polarized 

epithelial celIs, resulting in vectorial transport of drug substrates, and there is 

considerable (albeit not complete) overlap in substrate specificity between these 

transporters. Previous experiments in P-gp and BcrpI knockout mice indicated that 

these transporters can variously protect an organism against exogenous toxins and 

drugs by limiting penetration of substrates into brain, testis, and fetus , by restricting 

uptake of orally administered substrates, and by mediating excretion of substrates via 

liver and intestine. To extend these analyses we have initiated generation of Mrp2 

knockout mice, which will be crossed with the existing P-gp and BcrpI knockout 

mi ce in order to elucidate the separate and combined contributions of these 

transporters to pharmacological, toxicoiogical and physiological functions. 

Analysis ofthe BCRP/Bcrpl multidrug transporter The Breast Cancer 

Resistance protein (BCRP / ABCG2) was not previously identified as a source of 

resistance to epipodophyllotoxins. However, when we selected P-gp- and MrpIdeficient 

mouse celIlines for re si stance to etoposide, overexpression of wild-type 

BCrpI emerged as the dominant resistance mechanism. Resistance was accompanied 

by reduced intracellular etoposide accurnulation. Transduced wild-type BcrpI cDNA 

mediated resistance to etoposide and teniposide in fibroblasts, and trans-epithelial 

etoposide transport in polarized MDCKII cells. BcrpI-mediated etoposide resistance 

was reversed by two structurally different BCRP /BcrpI inhibitors, G FI20918 and 

KOI43- BCRP /BCrpI (inhibition) might thus impact on the antitumor activity and 

pharmacokinetics of epipodophyllotoxins. However, treatrnent of P-gp-deficient mice 

with GFI209I8 did not improve etoposide oral uptake, suggesting that BcrpI activity 

is not a major limiting factor in this process. In contrast, use of GFI20918 to inhibit 

P-gp in wild-type mice increased the plasma levels of etoposide after oral 

administration 4-5 fold. It may thus be worthwhile to test inhibition of P-glycoprotein 

in humans in order to improve the oral availability of etoposide. 

The food carcinogen 2-amino-I-methyl-6-phenylimidazo[4,5-b]pyridine (PhlP) is the 

most abundant heterocyclic amine found in various protein-containing foods. PhIP is 

mutagenic and carcinogenic in rodents, and it has been implicated in human breast 

carcinogenesis. H umans on a normal Western diet are exposed to PhIP on a daily 

basis. We investigated whether BcrpI could affect PhIP exposure of the body, as this 

could implicate BCRP activity in the cancer risk due to PhIP. Using polarized cell 

lines we found that PhIP is efficiently transported by murine BcrpI. In vivo 

pharmacokinetic studies in BCrpI knockout mice showed that BCrpI effectively 

restricts the exposure of mice to ingested PhIP, by decreasing its uptake from the gut 

lumen and by mediating hepatobiliary and intestinal elimination ofPhIP. Intra- or 

interindividual differences in BCRP activity in humans may thus also affect the 

exposure to PhIP and related food carcinogens, with possibie implications for cancer 

susceptibility.

Pharmacological function of the Organic cation transporters Octl and Oct2 

The polyspecific organic cation transporters I and 2 (Figure VIII.3) also transport a 

broad range of fairly small organic cations, including drugs, toxÏns and endogenous 

compounds. Their strategie localization in the basolateral membrane of epithelial 

cells in the liver, intestine (Octr) and kidney (Oct! and OCU) suggests that they play 

an essential role in removing noxious compounds from the body by mediating 

basolateral uptake of substrates from blood into excretory epithelia (Figure VIII.4). 

We previously showed that in OctI-!- mice, the hepatic uptake and intestinal excretion 

of organic cations is greatly reduced. Since OCtI and Om have extensively 

overlapping substrate specificities they rnight be functionally redundant. To 

investigate the pharmacologic and physiologic roles of these proteins we generated 

Oct2 single and OCtI/2 double knockout mice. Oct2-/- and OctI/2-/- mice are viabie 

and fertile, and display no obvious phenotypic abnormalities. Absence of OcU in 

itselfhad little effect on the pharmacokinetics of tetraethylammonium (TEA), but in 

OCtI/2-/- mice the renal secretion of this compound was completely abolished, 

leaving only glomerular filtration as a TEA clearance mechanism. As a consequence, 

plasma levels of TEA were substantially increased in Octr/2-/- rnice. This study shows 

that OCtI and OCt2 are together essential for renal secretion of (small) organic 

cations. A deficiency in these proteins may thus result in increased drug sensitivity 

and toxicity. 


Wang D-S, Kusuhara H, Kato Y, jonker jW, 

Schinkel AH, Sugiyama Y. Involvement of 

organic cation transporter 1 in the lactie 

acidosis caused by metformin. Mol 

Pharmacol2003; 63:844-8. 

Zeker N, Huisman MT, Reid G, 

Wielinga P, Kuil A, Breedveld P, 

Knipscheer P, Schellens jHM, 

Schinkel AH, Borst P. Evidence for two 

interacting ligand-binding sites in human 

MRP2 (ABCC2). J Biol Chem 2003; 

278:23538-44. 

Van Herwaarden AE, jonker jW, 

Wagenaar E, Brinkhuis RF, 

Schellens jHM, Beijnen jH, Schinkel AH . 

The brt-clSt cancer resistance protein 

(BcrpljAbCg2) restricts exposure to the 

dietary carcinogen 2-amino-l-methyl-6- 

phenylimidazo[4,5-b]pyridine. Cancer Res 

Nephron 

Proximal tubule 

(cross section) 

Liver section 

2003; 63:6447-52 . 

Glomeruius -~~~.~ 

ProximaJ tubule -f+~f-{c:: 

Distal tubule 

Henle's 

loop ---++-1 

Collecting duet -+ 1 

r.+x 

~ 

:=:sc:::.::... ~ 

Hepatocyte 

Figure VIII+ Localization ofthe OCTs (indicated by grey baU plus double-headed arrow) in the 

basolateral membrane of renal proximal tubules and ofhepatocytes. 

Branch 

portal vein 

jonker jW, Wagenaar E, Van Eijl S, 

Schinkel AH. De.ficiency in the organic 

cation transporters 1 and 2 (OctljOct2 

[SlcnaljSlcna2]) in mice abolishes renal 

secretion of organic cations. Mol Cell Biol 

2003; 23:7902-8. 

jonker jW, Schin kel AH . Pharmacological 

and physiological functions of the 

polyspecijic organic cation transporters 

OCT1, 2 and 3 (SLCnAl-3)· J Pharmacol 

Exp Ther (in press). 

CYP3A transgenie and knockout mice The cytochrome P450 3A (CYP3A) complex 

is a major factor in the metabolic breakdown of most drugs used today, and of 

various endogenous substrates, but also for the activation and inactivation of many 

(pre-)carcinogens. As CYP3A activity shows great inter- and intra-patient variability, it 

ean have a profound influence on variabie drug behavior (pharmacodynarnics) and 

drug toxicity. Moreover, its substrates overlap extensively with those of the drug 

transporters P-gp, BCRP and MRP2. To study this important system and its 

interactions with drug transporters in well-defined models, we have embarked on a 

project to generate CYP3A transgenic and knockout rnice. We have already generated 

several intermediate strains, but additional strains, currently under construction, are 

needed to begin informative studies on drug behavior and (pre-)carcinogen 

susceptibility. 

Figure VIII.3: Structure ofthe organic cation 

transporter OCTl.

CLINICAL AND PRECLINICAL PHARMACODYNAMICS OF 

ANTICANCER DRUGS 

::J 

en 

E 

Group leader Jan Schellens 

Jan Schellens MD PhD Group leader 

Jos Beijnen PhD Academic staff 

Alfred Schinkel PhD Academic staff 

Inge Meijerman External staff 

Karin Mohrmann PhD Post-doc 

NataHe Appels MSc Graduate student 

Pauline Breedveld MSc Graduate student 

Dick Pluim Technical staff 

Monique Van Eijndhoven Technical staff 

300 ,----------------------------, 

-e- Lv. NaCI 0.9% + Lv. MTX in WT 

T 

~ Lv. pantoprazole + Lv. MTX in WT 

~ 100 -" 

g 

~ 

c:: 

Cl) 

o 

c:: 

o 

co 

E 

ril 

co 

Q. 

>< 

I- 

::::E 

Cl 

0 

...J 

Lv. NaCI 0.9% + Lv. MTX in Bcrp1 k.o. 

Lv. pantoprazole + Lv. MTX In Bcrp1 k.o. 

10 

0.: 1 1 

0 10 20 30 40 50 60 70 80 90 100 

Time (min) 

Figure VIII.S: Pharmacokinetic interaction 

between pantopraz ole and methotrexate (MTX) in 

vivo in Beril' mice and wild type mice. Both in 

wild type mice pretreated with pantoprazole 

(40 mgjkg) and in BcrpI the clearance ofi.v. 

(85 mgjkg) was significantly decreased 1.8 to 

1.9fold compared to the clearance ofi.v. MTXin 

wild type mice. 

Role of BCRP and MRP2 in variability in clinical pharmacokinetics of 

anticancer drugs We focused on possible drug interactions with the antifolate drug 

methotrexate (MTX) . MTX is transported by BCRP (ABCG2) and MRP1-4 (ABCC1-4). 

In cancer patients, co-administration ofbenzimidazoles and MTX resulted in 

profound MTX-induced toxicity coinciding with an increase in serum concentrations 

ofMTX and its main metabolite 7-hydroxymethotrexate (7-0H-MTX). We 

hypothesized that benzimidazoles interfere with the clearance ofMTX andjor 

7-0H-MTX by inhibition of the ABC drug transporters BCRP andjor MRP2. First, we 

investigated the mechanism of interaction between benzimidazoles (pantoprazole 

and omeprazole) and MTX in vitro in membrane vesicles from Sf9 cells, infected 

with a baculovirus containing human BCRP, or human MRP2 cDNA. In Sf9-BCRP 

vesicles pantoprazole and omeprazole inhibited MTX transport (IC so 13 I-tM and 

36 I-tM, respectively) while pantoprazole did not inhibit MTX transport and at high 

concentrations (1 mM) it even stimulated MTX transport by MRP2 1.6-fold. Secondly, 

we studied the transport of pantoprazole in MDCKII monolayers transfected with 

mouse Berpl or human MRP2. Pantoprazole was actively transported by Berpl, but 

not by MRP2. FinaIly, the mechanism of the interaction was studied in vivo using 

Berp{/-mice and wild type mice. Both in wild type mice pretreated with pantoprazole 

and in Berpl -!- mice the clearance of i.V. MTX was significantly decreased 1.8 to 

1.9 fold compared that in wild type mi ce (Figure VIII.5). Thus, benzimidazoles 

differentially affect transport ofMTX mediated by BCRP and MRP2. Competitive 

inhibition for BCRP may explain the clinical interaction between MTX and 

benzimidazoles. These studies will guide future clinical trials to unravel the 

important interaction between benzimidazoles and MTX. Furthermore, we will 

explore the do se-range of MTX over which the interaction can be observed. Currently 

we are determining whether the oral bioavailability of MTX can be improved by coadministration 

of pantoprazole. 

We have shown that the efficacy of transport of MTX by BCRP may be significantly 

pH dependent. In Sf9-BCRP vesicles, MTX transport was 5-fold higher at pH 6.0 in 

comparison to pH 7.4- MTX is a weak acid (pKa 4.8-5.5) and the degree of ionization 

therefore greatly differs at the applied pH values. Follow-up studies should further 

unravel the mechanism of the pH dependency of transport of MTX and other 

relevant substrates by BCRP. 

Cellular distribution and membrane translocation of BCRP Mutations were 

produced in three sites localized in the outer membrane part ofhuman BCRP, 

namely, at position 418 (asn to ala) , 557 (asn to ala) and at position 596 (asn to ala). 

These three positions correspond to possible sites for glycosylation. In addition, 

double and triple mutants at these positions we re made. Mutated BCRP was 

transiently transfected into CHO and MDCK cells followed by immunoprecipitation 

and PNGase F treatrnent. Protein was detected with the MoAb BXP-21. Western bI ot 

analysis revealed that glycosylation was present at position 596 only. 

Immunofluorescent labeling of CHO and MDCK cells (using BXP-34 MoAb and 

fluorescent antibody Alexa 568) showed that the unglycosylated mutant (position 

596) did not appear to affect membrane localization ofBCRP. However, mutation at 

position 557 significantly reduced membrane staining of BCRP indicating that this 

position is essential for membrane localization of BCRP. Currently we are examining 

with double staining using an Ab against calnexin, whether BCRP mutated at 

position 557 accumulates in the endoplasmatic reticulum. 

In addition, we tested whether BCRP is phosphorylated under basal conditions. 

32P-Iabeling followed by immunoprecipitation and SDS-PAGE showed no 

phosphorylation, in contrast to MRP2. 

Generation of Mrp2-defrcient mice and compound Mrp2/Mrpl, 

M rp2/P-glycoprotein and M rp2/Bcrpl knockout mice To establish the role of 

MRP2 and its functional overlap with other transporters, we are generating 

constitutive Mrp2-deficient mice. This year the first homozygous Mrp2 deficient 

mi ce we re generated (project in close collaboration with Alfred Schinkel's group).

These mi ce are cross-bred with BcrpjMdnajMdnb knockout mice to generate 

double, triple and quadruple knockout strains. These models will enable the study of 

the functional role of MRP2 in vivo. In addition, the double and multi-knockouts are 

considered excellent models to explore the pharmacological behavior of substrate 

drugs that show overlap in affinity for MRPI, MRP2, P-gp and Bcrp. 

Support of clinical studies The support of clinical trials significantly increased this 

year. The clinical trials with the cisplatin-gemcitabine combination are ongoing. This 

combination is being explored in a schedule-intensive two-weekly combination in 

advanced non-small celllung cancer (NSCLC) as first-line treatment. Recent results 

reveal that the dose-intensity of cisplatin is 50% higher in this schedule than in the 

currently applied standard schedule in the clinic. This may re sult in higher clinical 

activity. 

In addition, the cisplatin-gemcitabine combination is the basis for a combination trial 

that we perform with LY317615, a PKC inhibitor. We continued to support the 

phase I trial combining carboplatin and topotecan. We deterrnine the 

pharmacokinetics ofboth drugs and pharmacodynamic endpoints, especially 

platinum DNA adducts, topoisomerase I expression and activity in white blood 

cells and tumor tissue. We also support the clinical mass balance studies with 

14C-labeled E7070' 14C-ET743, a DNA binding anticancer agent and the 

epothilone 14C-BMS 247550. E7070 is a sulphonurea derivative currently in clinical 

development. This year novel metabolic pathways were elucidated. The main 

metabolic pathway is glucuronidation, although hydroxylated metabolites were also 

identified. Recovery ofBMS 247550 was between 80 and 85%, which me ets FDA 

standards for these types of study. 

In close collaboration with the pharmacy department, pharmacodynamic endpoints 

for studies with farnesyltransferase inhibitors are being developed. The recently 

established LC-MSMS method to quantitate ras farnesylation is being validated using 

human and rodent tumor celllines, human peripheral white blood cells and buccal 

smears. Reproducible levels ofH-ras protein could be detected in buccal smears and 

tumor celllines. 

We have also embarked on a clinical trial with oral gemcitabine. As part of the 

pharmacodynamic evaluation we successfully validated a novel T-cell proliferation 

assay for which T-lymphocytes are collected from patients prior to start of treatment 

and during treatment. Cells are stimulated in CD3 coated dishes and CD4 and CD8 

cells are counted using flow cytometry. Reproducible ratios of CD4jCD8 were 

obtained in T-cell preparations ofhealthy volunteers in the first patients who were 

entered in the study. The aim is to determine the inhibiting effect of gemcitabine 

concentrations on the T-cell proliferation index. 


Crul M, Van Waardenburg RC, Bocxe S, 

Van Eijndhoven MA, Pluim 0, Beijnen JH, 

Schellens J H. DNA repair mechanisms 

involved in gemcitabine cytotoxicity and 

in the interaction between gemcitabine 

and cisplatin. Biochem Pharmacol 2003; 

6p75-82. 

Schellens JH, Planting AS, 

Van Zandwijk N, Ma J, Maliepaard M, 

Van der Burg ME, Boer-Dennert M, 

Brouwer E, Van der Gaast A, 

Van den Bent MJ, Verweij J. Adaptive 

intrapatient dose escalation of cisplatin in 

combination with low-dose vp16 in patients 

with nonsmaIl celllung cancer. Br ] Cancer 

2003; 88:814-21.

MOLECULAR PATHOLOGY 

Group leader Laura Van 't Veer 

laura Van 't Veer PhD Group leader 

Floor Van leeuwen MD Academic staff 

Sjoerd Rodenhuis MD PhD Academic staff 

lodewijk Wessels PhD Academic staff 

Annegien Broeks PhD Post-doc 

Marjanka Schmidt PhD Post-doc 

Dorien Voskuil PhD Post-doc 

Britta Weigeit MSc Graduate student 

Alina Vrieling MSc Graduate student 

larbia Afia Technical Staff 

Astrid Bosma Techn ical Staff 

Arno Floore Technical Staff 

Geert Van Haalem Technical Staff 

Angelina Huseinovic Technical Staff 

Siegina Klaver Technical Staff 

Ben Nota Technical Staff 

Arnout Van der Plas Technical Staff 

Anke Nooijen Research Assistant 

Renate Udo Research Assistant 

Wineke Bakker Student 

ArM heterozygous germline mutations contribute to breast cancer 

susceptibility The most important risk factor for breast cancer development is a 

family history of the disease_ Genes implicated in family history ofbreast cancer 

include the high penetrance genes BRCAl and BRCA2_ 5-10% of all breast cancer can 

be explained by germline mutations in these high-risk genes_ A larger part, - 10-30%, 

might be explained by mutations in low penetrance genes, for which candidates are 

ATM and CHEK2. The contribution of these genes might be explained by the role 

they play in the DNA damage control pathway. Radiation has been shown to be a 

strong risk factor for breast cancer and thus genetically predisposed individuals, 

especially women with inherited mutations in genes involved in DNA-damage repair 

and cell cycle control, may have an increased sensitivity to environmental exposures 

such as radiation. 

To evaluate the significance of germline mutations in ATM, CHEK-2 and BRCAl/2 

to the risk of (radiation-induced) contralateral breast cancer (clbc) , we assessed its 

mutation frequency in women who developed a clbc, with and without radiation 

treatment (RT) for the first breast tumor. Clbc patients were included if their first bc 

is diagnosed before age 50, and the interval between Iste and 2nd bc is at least l-year. 

We collected 64 patients who did not and 169 who did receive RT for their primary 

be. For each patient we obtained the full medical records for data collection. DNA 

was isolated from peripheral blood or paraffin tissue are (being) screened for all 

ATM gerrnline mutations, for one particular CHEK2'\"noode1C mutation and for 

48 BRCAl/2 founder and recurrent mutations. 

So far we determined 15 CHEK2 noodelC among 233 (6.4%) clbc patients, 

13 among 169 (7.7%) patients who did and 2 among 64 (3%) who did not receive RT. 

We identified 30 BRCAl/2 mutations among 188 (16%) clbc patients, 23 among 

128 (18%) patients who did and 7 among 60 (11.7%) who did not receive RT. ATM 

truncating gerrnline mutations were determined among 4 out of 188 clbc patients 

(2.1%) , all were detected among those who received RT. Overall 26% of all the clbc 

patients in this study carriers a germline mutation in one of the tested DNA 

damage rep air response pathway genes, 30% of the women who received RT 

carried a germline mutation versus 15% among those who did not receive RT, OR=2 

(95%CI 1.045-3-888 P=0.03)· Our results suggest that ionizing radiation treatrnent 

might be a risk factor for breast cancer development in these mutation carriers. The 

excess risk for heterozygotes to develop radiation induced contralateral breast cancer 

provides a scientific basis for mutation analysis and subsequently an intensified 

follow-up protocol for mutation carriers. 

Breast cancer patients following radiation treatrnent for Hodgkin's disease where 

approximately 90% of these tumors are estimated to be radiation-induced, did not 

reveal any ATM germline mutations. However, comparative genomic hybridization, 

does show us that these breast cancers do have aspecific pattern of genomic 

alterations. 

Molecular profiling for disease staging and therapy response in breast 

cancer We have established by gene expression proHling an expres sion pattem of 

70 marker genes in tumors predictive of a short interval to distant metastasis 

('prognosis signature') in young lymph node negative breast cancer patients (see 

Division XIII). In addition, we are now evaluating whether proHles determined in the 

primary tumors of patients that later metastasize are still present in these distant 

metastases. This knowledge is important for the use of the initial proHle in 

determining adjuvant treatrnent selection. 

Surgical specimens of primary breast tumors and their matching lymph node 

metastasis of 15 patients, and specimens of primary breast carcinomas and their 

matching distant metastasis from different localizations of eight patients were 

collected from the frozen tissue bank of our hospital. Fluorescently labeled cDNAs 

were hybridized to an 18k human micro array (Central Microarray Facility, 

Netherlands Cancer Institute). Gene clustering and tumor clustering were performed 

using an unsupervised hierarchical clustering algorithm (Pearson correlation 

coefficient). Our results show that the molecular program established in a primary 

breast carcinoma is not only highly preserved in its regionallymph node metastasis

ut also in its distant metastasis. These findings suggest that metastatic capability in 

breast cancer is an inherent feature, and is not based on clonal selections. The results 

further imply that neo-adjuvant treatment given to patients based on the response 

expres sion profiles of their primary breast tumor might also prevent the outgrowth of 

micrometastases. 

The next relevant question to answer is the prediction of treatment response. In 

patients treated in the context of a tamoxifen trial we are currently establishing a 

tamoxifen response profile. Prospective clinical trials are started to evaluate shortterm 

clinical responses (e.g. , doxorubicin, taxanes, aromatase-inhibitors). 

For the detection of minimal disease we identified a range of genes that we re 

expressed in breast cancer, but were absent in the profiles ofblood or bone marrow 

cells. Quadratic discriminant analysis using real-time quantitative PCR for four 

marker genes pIB, pS2, CKI9 and EGP2 provided us with a discriminant value with 

30% positivity in blood of the breast cancer patient group and no false positives 

among the healthy volunteers. The positive value is seen in a subgroup of patients 

that were known to have bone marrow metastases at the time ofblood sampling and 

is absent in patients with metastatic disease in other organs. Moreover, the MRD 

detection in peripheral blood seems indicative for a shorter survival interval and can 

identify micrometastatic disease in sentinal node biopsies. 

The Insulin-like Crowth Factor (ICF) system in relation to breast cancer and 

colorectal carcinogenesis Large prospective epidemiological studies have recently 

confirmed that the risk of several epithelial cancers is increased in individuals with 

relatively high serum concentrations ofInsulin-like Growth Factor-I. Experimental 

evidence shows that at the tissue level, IGF-I is a potent mitogenic and anti-apoptotic 

factor. Both lines of evidence point towards the IGF-system as a potential target for 

cancer prevention studies. The purpose of this project is to evaluate the effects of 

several dietary strategies on the circulating IGF-system, and to characterize the 

association between the circulating IGF-system and breast and colorectal tissue 

I G F -systems. 

In a preliminary series of 29 women who had undergone (prophylactic) mastectomy, 

. we performed analyses oflevels ofIGF-system components in serum 

(radioimmunoassays) and tissue (Real Time PCR and IHC). In this set of (normal) 

breast tissue and serum collected within one year of mastectomy no significant 

correlation was observed between tissue IGF-system components and serum levels of 

IGF-I. When limiting the analyses to serum samples taken on the day of surgery 

(N=7; less variation due to food intake, hormone levels, etc), the correlation 

coefficients we re much stronger (though not significant due to limited sample size). 

Possibly, serum and tissue IGF-systems have common deterrninants, or high levels 

of serum I G F -I may induce for instance a high' expres sion of I G F type I receptor in 

normal breast tissue. Recruitment of participants will continue (focusing mainly on 

blood sampling on day of surgery) until 100 matched samples of blood and normal 

breast tissue are available for study. 

Currently two dietary intervention studies are being conducted in high risk 

populations for breast cancer and colorectal cancer (in collaboration with other 

hospitals, e.g. Gelderse Vallei hospital, Ede). The effects oflycopene and isoflavones 

(2 food components) on the serum IGF-system are studied. A third study will start 

in 2004. A randomized, double-blind, placebo-controlled, cross-over design is used. 

All participants are asked to take dietary supplements (or placebo) during two periods 

for two months each, and visit the hospital4 times for dietary interview, 

anthropometric measurements, and to donate blood samples. 

The proposed research is expected to provide a better fundamental understanding of 

whether and how the IGF-system could be the target of cancer preventive measures. 

Ultimately, dietary prevention strategies will provide means of decreasing the risk of 

cancer development. 


Weigeit B, Bosma AJ, Hart AAM, 

Rodenhuis S, Van 't Veer LJ. Marker genes 

for circulating tumour cells predict survival 

in metastasized breast cancer patients. 

Br J Cancer 2003; 31:1°91-4. 

Broeks A, Urbanus J, De Knijff P, 

Nicke M, Klopper K, Dork T, Floore A, 

Van 't Veer LJ . IVSlO-6T>G: an ATM 

germline mutation frequently identified 

in breast cancer patients with an 

ancient common ancestry. Hum Mut 

2003; 21:521-8. 

Van 't Veer LJ, Weigeit B. Road map to 

metastasis. Nat Medicine 2003; 

9:999-1000. 

Broeks A, De Witte L, Nooijen A, 

Huseinovic A, Klijn JGM, 

Van Leeuwen FE, RusselI NS, 

Van 't Veer LJ. Excess riskfor bilateral 

breast cancer in CHEK2*l1oodeIC germline 

mutation carriers. Br Cancer Res Treat 

2003; 86:1-3· 

Voskuil DW, Bosma A, Vrieling A, 

Rookus MA, Van 't Veer LJ. Insulin-like 

Growth Factor (IGF)-system mRNA 

quantities in normal and tumor breast 

tissue of wo men with sporadic and familial 

breast cancer risk. Br Cancer Res Treat 

(in press).

MOLECULAR ANALYSIS OF BREAST CANCER 

Group leader Marc Van de Vijver 

Marc Van de Vijver MD PhD Group leader 

Harry Bartelink MD PhD Academie staff 

Rianne Oosterkamp MD Academic staff 

Hans Peterse MD Academic staff 

Sjoerd Rodenhuis MD PhD Academic staff 

Emiel Rutgers MD PhD Academic staff 

Juliane Hannemann MSc Graduate student 

Bas Kreike MD Graduate student 

Cathy Bosch Technician 

Hans Halfwerk Technician 

Pet ra Somer-Kristel Technician 


Faneyte IF, Schrama JG, Peterse JL, 

Remijnse PL, Rodenhuis S, 

Van de Vijver MJ. Breast cancer response to 

neoadjuvant chemotherapy: predictive 

markers and relation with outcome. 

Br ] Cancer 2003; 88:406-12. 

Robanus-Maandag EC, Bosch CA, 

Kristel PM, Hart AA, Faneyte IF, 

Nederlof PM, Peterse J L, 

Van de Vijver MJ. Association ofC-MYC 

amplification with progression from the in 

situ to the invasive stage in C-MYCamplified 

breast carcinomas. ] Pathol 2003; 

201(1):75-82. 

Genetic expression profiling in breast cancer to predict clinical behavior 

Gene expres sion profiling ofbreast cancer is being used to predict clinical behavior. 

We have previously identified a gene expres sion profile predicting the development 

of distant metastases. We are currently exploring whether gene expres sion profiles 

can be identified, corresponding to: 

:> Specific genetic alterations in the DNA of these breast carcinomas 

:> Histologie features of the tumors (including type and grade) :> Patterns 

of the formation of distant metastases :> Response to specific forms of 

systemic therapy :> Locoregional recurrenee of disease 

Response to specific forms of systemic therapy Patients with locally advanced 

breast (LABC) cancer often undergo neoadjuvant chemotherapy treatment. At 

present, commonly used regimens of neoadjuvant chemotherapy include the 

combination of adriamycin and cyclophosphamide (AC). Good responses have also 

been observed with adriamycin and docetaxel (AD). It is currently not possible to 

predict sensitivity or resistance of tumors to specific drugs. Some patients may 

therefore undergo the toxicity of these drugs, but do not benefit from the therapy. 

We started a prospective phase 111 trial for patients diagnosed with locally advanced 

breast cancer, randomizing between six courses of adriamycine (60 mgjm 2 ) and 

cyclophosphamide (600 mgjm 2 ) or adriamycin (50 mgjm 2 ) and docetaxel 

(75 mgjm 2 ) respectively. Chemotherapy was administered every three weeks. Total 

RNA was isolated from a frozen 14 G co re needle biopsy obtained from the tumor 

before treatment. All patients underwent surgery af ter completing chemotherapy. If 

there was residual tumor after completion of chemotherapy, RNA was isolated from 

frozen tissue sections. Amplified mRNA was hybridized on human 18k cDNA 

rnicroarrays obtained from the NKI micro array facility. Supervised and unsupervised 

classification have been used to analyze differences between gene expression before 

and af ter treatment and to correlate gene expression profiles to patient's response to 

the chemotherapy administered. 

Thus far 62 patients with LABC have been randomized in the study. Good quality 

RNA from tissue with more than 50% tumor cells was obtained from 46 biopsies 

and 18 tumors. In total, data from 49 patients (three tumors without biopsy) could be 

included into the analysis. From these patients 25 were treated in the AC arm, 24 in 

the AD arm of the study. 

Preliminary analysis indicates that there are significant differences in gene expression 

in the tumor before and after chemotherapy treatment. There does not appear to be a 

major difference in gene expression between tumors from patients with a complete 

pathological remission compared to all other patients. There are subtle differences in 

gene expression between these two groups, but the identification of areliabIe 

'response signature' probably requires a higher number of patients for analysis. 

Locoregional recurrence of disease Af ter breast conserving therapy for breast 

cancer, 5-30% of patients will develop alocal recurrence. A limited number of risk 

factors for local recurrenee has been identified; the most important risk factors appear 

to be incomplete resection of the tumor and young age. The identification of additional 

risk factors would be very useful in guiding optimal therapy and also improve 

understanding of the mechanisms underlying local recurrence. Of 60 breast ca neer 

patients younger than 51 years at diagnosis, treated with breast conserving treatment, 

RNA was isolated from frozen tumor material from the primary invasive carcinoma. 

The patients we re selected for having developed alocal recurrenee (n=26); controls 

(n=34) we re free oflocal recurrence at least 5 years after treatment; of 10 patients, RNA 

from the local recurrenee was also available for analysis. Gene expres sion profiling was 

performed using RNA from these 70 tumors, using a glass array containing 18,000 

cDNA's. Using two-dimensional hierarchical cluster analysis, the tumors from 

patients with and without local recurrenee could not be distinguished. Supervised 

analysis revealed a possible classifier consisting of three genes; these data need to be 

validated. The local recurrences and the primary tumors from the same patients coclustered 

in all cases. Paired-data analysis showed no set of genes that is consistently 

different in expres sion between primary tumors and recurrences.

IX DIVISION OF RADIOTHERAPY 

The year 2003 was especially rewarding for the Radiotherapy Department, as we 

were the first in the world to introduce a cone beam CT linear accelerator into clinical 

practice. With this new volume imaging technique, we are able to obtain critical 

information before and during treatment about precise tumor position relative to 

normal structures. This new imaging system also gives us vital information prior to 

and during treatment about tumor and norm al organ motion. The utilization of the 

information on tumor location and organ motion now allows us to deliver imagedguided 

radiotherapy. Additionally, with the introduction of the cone beam CT linear 

accelerator, we now can obtain maximum benefit from our in-house software aimed 

at improving the delivery techniques of intensity-modulated radiotherapy for several 

tumor sites. 

More insight has also been obtained in the mechanisms involved in radiation 

induced cell death following the apoptotic pathway. A novel imaging technique with 

Annexin scintigraphy allowed us to demonstrate in vivo apoptosis, which appeared to 

be related to the very early response oflow dose irradiation in patients with 

Hodgkin's lymphoma. 

IMAGE ACQUISITION AND PROCESSING 

A Betgen, J De Bois, B Brand, J Van Dijk, J Duppen, I Fitton, M Frenay, J Van der Geer, H Lotz, 

D Jaffray, SMuller, P Nowak, T Nuver, F Pos, C Rasch, P Remeijer, C Schneider, MHP Smitsmans, 

JJ Sonke, R Steenbakkers, M Witte, J Wolthaus, L Zijp, JV Lebesque, M Van Herk 

The image acquisition and processing group has had an exciting year in which the 

most important highlight is the clinical introduction of a linear accelerator equipped 

with a cone-beam CT imaging system that allows on-line collection of three and fourdimensional 

patient data just prior to treatment. Also, much progress has been made 

concerning collection and analysis of four-dimensional (lung) images including PET, 

respiration correlated CT, respiration-correlated megavoltage images and respiration 

correlated co ne beam CT. 

Figure IX.1: Linear accelerator with 

integrated cone-beam CT s}'stem for 

high precision soft-tissue localization. 

Perpendicular to the beam line, a kV 

X-ra}' source and a flat panel imager 

are mounted. In a single gantry 

rotation (100 sj, sufficient information 

is obtained for reconstructing a high 

quality JD CT data set. 

Division Head, Group leader Harry Bartelink 

Berthe Aleman MD Academic staff 

Harry Bartelink MD, PhD Academic staff 

)ose Beiderbos MD Academic staff 

Liesbeth Boersma MD PhD Academic staff 

Eugenè Damen PhD Academic staff 

Roei De Boer PhD Academic staff 

Katrien De Jaeger MD Academic staff 

Luc Dewit MD PhD Academ ic staff 

Riek Haas MD Academic staff 

Guus Hart MSc Academic staff 

Wi/ma Heemsbergen MSc Academic staff 

Edwin Jansen MD Academic staff 

Joos Lebesque MD PhD Academic staff 

Harry Masselink MD Academic staff 

Ben Mijnheer PhD Academic staff 

Luc Moonen MD PhD Academic staff 

Coen Rasch MD PhD Academic staff 

Peter Remeijer PhD Academic staff 

Nieola RusselI MD PhD Academic staff 

Govert Salverda MD Academic staff 

Christoph Schneider PhD Academic staff 

Joep Stroom phD Academic staff 

Bart Van Bunningen MD Academic staff 

Marcel Van Herk PhD Academic staff 

Marcel Verheij MD PhD Academic staff 

Fritts Wittkämper PhD Academic staff 

Nina Bijker MD PhD Temporary staff 

Gerben Borst Temporary Staff 

Patricia De Haan MD Temporary staff 

Frank Hoebers MD Temporary staff 

Bas Kreike MD Temporary staff 

Dimitry Nuyten MD Temporary staff 

Floris Pos MD Temporary staff 

Roei Steenbakkers MP Temporary staff 

Caroline Tissing-Tan MD Temporary staff 

Fred Ubbels MD Temporary staff 

Clinical implementation of cone-beam CT guided radiotherapy Recently, we 

integrated cone-beam CT (Elekta Synergy) on a linear accelerators in our hospital 

(Figure IX.I). The system allows contact-less and fast localization of soft-tissue 

structures. Acquisition of cone-beam CT with 320 projections requires a single 

gantry rotation of about IOO s. In-house developed high-speed CT reconstruction 

code provides a 256x256x256 volume (with I mm cubic voxel size) in about 20 s on a 

2.8 GHz Pc. The clinical image quality is adequate for differentiation of soft tissue 

structures such as bladder and rectum (Figure IX.2). We integrated the cone-beam 

Figure IX.2: Example of a prostate scan made with 

the cone-beam CT system shown in Figure 1. Note 

the good visualization of soft tissue structures.

econstruction code with image registration software for automated localization of 

bony anatomy, and automated tracking algorithms for the pro state position. Initially, 

the system is being evaluated as portal-imaging replacement, by registering bony 

anatomy. 

Corine Van Vliet-Vroegindeweij PhD 

Temporary staff 

Conny Vrieling MD PhD Temporary Staff 

luc Bos PhD Post-doe 

Mischa Hoogeman PhD Post-doe 

Rob louwe PhD post-doe 

Yvette Seppenwoolde PhD Post-doe 

Jan-Jakob Sonke PhD Post-doe 

Bram Van Asselen PhD Post-doe 

leah McDermott Ba/Bs Graduate student 

Agnieszka Olszewska MSc Graduate Student 

lennert Ploeger MSc Graduate student 

Marco Schwarz MSc Graduate student 

Monique Smitsmans MSc Graduate student 

Anja Betgen Technical staff 

Bob Brand Technical staff 

Josine De Bois Technical staff 

JooP Duppen Technical staff 

Michel Frenay Technical staff 

Tom Minderhoud Technical staff 

Pietje Muller Technical staff 

Kenneth Pengel Technical staff 

Maddalena Rossi Technical staff 

René Tielenburg Technical staff 

Peter Van de Ven MSc Technical staff 

Lambert Zijp Technical staff 

John Cho MD Guest 

Avraham Eisbruch MD G uest 

Heather Jones MD Guest 

Mark Kessler MD Guest 

Stefanie Peeters MD Guest 

Respiration correlated cone-beam CT Respiration motion causes artifacts in the 

reconstructed data. We therefore developed a method for reconstructing CT in 

certain phases ofbreathing. To derive at which phase each raw X-ray projection was 

taken, a method was developed that extracts the breathing phase from the X-ray 

images themselves. The method enhances steep gradient features in all X-ray images, 

and makes use of the fact that the position of the diaphragm is NOT stationary in 

time, while most other features are. Using the respiration signal, X-ray projection 

images that are close to a selected respiration phase are selected, and cone beam 

CT reconstruction is performed with only the selected data. Typically, 8 to 

16 respiration phases can be reconstructed while maintaining sufficient image 

quality to visualize a moving lung tumor (Figure IX.3). 

Figure IX.j: Respiration correlated cone beam CT. Structures that move due to respiration show blurred in 

the cone-beam reconstrUctions (lower panels). By including only projection data made from a selected 

respiration phase, a sharp im~ge of the moving tumor is obtained. Typically, we reconstruct the respiration 

movement in 16 phases, giving a smooth high quality movie. 

Figure IX+ Example of automatic prostate 

localization on cone-beam CT data. The image on 

the left is the planning CT. The image on the right 

is the cone-beam CT. The software has 

automatically aligned both prostates to quantify 

the prostate position. This technique allows 

automatic on-line correction for prostate motion. 

Automatic prostate localization using cone beam CT images We previously 

developed an automatic pro state localization method based on 3D gray-value 

matching. This method was already tested on conventional CT scans. The purpose of 

the study was to test the algorithm on images acquired with the cone beam system. 

In this study we used a conventional planning CT scan and 2-8 'on-line' cone beam 

CT-images of 5 patients. The cone beam CT-images were acquired prior to each 

treatrnent fraction. We started by matching the bony anatomy. Then, we registered a 

region of interest of the planning CT scan, which was generated from the delineated 

contour of the planning CT scan, to the on-line co ne beam CT-images using 3D grayvalue 

matching. The results were evaluated visually. Results: Out of 26 matches, 

21 matches were visually correct (Figure IX.4). This study shows that the automatic 

gray-value matching algorithm can be used with cone be am CT-images to correct online 

for pro state movement.


Image acquisition and processing 

Dekker N, Ploeger LS, Van Herk M . 

Evaluation of cast fonctions for gray value 

matching of two-dimensional images in 

radiotherapy. Med Phys 2003; 3°:778-84. 

Figure IX.5: Example of registration of 4D CT data and 4D PET data. The underlying image shows the 

CT data set of a moving tumor in 16 phases. The contours were derived from 4D PET data that was 

registered using grey value matching. Note how weU the motion ofthe tumor in PET and CT correspond. 

Respiration correlated image fusion of CT, PET and portal images Previously 

we developed a method to retrospectively obtain 4D data using arespiration 

correlation algorithm applied to images from our CT scanner. This year, we 

developed a method for respiratory-gated PET acquisition (using the cardiac mode of 

our mobile PET scanner with in-house developed gating hardware). In addition, we 

developed a method to acquire respiration correlated me ga-voltage images. Our 

software packages for visualization and image registration were extended to work 

with 4D data. For instanee, Figure IX.5 shows a time sequence of respiration 

correlated CT with a registered time sequence of PET. We are currently using these 

tools to study the motion pattem oflung tumors, not only prior to treatrnent, but 

specifically how it changes during the course of radiotherapy. In the future this will 

allow safe application of small margins for lung cancer patients and allow further 

dose escalation. 

Adaptive radiotherapy for invasive bladder cancer Patients with solitary 

bladder cancer not fulfilling the criteria for brachytherapy are asked to participate in 

the N02-PBI trial where the bladder tumor is selectively irradiated to a dose of 60 Gy 

(25x2.4) . Because of the extreme mobility of the bladder, we developed and an 

adaptive margin strategy. This method uses multiple CT-scans acquired in the first 

week of treatment to define the tumor volume and the extent of tumor movement to 

guarantee adequate treatrnent. A software tooI was developed to all ow simultaneous 

delineation in up to 10 scans reducing the workload and increasing the consistency 

of delineation. 

A model for predicting short-term bladder shape changes Using repeat MRI of 

healthy volunteers and volunteer patients, the short-term shape change of the bladder 

due to urinary inflow wàs quantified. The purpose of this study is to all ow predictions 

of bladder shape change during the period between imaging and treatrnent during 

on-line image guided radiotherapy. A shape model derived from repeat CT data of 

pro state cancer patients was applied to predict the bladder shape IS minutes af ter 

measurement, which is the expected time delay in image guided radiotherapy. For 

some volunteers, a bladder wall motion of up to 2.5 cm was seen in this period. 

Using the model the maximum error in bladder wall position was reduced to 0.5 cm. 

Furthermore it was found that changes in rectum filling caused a shift in bladder 

position, while hardly influencing the bladder shape. 

Ploeger LS, Frenay M, Betgen A, 

De Bois JA, Gilhuijs KG A, Van Herk N. 

Application of video imagingfor 

improvement of patient set-up. Radiother 

Oncal; 68 :277-84. 

Ploeger LS, Betgen A, Gilhuijs KG, 

Van Herk M. FeasibiLity ofgeometrical 

verification of patient set-up using body 

con tours and computed tomography data. 

Radiother Oncol. 2003; 66:225-33. 

Sonke J). Ploeger LS, Brand B, 

Smitsmans MHP, Van Herk M . 

Leaf trajectory veTjication during dynamic 

intensity modulated radiotherapy using an 

amorphous silicon flat panel imager. 

Med Phys 2003 (in press). 

Sonke J). Brand B, Van Herk M . Focal spot 

motion oflinear accelerators and its effect 

on portal image analysis. Med Phys. 2003; 

3°:1067-75. 

Steenbakkers RJHM , Deurloo KEI , 

Nowak PJCM , Lebesque JV, Van Herk M , 

Rasch CRN . Reduction of dose delivered 

to the rectum and bulb of the penis using 

M RI delineation for radiotherapy of the 

prostate. Int] Radiat Oncol Biol Phys; 

57 (5):1269-79. 

Van Herk M , Witte M, Van der Geer ). 

Schneider C, Lebesque Jv. Biological and 

physical fractionation effects of random 

geometrical errors. Int] Radiat Oncol Biol 

Phys 2003; 57:1460-71. 

Van Herk M . Errors and margins in 

radiotherapy. Sem Rad Oncol 2003 

(in press). 

Attenuation correction using planning CT for detection of lung cancer For 

PET scanners without a built-in CT, transmission PET images (ACPET) are used for 

photon attenuation correction. In this study we compared attenuation correction 

using transmission PET and retrospective correction using planning CT. We 

implemented a method based on a Chang algorithm using an attenuation estimate 

from the spatially registered CT data. Visually the attenuation corrected images from 

CT (AC CT) show a better discrimination of the tumors and the lyrnph nodes from 

the background. They appear with a higher intensity and it is possible to detect them 

more easily and earlier in the sequence of images. Quantitatively the improvement of

the contrast between a lung tumor and its neighborhood on ACCT images compared 

with those on ACPET images are on average 13.9% (15.6%). Thus, lung tumors and 

lymph nodes are better differentiated from the background of the PET images. 

A 'Big Brother' evaluation of physician-computer interaction during 

deli neation As a part of a multi-institutional delineation study, CT scans of the 

thoracic region we re distributed of 22 non-small-cell-lung-cancer patients and were 

delineated by II expert observers. Software called 'Big Brother' recorded every action 

during delineation allowing detailed analysis of delineation 'style' and outcome. The 

location of all corrections (moving or erasing a point) was detected. The corrections 

we re projected in 3-D on the average delineated target of each patient. Delineation 

variation was expressed in terms of a SD and compares with the corrections. The 

mean delineation variation was 0.74 cm SD. The mean number of corrections was 

39, and depended both on patient and observer. For patients with a target mostly 

surrounded by lung tissue (delineation variation 0.33 cm SD) and mediastinum 

(delineation variation 0.76 cm SD) we found a positive correlation between the 

number of corrections and the delineation variation. For patients with atelectasis 

(delineation variation I.I2 cm SD), we found a negative correlation. Bases on these 

results we developed an improved delineation software package that uses coupled 

delineation windows and PET information to re duce these large variations. 

Modeling the effect of geometrie uncertainties on tumor control In this study, 

simulations of pro state radiotherapy were performed using various distributions of 

the tumor cell density inside the target, while the total number of tumor cells was 

kept constant. Typieal geometrical uncertainties result in about 1% loss ofTCP for 

uniform cell density. When the tumor cells are concentrated in a small node on one 

side of the target (typieal for pro state) or when there is a rind oflower cell density 

around the target (typical for head and neck) the TCP loss decreases. The effect 

stabilizes when the ratio of tumor cell densities exceeds roooo. The results indicate 

that the penumbral dose is adequate to eradicate low tumor cell density regions even 

in the presence of geometric errors. 

Effect of target size and tissue density on the margin required for random 

errors Most margin algorithms assume a Gaussian shape of the penumbra of the 

dose distribution. In this study, margins were derived for random errors based on 

realistic dose profiles in water and in lung. It was found that the required mar gin for 

random errors increased with increasing tissue density and decreased with 

increasing target size. The 'classical' margin of 0.7 times the standard deviation of 

the random errors is, however, safe for most situations except for very small targets 

in water-equivalent tissue. 

DOSIMETRY 

B Brand, C De Wagter\ S Gillis\ R Louwe, L McDermott, A Olszewska, E Smit, M Van Herk, 

M Wendling, BJ Mijnheer 

Portal dosimetry A study was performed to determine the long-term stability and 

temperature dependence of two types of electronic portal imaging devices (EPID): 

liquid-filled matrix ionization chamber (LiFi-type) and amorphous silicon-based flat 

panel (aS i-type) imagers. In addition, the stability of the response of one LiFi-type 

imager was assessed by comparing the results of portal dosimetry and in-vivo 

dosimetry using diodes during the treatment of 31 prostate patients. For two LiFi-type 

imagers, a reduced dosimetrie stability was observed, whieh was correlated with 

either radiation damage or with performance of the electronie hardware. The longterm 

stability of these imagers could be considerably improved by correcting for 

room temperature fluctuations and gradual changes in response due to radiation 

damage. As aresult, the reproducibility was better than 1% (1standard deviation, SD) 

over a period of two years. For the aSi-type imagers, the radiation history could be 

, University Hospital, Ghent, Belgium

emoved by updating the dark-field correction af ter a period of EPID inactivity. Such 

a dynamic dark-field correction resulted in a reproducibility of 0.5% (rSD) over a 

period of one year. By extrapolation it was estimated that dose to the electronics must 

be somewhere between 9 Gy and r7Gy. The center of the detector received 580 Gy. 

The average difference between the measured and prescribed dose during pro state 

patient treatment was -0.7% ± I.5% (rSD), and -0.6% ± I.r% (rSD) for EPID and 

diode in-vivo dosimetry, respectively. It can be concluded that a high stability ofthe 

response can be achieved for both types of EPID, which makes them suitable for 

accurate dose measurements. 

In a second study we investigated the dose-response characteristics, including 

ghosting effects, of an amorphous silicon-based electronic portal imaging device 

(a-Si EPID) under clinical conditions. EPID measurements were performed using 

one prototype and two commercial a-Si detectors (Elekta Oncology Systems, Crawley, 

UK) on two linear accelerators; one with 4 and 6 MV and the other with 8 and r8 MV 

x-ray beams. First, the EPID signal and ionization chamber measurements in a miniphantom 

we re compared to determine the amount ofbuild-up required for EPID 

dosimetry. Subsequently, EPID signal characteristics were studied as a function of 

dose per pulse, pulse repetition frequency (PRF) and total dose. There was an overresponse 

of the EPID signal compared to the ionization chamber of up to r8%, with 

no additional build-up layer. The addition of a 2.5 mm thick copper plate sufficiently 

reduced this over-response to within r% at clinicaUy relevant patient-detector air 

gaps. The response of the EPIDs varied by up to 8% over a large range of dose per 

pulse values, PRF values and number of monitor units. EPID response showed an 

under-response at shorter beam times due to ghosting effects, which depended on 

the number of exposure frames for a fIXed frame acquisition rate. With an 

appropriate buildup layer and corrections for dose per pulse, PRF and ghosting, the 

variation in the a-Si EPID response can be reduced to weU within ±r% (rSD). 

(Figure IX.6) 


Dosimetry 

Louwe RJ , Damen EM, Van Herk M , 

Minken AW, Torzsok 0 , Mijnheer BJ . 

Three·dimensional dose reconstruction of 

breast cancer treatment using portal 

imaging. Med Phys 2003; 3° :2376.89. 

McDermott LN, Louwe RJW, Sonke)J, 

Van Herk M, Mijnheer BJ. Dose-response 

and ghosting efficts of an amorphous 

silicon electronic portal imaging device. 

Med Phys 2003 (in press). 

Figure IX.6: EPID response with and 

without corrections for ghosting effects. 

For varying number of monitor units, 

the original response for 4 and 18 MV 

(solid lines) is shownwith the corrected 

response (dashed lines). 

Pre-treatment verification of I M RT Because of their high stability and before 

mentioned dosimetric characteristics, a-Si EPIDs are promising devices for a number 

of dosimetric applications, including pre-treatment verification of IMRT. For that 

purpose we examined the performance of an Elekta a-Si EPID in an r8 MV photon 

beam. After calibrating the EPID in a large field relative to the dose distribution 

measured with a calibrated ionisation chamber positioned in a mini-phantom, EPID 

beam profiles were compared with ionisation chamber measurements in fields of 

various sizes. Beam profiles measured with both devices agreed in the high-dose 

region within ab out 5% and ro% for field sizes down to 5x5cm2 and rxrcm 2 , 

respectively. In the penumbra region deviations up to 2.0 mm occurred. 

Qualitatively, these differences can be explained by the difference in electron 

transport and size between the EPID and the mini-phantom. Consequently, several 

kernel components were designed to correct for the EPID field-size dependence, 

resulting in differences in dose smaller than 2.5% for the smaUest field size. It can be 

concluded that the a-Si EPID performs weU in measuring dose profiles for a wide


Treatment planning 

range of field sizes af ter applying such a field-size correction, thus allowing pretreatrnent 

verification of very small IMRT fields. 

Bos L, Schwarz M, Baer W, Alber M , 

Mijnheer BJ, Lebesque jV, Damen EMF. 

Comparison between manual and 

automatic segment generation in step-andshoot 

IMRT for prostate cancer. Med Phys 

(in press). 

Engelsman M, Remeijer P, Van Herk M, 

Mijnheer B, Damen E. The theoretical 

benefit ofbeam fringe compensation and 

field size reduction for iso-normal tissue 

complication probability dose escalation in 

radiotherapy oflung cancer. Med Phys 

20 03; 30:1086-95· 

Schwarz M , Bos L, Mijnheer BJ, 

Lebesque jV, Damen EM F. Importance of 

accurate dose calculations outside the 

segment edges in intensity modulated 

radiotherapy treatment planning. Radiother 

Onco1 2003; 69: 3°5-14. 

TREATM ENT PLAN N I NG 

jSA Beiderbos, Lj Bos, A Eisbruch, K De jaeger, BA Fraass, M L Kessler, jV Lebesque, DL McShan, 

Tj Minderhoud, CRN Raseh, CJ Schneider, M Schwarz, B. Van Asselen , C Van Vliet-Vroegindeweij, 

Bj Mijnheer, EMF Damen 

The effect of geometrical uncertainties on dose distributions for prostate 

Intensity-Modulated Radiotherapy (I MRT) A study was performed of the effects 

of geometrical uncertainties on the dose distribution of IMRT treatrnent plans for 

prostate irradiation. For five patients, three different treatment plans were designed, 

using a five-field IMRT technique. The CTV was defined as the pro state plus seminal 

vesicles. The three plans were: a reference plan delivering 78 Gy (78-Gy plan) to 

PTVI (CTV+ 10 mm margin); a sequential boost plan, irradiating PTVI to 68 Gy, and 

boosting PTV2 (CTV+ an anisotropic margin of 0 to 5 mm) to 78 Gy, and a 

simultaneous integrated boost (SIB) plan delivering 68 Gy to PTVI and 

simultaneously 78 Gy to PTV2. The sensitivity of each plan for geometrical 

uncertainties was evaluated with computer simulations, using statistics on organ 

motion and set-up uncertainties specific for our institution. Results showed that the 

effect of geometrical uncertainties was very small for the reference and sequential 

boost plans. For the SIB technique, the NTCP was reduced by 5.6% to 12.4%, 

compared to the 78-Gy plans. From this study we concluded that a reduction of the 

margins for a part of the treatrnent (sequential boost) resulted in a similar TCP 

compared to the 78-Gy plans, and a minor reduction in NTCP. The SIB technique 

resulted in a small reduction of coverage probability of the CTV, but reduced the 

NTCP considerably. For all plans, the NTCP determined during treatment planning 

is an accurate predictor for the NTCP af ter incorporation of organ motion and set-up 

uncertain ties. 

100 

90 

80 

70 

~ 60 

~ 50 

:> 

~ 

40 

30 

20 

10 

IC 

n=0.08 

n=0.12 

- n=0.25 

- n=0.5 

--_ .. _. n=l 

10 20 30 40 50 60 70 80 

Oose(Gy) 

Figure IX. 7: DVH curves ofthe rectal wal! 

for an IMRT treatment of a patient with 

prostate cancer for five va lues of n. 

The use of Equivalent Uniform Oose (EUO) in the optimization of prostate 

I M RT The concept of EUD has recently gained interest as metric to drive the 

optimization of IMRT treatment plans. 

N n 

1 l/ n 

The EUD is defined as EUD = N L d i (where N is the number of voxels of the 

i=I 

anatomical structure of interest, di the dose associated with the i-th voxel and n a 

parameter that, for every organ, describes the volumetric dep enden ce of the doseresponse 

relationship) incorporates a parameter n that describes the volume 

dependence of the irradiated tissue. A study was started to investigate how the 

resulting dose distribution depends on the value of n when the EUD is used to 

control the dose to the rectal wall. For 5 patients a full fluence optimization was 

performed for the clinically applied five-field SIB technique as described in the 

previous section. A co st function was defined, which aims at minimizing the EUD in 

the rectal wal!, while ensuring a specific dose coverage of the PTVs, and limiting the 

dose in the other OARs. Treatrnent plans for the five patients were optimized for five 

different values of n (I, 0.5, 0.25, 0.12 and 0.08). The results showed that different 

values of n lead to very similar dose distributions in the PTVs (differences in mean 

dose < 1%, differences in dose given to 99% ofthe volume < 3 %.). For the rectal wall 

the following observations were made (Figure IX.7): a) All cumulative DVH curves 

crossed each other around 60 Gy; b) The volume receiving doses between 60 and 

65 Gy was not influenced by the value of n; c) The rectal wall volume receiving 30 Gy 

and 45 Gy could change by 45% and 30%, respectively, depending on the value of n; 

d) For doses higher than 65 Gy the differences were always within 10%. Different 

values of n affected also the position of isodose surfaces. The dose gradient width, 

defined as the di stance between the 70 Gy and the 30 Gy isodose curves, changed in 

the AP direction by a factor of 3 when n decreased from I to 0.08. High values of n 

were associated with less dose conformity and a larger volume (at least 20%) of 

normal tissues receiving 50 Gy or more. The choice of n, finally, affected the 

weighting of the five be am directions: lower values of n favored orientations with a

larger component in the AP direction. In general, the most significant changes were 

seen for values of n between 0.08 and 0.5. A recent Italian multicenter study 

suggests a value of n = 0.26 when both serious and mild complication are taken into 

account and a value of n = 0.06 when only serious rectal side effect are taken into 

account. 

Prevention of dysphagia and aspiration following intensive ehemo-RT of 

head and neek eaneer Severe late dysphagia and aspiration are the main barriers 

for intensification of chemo-RT /accelerated RT. It is therefore of interest to analyze 

whether new irradiation techniques, like IMRT, have the possibility to re duce the 

dose to the anatomical structures involved in swallowing without affecting the dose 

coverage of the PTV and the possibility of sparing other organs at risk, in particular 

spinal cord and parotid glands. This study, carried out in collaboration with Dr. 

Avraham Eisbruch from the University of Michigan (Ann Arbor, MI, USA) started 

with a joint effort between radiation oncologists and radiologists aimed at defining a 

proper method of outlining the structures of interest from the CT scans. The 

structures determined by the panel were delineated on the planning CT of 14 patients 

with advanced head and ne ck cancer, and 3 RT techniques were compared: I) 

'standard 3D' (3D), 2) 'standard' IMRT (stIMRT), and 3) 'dysphagia specific' IMRT 

(dsIMRT). In the latter technique the cost function during optimization aimed at 

minimizing the doses to the swallowing structures. All techniques utilized RTOG 

IMRT protocol 0022 guidelines for target and critical structure doses. Because the 

lowest dose causing local VF-observed abnormalities in the previous UM study was 

50 Gy, the percentage volume of each structure receiving ~ 50 Gy (Vso) was the 

metric for comparison. Results showed that stIMRT significantly reduced Vso of the 

constrictors compared with 3D (mean 82% vs. 90%, respectively; p=0.02). 

Significant additional sparing was achieved by dsIMRT (mean 70.5%, P=O.OOI 

compared to stIMRT). The main benefit of dsIMRTwas in oropharyngeal cancers 

where Vso was reduced on ave rage by 23% compared with stIMRT. Similar findings 

were noted in the Vso ofthe supraglottic larynx and suporahyoid muscles. Target 

irradiation and parotid and glottic sparing were equivalent between stIMRT and 

dsIMRT; however, the dose fall-off outside the targets was steeper in the dsIMRT 

plans. dsIMRT is therefore a promising technique for the patients who are most 

likely to be affected by dysphagia problems; further study will be aimed at assessing 

the clinical relevanee of such sparing. 


Studies of radiation response in 

normal tissue 

De jaeger K, Seppenwoolde Y, 

Kampinga HM, Boersma LJ, 

Beiderbos jSA, Lebesque jv. Significanee 

of plasma transforming growth factor-f3 

levels for radiotherapy of non-small cell 

lung cancer. Int] Radiat Oncol Biol Phys 

(in press). 

De jaeger K, Hoogeman MS, 

Engelsman M, Seppenwoolde Y, 

Damen EM, Mijnheer Bj , Boersma Lj, 

Lebesque jv. Incorporating an improved 

dose-calculation algorithm in conformal 

radiotherapy oflung cancer: re-evaluation 

of dose in normallung tissue. Radiother 

Oncol 2003; 69:1-10. 

De jaeger K, Seppenwoolde Y, 

Boersma Lj, Muller SH, Baas P, 

Beiderbos jS, Lebesque jV. Pulmonary 

fonction following high-dose radiotherapy 

of non-small-celllung cancer. Int] Radiat 

Oncol Biol Phys 2003; 55:1331-4°. 

Seppenwoolde Y, Lebesque jV, 

De jaeger K, Beiderbos jS, Boersma LJ, 

Schilstra C, et al. Comparing different 

NTCP models that predict the incidence of 

radiation pneumonitis. Int] Radiat Oncol 

Biol Phys 2003; 55724-35. 

STUDIES OF RADIATION RESPONSE IN NORMAL TISSUE 

jSA Beiderbos, Lj Boersma, EMF Damen, K De jaeger, W Heemsbergen, M Hoogeman, 

HH Kampinga 2 , Bj Mijnheer, K Pengel, M Rossi, Y Seppenwoolde, jV Lebesque 

Detailed knowledge of the radiation response of organs at risk, as a function of the 

3D dose distribution is essential for computer optimization of radiotherapy plans. 

These responses are being studied for a number of organs at risk. 

Lung: radiation pneumonitis and transforming growth factor-beta (TGF-tll) 

levels In dose-escalation studies of radiotherapy (RT) for non-small celllung cancer 

(NSCLC), radiation pneumonitis (RP) is the most important dose-limiting 

complication. TGF-~I has been reported to be associated with the incidence ofRP. It 

has been proposed that serial measurements of plasma TGF-~I during treatment can 

be valuable for estimating the risk of RP and for decideing whether further doseescalation 

can be safely applied. To test this hypothesis, the time course ofTGF-~I 

levels was evaluated in patients irradiated for NSCLC in relation to the development 

of RP and dose-volume parameters. 

Plasma samples were obtained in 68 patients irradiated for NSCLC before and at 4, 

6 and 18 weeks after the start of RT. Plasma TGF-~I levels were deterrnined using a 

bio-assay based on TGF-~I-induced plasminogen activator inhibitor-I expres sion in 

2 Department of Radiation and Stress (eli Biology, University of Groningen, Groningen, 

The Netherlands.


Intensity-modulated radiotherapy (IMRT) 

Beiderbos jS, De jaeger K, 

Heemsbergen WD, Seppenwoolde Y, 

Baas P, Boersma LJ, Lebesque jV. First 

results of a phase IjIl dose escalation trial 

in non-small celllung cancer using threedimensional 

conformal radiotherapy. 

Radiother Oncol2oo3; 66:119-26. 

Koper PCM, Heemsbergen WD, 

Hoogeman M, jansen PP, Hart AAM, 

Wijnmaaien Aj, Van Os M, Boersma LJ, 

Lebesque jV, Levendag P. Impact of 

volume and location of irradiatied rectum 

wall on rectal blood loss aJter radiotherapy 

of prostate cancer. Int] Radiat Oncol Biol 

Phys (in press). 

mink lung cells. Chest CT-scans we re made of all patients prior to RT and repeated 

at 18 weeks post-RT. CT data we re used to calculate the mean lung dose (MLD) and 

to score radiation-induced radiological changes. RP was defined either based on the 

presence of these radiological changes, or based on clinical symptoms. Symptomatic 

RP was scored according to the CTC criteria (~grade I) and the SWOG criteria 

(~grade 2). 

Pre-RT TGF-~I levels were increased as compared to healthy individuals. On average, 

the TGF-~I levels normalized towards the end oftreatment and remained stabie 

until 18 weeks af ter RT. In 29 patients, however, TG F-~I increased at the end of RT. 

The multivariable analyses revealed that the MLD was the only variabie significantly 

correlated with the risk ofboth radiographic RP and symptomatic RP. The TGF-~I 

level at the end of radiation treatment was significantly associated with the MLD 

(p20 Gy (V20), the average difference was 12%. For both 

parameters, a strong correlation was found between the EPL and CS algorithms 

yielding a straightforward conversion procedure. Re-evaluation of the dose-effect 

relations showed that lung complications occur at a 12-14% lower dose. The values of 

the TDso parameter for local perfusion reduction and radiation pneumonitis 

changed from 60.S Gy and 34.1 Gy to Sr.I Gy and 29.2 Gy, respectively. 

00 L TORA 

T 

E Y (I 

SA Beiderbos, Lj Boersma, R De Boer, JJP De Goede, K De jaeger, WD Heemsbergen, P Koper 3 , 

M Hoogeman, EA Lamers, T Nuver, C Raseh, P Remeijer, M Van Herk, HP Vijlbrief, jV Lebesque 

In 2003, there was a significant increase in the number of 'high dose high precision' 

conformal radiation treatments. This number increased from 300 (2002) to 400 

patients in 2003. This increase can be mainly attributed to the number of patients 

treated for lung ca neer (+4S) and for head & neck cancer (+30). In the near future we 

expect a further increase in the number of treatments, due to the ongoing expansion 

of the treatment capacity at our department. 

Prostate ca neer The recruitment phase of the dose escalation trial (68 Gy vs. 78 Gy) 

was ended in January 2003. In total669 patients have been included, balanced 

over the treatment arms. Statistical analysis of acute and late toxicity have been 

performed (see section studies of radiation response in normal tissue). Evaluation 

of the main endpoints (survival, freedom from failure) is planned for the second half 

of2004· 

3 Erasumus MC- Daniel den Hoed Cancer Center, Rotterdam, The Netherlands.

____________________________________________________________ 4---------------~:'~~~l.~I~:f~.~.~~.'l~ ____ ~ 

Lung cancer Based on a phase I study performed we have developed a protocol 

for a phase III multi-center trial of conventional versus high-dose radiotherapy 

for inoperable stage I non-small celllung cancer (67.5 Gy vs. 87.75 Gy). 

Permis sion for this study has been obtained and the trial is expected to start in 

January 2004- We aim at the inclusion of 340 patients within 5 years (annual 

accrual rate of70-85 patients), balanced over the treatrnent arms. The primary 

endpoint of interest is the competing risk cumulative incidence of 'treatrnent 

failure' within 2 years. 

BREAST CANCER 

N Antonini, LBoersma, J Cho, E Damen, AAM Hart, H Jones, B Kreike, B Mijnheer, N Russeli, 

L Van 't Veer, M Van De Vijver, C Vrieling, H Bartelink 


Breast cancer 

Cho Bq, Mijnheer BJ, Bartelink H. 

Determining optimal two-beam axial 

orientations Jor left-sided breast cancer 

patients. Med Phys 2003 (in press). 

Cho BJC, Schwarz M, Mijnheer Bj. 

Bartelink H. Simplified intensity modulated 

radiotherapy using predefined segments to 

reduce cardiac. Radiother Oncol 2003 

(in press). 

The previous published boost versus no boost trial, which included more than 

5000 patients were included, has been updated. These early stage breast cancer 

patients all underwent breast-conserving therapy and were randomized between no 

boost and 16 Gy boost. A detailed analysis was performed to investigate prognostic 

factors that could explain the higher local recurrence rate in young patients. 

However, none of the known clinical and pathological prognostic factors could fully 

explain this age dependent relationship. To further investigate the age dependent 

association to the risk oflocal recurrence, a pioneering effort has began to search for 

a genetic profile with microarrays (in close collaboration with the Division XIII 

group). We believe that genomic information could be used for predicting which 

patients will benefit from breast conserving therapy or which patients will require 

radical mastectomy. 

Vrieling C, Collette L, Fourquet A, 

Hoogenraad WJ, Horiot JC, Jager Jj. 

Bing Oei S, Peterse HL, Pierart M, 

Poortmans PM, Struikmans H, 

Van den Bogaert W, Bartelink H. Can 

patient-, treatment- and pathology-related 

characteristics explain the high local 

recurrence rate Jollowing breast-conserving 

therapy in young patients? EurJ Cancer 

2003; 39:932-44. 

Breast I M RT The optimal fluence profile of a beam depends on the profiles of 

other beams but most optimizations assume fIXed beam orientations, a priori. 

A study was started to determine the optimal 2-beam orientation that best spares 

the heart for left-sided breast cancer patients. These patients were planned using 

a conformal (3DCRT) and a simplified intensity modulated (sIMRT) technique 

using predefined segments, and different 2-beam orientations. Optimal segment 

weights were determined by minimizing aquadratic objective cost function. It 

could be shown that optima13DCRT 2-beam orientations for the breast exist and 

they correspond to a hinge angle of approximately 188°, i.e. the clinically applied, 

almost opposing tangential medial and lateral beams. Optimal sIMRT 2-beam 

orientations for left-sided breast cancers are bimodal, containing hinge angles 

around 160° and 210°. IMRT techniques are less sensitive to beam orientation 

compared to uniform intensity beam techniques, and re sult in significantly 

improved heart sparing, but at a cost of slightly compromised PTV coverage. 

In a second study we quantified the benefits of the simplified IMRT solution by 

comparing it with a conformal and fun fluence IMRT approach, for clinical and 

optimal beam orientations (the latter obtained from the former study). In a group 

ofleft-sided breast cancer patients having a maximum heart distance of at least 

2.0 cm, plans were made using different conformal and IMRT techniques, as weIl 

as different beam orientations. Dose-volume histograms and radiobiologie 

modeling were used for plan evaluation. For a given beam orientation, fun 

fluence IMRT plans are the best and conformal plans are the worst. The dose 

distributions are better conformed around the PTV with more optimal beam 

orientations, resulting in better sparing of adjacent organs at risk. For clinica12-beam 

orientations, significant heart sparing is possible with the addition of intensity 

modulation but at the expense of worsening target coverage. Simplified IMRT can, 

for all intents, be substituted for full IMRT with clinical beam orientations. Applying 

more optimal IMRT beam orientations, as obtained from the former study, improves 

PTV coverage while maintaining significant he art sparing, but increases the PTV 

dose heterogeneity.


Mechanisms, modulation and prediction 

oJ radiation-induced cell death 

Haas RLM , Valdés Olmos RA, 

Hoefnagel CA, Verheij M, De Jong D, 

Hart AAM, Bartelink H. Thallium-201- 

chloride scintigraphy in staging and 

monitoring radiotherapy response in 

Jollicular lymphoma patients. Radiother 

Oncol2003; 69:323-28. 

Haas RL, Poortmans P, De Jong D, 

Aleman BM, Dewit LG, Verheij M, 

Hart AA, Van Oers MH, Van der Hulst M, 

Baars JW, Bartelink H. High response rates 

and lasting remissions after low-dose 

involved field radiotherapy in indolent 

lymphomas. ] Gin Oncol2003; 21:2474-80. 

Ruiter GA, Zerp SF, Bartelink H, 

Van Blitterswijk WJ, Verheij M. Anti-cancer 

alkyl-Iysophospholipids inhibit the 

phosphatidylinositol 3-kinase-Aktj P KB 

survival pathway. Anticancer Drugs 2003; 

14:167-73. 

Van Blitterswijk W). Van der Luit A, 

Veldman R). Verheij M, Borst J. Ceramide: 

second messenger or modulator oJ 

membrane structure and dynamics? 

BiochemJ 2003; 369:199-211. 

Van der Luit AH , Verheij M, 

Van Blitterswijk WJ. Raft lipid metabolism 

in relation to alkyl-Iysophospholipid-induced 

apoptosis. In: Membrane Microdomain 

Signaling: Lipid Rafts in Biology and 

Medicine. Editor MP Mattson, Humana 

Press Inc 2003 (in press). 

Van der Luit AH, Budde M, Verheij M, 

Van Blitterswijk WJ. Different modes oJ 

intemalization oJ apoptotic alkyllysophospholipid 

and cell-rescuing 

lysophosphatidylcholine. Biochem ] 2003; 

374747-53· 

Veldman RJ, Zerp S, Van Blitterswijk WJ, 

Verheij M. N-hexanoyl-sphingomyelin 

potentiates in vitro doxorubicin cytotoxicity 

by enhancing its cellular inftux. BritishJ 

Cancer 2003 (in press). 

MECHANISMS, MODULATION AND PREDICTION OF 

RADIATION-INDUCED CELL DEATH 

D De Jong, R Haas, FJP Hoebers, JH Schellens, RA Valdés Olmos, WJ Van Blitterswijk, S Vink, 

SF Zerp, H Bartelink, M Verheij 

Our line of research continues to focus on (I) novel pharmacological strategies to 

increase the cytotoxic effect of radiation and (2) new endpoints to quantify and 

predict the efficacy of new combination therapies. A stepwise program of sequential 

in vitro, preclinical and clinical screening tests has been set up to identify novel 

promising drugs for combined modality treatment. 

Alkyl-Iysophospholipids (ALPs) ALP such as Perifosine) represent a flIst group of 

compounds that have become available for clinical use along this approach. These 

synthetic anti-tumor agents are known to accumulate in cellular membranes where 

they disturb the lipid microenvironment and interfere with lipid-mediated signaling. 

In combination with radiation, ALPs cause a synergistic apoptotic effect and reduce 

clonogenic cell survival. Besides these radiosensitizing properties, ALPs show potent 

anti-angiogenic effects in vitro. In two other projects (in collaboration with W Van 

Blitterswijk, Division 111) we investigate mechanisms of cellular ALP uptake and 

transport, and address the functional implications of an altered membrane lipid 

composition on cellular drug uptake and radiosensitivity. 

Following their in vitro characterization, ALPs were subsequently tested in vivo. In 

two different mouse xenograft tumor models (Ä43I and HNXOE) the stability, 

biodistribution and tumor up take of Perifosine was determined. Following oral 

administration no significant degradation of this ALP was measured. Maximal 

plasma concentrations were reached between 8 and 16 hours, and clinically relevant 

intratumoral drug concentrations were detected from 8 hours onwards, reaching 

stabie levels after 48 hours. In 2002, a clinical phase I study was initiated, testing the 

feasibility and tolerability of combined modality treatment consisting of oral 

administration of Perifosine and radiotherapy in patients with locally advanced 

inoperable solid tumors. Twenty-two patients were entered in this study. Based on 

the observed dose limiting toxicity (nausea and vomiting), the recommended dose for 

a subsequent phase 11 study is 150 mg/day. 

Prediction of tumor response In vivo apoptosis imaging. To evaluate whether 

treatment-induced apoptosis can be visualized in vivo and whether it predicts clinical 

outcome, we introduced a novel in vivo apoptosis imaging technique in collaboration 

with the department of Nuclear Medicine (Valdés Olmos, Division XIII) This 

99mTc-Annexin V scintigraphy (TAVS) is based on the exposure of 

phosphatidylserine (PS) at the outer leaflet of the plasma membrane lipid bilayer 

during the early phase of apoptosis_ The human endogenous protein annexin V has a 

high affmity for PS, providing a detection method for apoptotic celis in vitro and in 

vivo. As proof-of-principle, TAVS was perfonned in follicular lymphoma, an 

extremely radiation- and apoptosis-sensitive malignancy. These patients show a high 

response rate of 90% after low-dose (2X2 Gy) involved field irradiation. In 12 patients 

a baseline and 24 hours post-radiotherapy TAVS were perfonned. In addition, fine 

needle aspiration cytology (FNAC) was carried out before and after radiation for 

cytomorphological verification. FNAC confrrmed the apoptotic nature of the response 

in all patients. In n/I2 patients the FNAC and TAVS correlated with the type and 

ons et of the clinical response. Whether TA VS can be used to monitor treatmentinduced 

apoptosis in vivo in solid tumors (NSCLC, SCLC, HNSCC, breast cancer) 

as welI, is under investigation. 

Cisplatin-ONA adducts The amount of cisplatin-DNA adducts has been shown to 

correlate with treatment outcome in NSCLC and may provide a valuable tooI to 

select patients for cisplatin-based combined modality treatment. In collaboration with 

the Division VIII (Begg and Schellens) we measure levels of cisplatin-DNA adducts 

in tumor and nonnal tissue obtained from patients with stage IV head and neck 

cancer treated by (conventional i.v or supradose i.a) cisplatin-based chemoradiation 

(RADPLAT). So far, adduct levels have been measured in 16 patients; 10 after i.V. 

and 6 after i.a. cisplatin infusion. N onnal tissue samples were obtained from all

16 patients; primary tumor from 10 patients with tumors. Cisplatin-DNA adduct 

levels in primary tumor of H&N cancer are 4-fold increased compared to leul

X DIVISION OF MEDICAL 

ONCOLOGY 

Division head, Group leader Sjoerd Rodenhuis 

Sjoerd Rodenhuis MD PhD Head 

Joke Baars MD PhD Academic staff 

Paul Baas MD PhD Academic staff 

Evert Bais MD Academic staff 

Jos Beijnen PhD Academic staff 

Willem Boogerd MD PhD Academic staff 

Henk Boot MD PhD Academic staff 

Annemieke Cats MD PhD Academic staff 

Jan Paul De Boer MD PhD Academic staff 

Bert De Gast MD PhD Academic staff 

Emine Göker MD PhD Academic staff 

John Haanen MD PhD Academic staff 

Helgi Helgason MD PhD Academic staff 

Alwin Huitema PhD Academic staff 

Martijn Kerst MD PhD Academic staff 

Sabine Linn MD PhD Academic staff 

Herman Neering MD PhD Academic staff 

Bastiaan Nuyen PhD Academic staff 

Rianne Oosterkamp MD PhD Academic staff 

Hilde Rosing PhD Academic stafff 

Jan Schellens MD PhD Academic staff 

Jan Schornagel MD PhD Academic staff 

Ank Sonnenberg MD PhD Academic staff 

Babs Taal MD PhD Academic staff 

Wim Ten Bokkei Huinink MD PhD 

Academic staff 

Jaap Van der Sande MD PhD Academic staff 

Marchien Van der Weide MD PhD 


Roei Van Gijn PhD Academic stafff 

Rianne Van Maanen PhD Academic staff 

Laurence Van Warmerdam MD PhD 


Nico Van Zandwijk MD PhD Academic staff 

Ans Vielvoye-Kerkmeer MD PhD Academic staff 

Hanneke Zuetenhorst MD PhD Academic staff 

Karin Mohrman Post-doc 

Natalie Appels Graduate student 

Jan Hendrik Beumer Graduate student 

Tessa Bosch Graduate student 

Elke Brouwers Graduate student 

Kristel Crommentuyn Graduate student 

Milly De Jonge Graduate student 

Monique Den Brok Graduate student 

Judith Engwegen Graduate student 

Suzanne Frankfort Graduate student 

Marie-Christine Gast Graduate student 

Markus Jörger Graduate student 

Bregt Kappelhof G raduate student 

Marleen Kemper Graduate student 

Marjolein Klous Graduate student 

Isa Kuppens Graduate student 

CU N ICAL PHARMACOLOGY 

Jan Schellens, Jos Beijnen, Wim Ten Bokkei Huinink, Paul Baas, Evert Bais, Sjoerd Rodenhuis, 

Jan Schornagel, Nico Van Zandwijk 

Main research interests The research program includes (i) optimizing oral 

bioavailability through temporary inhibition of drug transport proteins in the gut 

epithelium and drug metabolizing cytochrome P 450, (ii) phase land 

pharmacological studies, (iii) modeling of sparse pharmacokinetic and 

pharmacodynamic data using NONMEM and (iv) studies to unravel the metabolism 

of anticancer drugs. We also explore (v) the feasibility and safety of chemoradiotherapy 

using etherlipids as radiosensitizers. Furthermore, we develop and 

implement (vi) methods for pharmacogenotyping of patients. 

Ph ase I studies and drug scheduling The number of active studies has 

increased since last year. At present, 26 phase Ijll and pharmacologic studies are 

being conducted, of which 18 studies are open for accrual. One of the major 

translational research activities continues to be the development of an oral treatment 

schedule for paclitaxel and docetaxel in combination with oral cyclosporin A (CsA). 

In current studies we explore the feasibility of a capsule formulation to deliver oral 

paclitaxel. 

We have shown that gut epithelial BCRP plays an important role in the absorption of 

oral topotecan. We have shown that IOO mg of elacridar is sufficient to achieve a 

maximal increase in the apparent oral bioavailability of topotecan. Both drugs can be 

administered simultaneously, which greatly increases the practical feasibility of this 

oral strategy. This study is executed in collaboration with the University Medical 

Center Utrecht (head: professor EE Voest). A dose-escalation study of topotecan plus 

a fIXed dose of 100 mg elacridar has reached dose-limiting toxicity (DLT) at 2.5 mg 

topotecan at a daily 7( 5 schedule. The advised dose is 2.0 mg at this schedule. 

DLTs were leucocytopenia and neutropenia. 

We started a new study with oral docetaxel together with ritonavir with the aim to 

enhance the oral bioavailability of docetaxel by blockade of CYP3A4, the main 

cytochrome P 450 enzyme involved in metabolic degradation of docetaxel. The 

increase in bioavailability was low, which has resulted in an amendment of the 

protocol. We now test the oral dose of IOO mg docetaxel plus 600 mg of ritonavir. 

In the phase I study with the farnesyltransferase inhibitor zamestra (RII5777) in 

combination with cisplatin and gemcitabine, DLT was reached at the level of 

IOO mgjm 2 cisplatin day I, IOOO mgjm 2 gemcitabine days land 8 and zarnestra 

200 mg days 1-7, q three weeks. The main toxicities are myelosuppression, nausea 

and vomiting. A new pharmacological study with zarnestra was started in patients 

with mild or moderate liver dysfunction. Patient inclusion has continued af ter 

am en ding the protocol, which limited recruitment to patients with mild liver 

dysfunction as defined by a Child-Pough score lower than 7 and to patients with 

normalliver function. The toxicity in patients with moderate liver dysfunction was 

too pronounced to continue with this patient group. 

The two-center phase land pharmacologic study of oral CPTII plus capecitabine 

daily7(I4 was completed. Toxicities observed we re mainly diarrhea and nausea. 

The randomized phase I study with weekly or two-weekly gemcitabine and cisplatin 

in patients with NSCLC has recruited to date 94 patients. The dose-intensity is 

highest in the two-weekly schedule and was reached at 105 mgjm 2 of cisplatin plus 

15°0 mgjm 2 of gemcitabine. The dose-intensity of cisplatin is 50% higher than that 

in the standard schedule of this combination. In the reversed order of administration 

of cisplatin and gemcitabine, both given on one day, DLT was reached at 97.5 mgjm 2 

cisplatin and 15°0 mgjm 2 gemcitabine and consisted of hearing loss due to cisplatin. 

Currently, we explore the safety of the extended cohort of 90 mgjm 2 cisplatin and 

15°0 mgjm 2 gemcitabine. 

The study to unravel novel metabolites ofE7070 is ongoing. We have identified new 

metabolites in the mass balance study with 14C-labeled E7070. One of the major

pathways is glucuronidation. Also, we have identified that hydroxylation results in 

new metabolites. 

The phase I study with Kahalalide F (KF), a novel dehydroaminobutyric acidcontaining 

peptide isolated from the Hawaiian herbivorous marine species of 

mollusk Elysia rufescens, in androgen resistant pro state cancer is ongoing. Twentyone 

patients have been recruited thus far and doses could be increased rapidly from 

20 mgjm 2 jday on a daily1(5 schedule q three weeks to 930 mgjm 2 jday. At this level, 

the D LT was encountered, which consisted of reversible grade 4 transaminase 

increase. PSA marker reduction was seen in four patients coinciding with 

meaningful clinical improvement in one patient during six months. Currently, 

the activity of KF is explored at the level of 560 Jlgjm 2 jdaY*5, q day 21. The level of 

7°° Jlgjm 2 jdaY*5 turned out to be too high as more than one-third of the patients 

developed temporary CTC grade 3-4liver dysfunction. 

The combination study of carboplatin and topotecan is still ongoing. Tumor biopsies 

have been collected in 24 out of 26 recruited patients to measure platinum-DNA 

adducts and catalytic activity and expres sion of topoisomerase I. The DLT is 

myelosuppression. 

A two-center phase I study with a new polymer platinum compound AP5346 was 

started. Doses were rapidly increased from the starting dose-Ievel of 40 mgjm 2 to 

160 mgjm 2 • One allergie reaction was observed at the level Of160 mgjm 2 • 

The phase I study in advanced breast cancer employing the combination of oral 

vinorelbine on days one and eight and cyclophosphamide on day 1-14 was closed 

for recruitrnent after the DLT had been reached. The DLT is myelosuppression. 

Overall, oral tolerability is good. 

The phase I study with the combination of the radiosensitizer perifosine and 

radiation is ongoing. Perifosine is an ether lipid previously explored by us in a phase 

I study. The dose was increased to 200 mg daily during radiotherapy, but needed to 

be reduced to 150 mg per day in combination with standard dosed radiation due to 

DLT. The DLT was nausea and vomiting. 

A mass balance study was performed with the marine derived anticancer drug 

ET743 (trabectamin). In total8 patients were recruited. Total recovery of radioactivity 

varied significantly between patients and was 35-85%. Currently, we explore whether 

metabolites can be identified. We also performed a mass balanee study with the novel 

antimitotic agent BMS247550, which is an epothilone B derivative. Recovery reached 

80-85% of the iv administered dose and the current analysis is focused on 

identification of metabolites. 

We started a new phase I study with the combination of the EG FRl and 2 antagonist 

GW572016 in combination with oxaliplatin-gemcitabine (FOLFOX-4). Main toxicities 

associated with the addition of GW572016 to this standard combination consist of 

mild to moderate diarrhea and nausea. GW572016 is adrninistered daily in a capsule 

formulation in this two center study together with the Academie Medical Center, 

Amsterdam (head: professor DJ Richel). 

We perform a phase I study with the combination of the cell cycle inhibitor 

E7070 plus capecitabine. E7070 is given iv as a short infusion every 3 weeks and 

capecitabine orally days 1-14, q 3 weeks. The combination is weU tolerated. Nine 

patients were recruited. 

We started a three-center phase I study with the CDK2 inhibitor CYC2020 in 

combination with standard cisplatin-gemcitabine in patients with advanced nonsmall 

ceUlung cancer. In total 6 patients were recruited in three centers. Main 

toxicities thus far were grade 3 transaminase, alkaline phosphatase and GGT 

elevation. The current dose-Ievel is 800 mg of CYC202 taken orally as capsules. 

Wandena Lakhai Graduate student 

Jeany Rademaker-Lakhai Graduate student 

Liesbeth Rook Graduate student 

Linda Silvertand Graduate student 

Ellen Stokvis Graduate student 

Sabien Van der Schoot Graduate student 

Anthe Zandvliet Graduate student 

Carolien Alderen Technical staff 

Esther Boerhorst Technical staff 

Michel Hillebrand Technical staff 

Ciska Koopman-Kroon Technical staff 

Lianda Nan-Offeringa Technical staff 

Mariët Ouwehand Technical staff 

Bas Thijssen Technical staff 

Mathijs Tibben Technical staff 

Selma Van Dam Technical staff 

Annelies Boekhout Nurse practitoner 

Ria Dubbelman Nurse practitioner 

Joke Foekema Nurse practitioner 

Marjo HoJtkamp Nurse practitioner 

Marianne Keessen Nurse practitioner 

Cora Kfaasse Bos Nurse practitoner 

Henk Mallo Nurse practitioner 

Annemarie Nol Nurse practitioner 

Margaret Schot Nurse practitioner 

Martha Swart Nurse practitioner 

Clinical proteomies Prospective studies were initiated in patients with colorectal 

cancer, gastric cancer and breast cancer who are being treated with standard 

chemotherapy. The aim is to explore correlations between the proteomics proflle 

prior to start of therapy and treatrnent response in these patient cohorts. Colorectal 

cancer patients may be treated oxaliplatin plus capecitabine or capecitabine alone, 

gastric cancer patients with the combination ECF (epirubicine, cisplatin, SFU) or 

ECC (capecitabine instead of SFU). Breast cancer patients may be treated with 

capecitabine in second or third line of therapy. Recruitrnent is rapid in all three 

studies. In total a minimum of 25 patients will be included per protocol.


Clinical Pharmacology 

Callies S, De Alwis DP, Harris A, Vasey P, 

Beijnen JH, Schellens JH, Burgess M, 

Aarons L. A population pharmacokinetic 

model for paclitaxel in the presence of a 

novel P-gp modulator, Zosuquidar 

Trihydrochloride (L Y335979) . Br ] Clin 

PharmacoI2oo3;56:46-S6. 

Crul M, Schoemaker NE, Pluim D, 

Maliepaard M, Underberg RW, Schot M, 

Sparidans RW, Baas P, Beijnen JH, 

Van Zandwijk N, Schellens JH . 

Randomized phase I clinical and 

pharmacologic study of weekly versus 

twice-weekly dose-intensive cisplatin and 

gemcitabine in patients with advanced 

non-small celllung cancer. Clin Cancer 

Res 20°39(10, pt 1):3526-33. 

Schellens JHM, Planting ASTh, 

Van Zandwijk N, Ma J, Maliepaard M, 

Van der Burg MEL, De Boer-Dennert M, 

Van der Gaast A, Van den Bent Mj. 

Verweij J. Adaptive intrapatient doseescalation 

of Cisplatin in combination 

with low dose VP16 in patients with nonsmall 

celllung cancer. Br ] Cancer 2003; 

88:814-21. 

Schoemaker NE, Rosing H, Jansen S, 

Schellens JHM, Beijnen JH. High 

Performance Liquid Chromatographic 

Analysis ofthe Anticancer Drug lrinotecan 

(CPT-11) and lts Active Metabolite SN-38 

in Human Plasma. Ther Drug Monitoring 

2003; 25:120-4. 

Retrospective studies were initiated in patients with breast cancer, gastric cancer and 

non-smalllung cancer who were treated with oral paclitaxel as single agent in first or 

second line of therapy. 

Clinical Pharmacogenetics We continued this research line with the aim to 

determine the genetic profile of patients for drug oxidizing and conjugating enzyme 

systems and drug transporters. The genetic profile will be correlated with exposure 

levels of anticancer drugs and toxicity and efficacy parameters in patients. This 

approach may identify patients at risk for severe overexposure or undertreatment. We 

currently determine single nucleotide polymorphisms (SNPs) of drug metabolizing 

and transporting systems using RT-PCR and sequencing assays. In total more than 

45 SNPs can routinely be identified in the CYP's 2D6, 3A4, 2C9, 2C19 and in UGT, 

GSH, DPD, P-gp, BCRP and MRP2. This methodology is currently being applied in 

two multicenter clinical trials in which patients are treated with docetaxel or CPTII 

and in two'single center studies in patients with advanced colorectal cancer treated 

with oxaliplatin/capecitabine and gastric cancer treated with ECC/ECF. 

PHARMACY 

Jos Beijnen, Jan Schellens 

The research programs of the Department of Pharmacy & Pharmacology comprise 

the pharmaceutical formulation and drug level monitoring of (investigational) 

anticancer drugs to support pre clinical (see Division XIII, in collaboration with Olaf 

van Tellingen) and clinical pharmacologic research. New initiatives have been started 

in the fields of onco-proteomics (in collaboration with the departrnent of Clinical 

Chemistry, Hans Bonfrer) and pharmacogenetics (in collaboration with the 

departrnent of Molecular Biology, Slotervaart Hospital, Paul Smits and Utrecht 

University, Irma Meijerman). Research is conducted in the setting of the foundation 

NLADF (established in 1990) which is a collaboration between The Nethedands 

Cancer Institute and Slotervaart Hospital. Drug formulation and bioanalytical studies 

are covered by our GMP (Good Manufacturing Practice) and GLP (Good Laboratory 

Practice) licenses, respectively. In 2003 an ISO certification (9°01:2000) was 

acquired in the scope: purchasing, storage and distribution of drugs for world-wide 

clinical investigations (Good Distribution Practice "GDP"). 

Formulation Formulation research with the following investigational anticancer 

agents has been performed over the past year: AP-5346, C13II, E09 (Eoquin TM), 

epothilone D, ES-285.HCl, imexon, kahalalide F, the sacidins PMOOI04 and 

PMOII69, and the campthotecin-derivatives II24 and 417. Furthermore, an oral 

dosage form of thalidomide has been developed and manufactured. The poody watersolubie 

marine compound ES-285.HCI has been formulated as a lyophilized inclusion 

complex with 2-hydroxypropyl-fS-cyclodextrin (HPfSCD) to be reconstituted and further 

diluted in an infusion fluid before intravenous administration. Characterization of the 

ES-285.HCI-HPfSCD complex was performed using differential scanning calorimetry, 

X-ray diffraction analysis, and various solid state 13C nuclear magnetic resonance 

(CP IMAS, HP IDEC) techniques. ES-285.HCI has recently entered phase I clinical 

trials. On the analogy of AP-5280, the first hydroxypropylmethacrylamide (HPMA) 

polymer bound platinum compound formulated at our departrnent, a lyophilized 

intravenous formulation of AP-5346 was developed. A set of analytical test methods 

were developed for the quality control of the active drug substance and final product. 

Clinical trials with AP-5346 are currently ongoing in France and in the institute. The 

bladder instillation of Eoquin TM, formulated and manufactured at our site has shown 

promising results in the phase I clinical study in the UK. Phase 11 studies will be 

initiated. The macrolide microtubulin inhibitor epothilone D was re-formulated in 

order to avoid the use of Cremophor EL, which is present in the current dosage form. 

A prototype-formulation composed of HPfSCD, ethanol, and polysorbate 80 was 

developed using design-of-experiment modelling. Freeze-drying from t-butanol was 

applied in order to stabilize the compound. Lyophilized produets were also developed 

for the highly unstable compounds C13II and imexon. Phase I clinical studies ofboth 

compounds will be initiated the first quarter of 2004.

__________________________________________________________________-r----------~I~I~~(.~I 

I , ' 

.1..:. 

~~l.~~.~Ir'A .• ~~ 

____ 

~ 

Bioanalysis and clinical pharmacology Bioanalytical services are provided for 

numerous preclinical and clinical studies in-and outside the Institute. This concerns 

the following (investigational) anticancer drugs and metabolites: platinum 

compounds (cisplatin, carboplatin, oxaliplatin, AP-5280, AP-5346), topoisomerase-I 

inhibitors (irinotecan/SN-38, topotecan, DRF-r644), taxoids (paclitaxel, docetaxel), 

marine compounds (trabectedin, ES-285, kahalalide F, aplidine), fludarabine, 

gemcitabine (+ dFdU), cyclophosphamide (+ 4-hydroxycyclophosphamide, 

2-dechloroethylcyclophosphamide), thiotepa (+TEPA), D-2485r, E7070 (+Mr), 

perifosine, melphalan, lonafarnib. 

We have adapted our LC/MS/MS assay for trabectedin (ET-743) by developing an 

automated, on-line sample concentration and column switching procedure. This has 

reduced costs and the time needed for sample pretreatrnent of the method 

substantiaIly. 

To support a phase I study with ES-285 ((2S,3R)-2-amino-3-hydroxy-octadecane), a 

novel, investigational marine anticancer agent pharmaceutically developed and 

formulated by us, it was necessary to increase the sensitivity of the available 

LC/MS/MS assay ten-fold (lower limit of quantitation: IQ ngjmL 1t r ng/mL). This 

could be achieved by using a more sensitive MS detection system. With this change it 

became possible to monitor ES-285 levels during several hours af ter intravenous 

drug administration, in plasma of patients treated with the starting dose (4 mg/m 2 ) 

of the phase I trial. 

Elacridar (GFr209r8), the potent P-gp and BCRP blocking agent, is analyzed by an 

LC/MS/MS method developed in our laboratory. The assay has been successfully 

applied to support clinical studies where the drug was tested to enhance the oral 

bioavailability of topotecan. 

Kahalalide F is analyzed by LC/MS/MS. When a stabie isotopically labeled internal 

standard became available we could further increase the accuracy of the assay. This 

method exemplifies the utility of a stabie isotopically labeled analogue of the analyte 

as an intern al standard in quantitative LC/MS/MS technology. 

Mass balance studies utilizing carbon-r4labelled drugs were performed for 

trabectidin (ET-743; Yondelis®) and the synthetic epothilone BMS-247550. Af ter 

intravenous administration, blood samples were taken and urine and faeces were 

collected for the determination of total radioactivity using liquid scintillation 

detectioh and for measurements of the levels of unchanged drug using high 

performance liquid chromatography (HPLC) equipped with tandem MS detection or 

ultraviolet detection. Thereafter mass balance was calculated for these experimental 

drugs. Only nanoCi levels of radioactivity could be used in the epothilone BMS- 

247550 because the drug is not stabie after incorporation of higher radioactivity. 

Samples for low level radioactivity measurements were send to the UK to be analyzed 

by the sophisticated, ultrasensitive Accelerator Mass Spectrometry (AMS) technology. 

AMS is a nuclear physics technique that uses the high energy of a tandem Van de 

Graaff accelerator to measure very small quantities oflong-lived isotopes. 

Radioactivity recovery oflabeled BMS-247559 was between 80 and 85%. The 

metabolic fate of trabectidin was investigated, leading to the discovery of a number of 

metabolites including ET-745 and ET-73I. 

We further implemented pharmacokinetically guided adaptive dosing strategies in 

high dose chemotherapy for cyclophosphamide, thiotepa and carboplatin (CTC) as 

weIl as for paclitaxel. Limited sampling strategies are being developed improving 

the feasibility of pharmacokinetically guided dosing in the CTC regimen. To refine 

dose individualizations for cyclophosphamide, we further focused on describing 

the pharmacokinetics ofboth its active and inactive metabolites 

4-hydroxycyclophosphamide, 2-dechloroethylcyclophosphamide and phosphoramide 

mustard. Moreover, a method of analysis is being developed for the determination of 

cyclophosphamide and five ofits metabolites in a single analytical LC/MS/MS run. 

The assay for cyclophosphamide also appeared useful for the simultaneous 

quantification of fludarabine. A combined LC/MS/MS assay for fludarabine and 

cyclophosphamide has been fully validated in compliance with recent FDA 

guidelines. 

At the request of the EORTC, an LC/MS/MS method has been set up and validated 

for the analysis of the new farnesyl transferase inhibitor, lonafarnib, in biological 

matrices. 


Pharmacy 

Crul M, Van Waardenburg RC, Bocxe S, 

Van Eijndhoven MA, Pluim 0, Beijnen JH, 

Schellens JHM. DNA repair mechanisms 

involved in gemcitabine cytotoxicity and in 

the interaction between gemcitabine and 

cisplatin. Biochem Pharmacol2003; 

6p75-82. 

De Jonge ME, Mathot RAA, Van Dam SM, 

Rodenhuis S, Beijnen JH. Sorption of 

Thiotepa to polyurethane catheter causes 

falsely elevated plasma levels. Ther Drug 

Monit 2003; 25:261-3. 

Kemper EM, Van Zandbergen AE, 

Cleypool C, Mos HA, Boogerd W, 

Beijnen JH, Van Tellingen 0. Increased 

penetration of Paclitaxel into the brain by 

inhibition of p-glycoprotein. Clin Cancer 

Res 2003; 9:2849-55. 

Stokvis E, Nan-Offeringa L, Rosing H, 

Lopez-Lazaro L, Acena JL, Miranda E, 

Lyubimov A, Levine BS, D'Aleo C, 

Schellens JHM, Beijnen JH. Quantitative 

analysis of ES-28 5, an investigational 

marine anticancer drug, in human, mouse, 

rat, and dog plasma using coupled liquid 

chromatography and tandem mass 

spectrometry.] Mass Spectrom 2003; 

38:548-54. 

Van Tellingen 0, Buckle T, Jonker JW, 

Van Der Valk MA, Beijnen JH . 

P-glycoprotein and Mrp1 collectively protect 

the bone marrow from vincristine-induced 

toxicity in vivo. Br] Cancer 2003; 

89:1776-82. 

Bouma M, Nuijen B, Jansen MT, Sava G, 

Picotti F, Flaibani A, Bult A, Beijnen JH. 

Development of a IC method for 

pharmaceutical quality con trol of the 

antimetastatic ruthenium complex 

NAMI-A] Pharm BiomedAnal2003; 

31:215-28.

Bioanalytical services have been provided to the groups of Alfred Schinkel 

(midazolam and cyclosporin measurements in cytochrome P450 3A transgenic 

mice), Jan Scheliens (analysis ofbenzimidazoles) and Piet Borst (morphine, 

morphine-3-glucuronide and morphine-6-glucuronide measurements in MRPs 

transfected celi modeIs). Morphine and its glucuronide-metabolites were quantified 

bya novel LC/MS/MS method developed in our laboratory. 

A new sensitive LC/MS/MS assay for docetaxel has been developed. High sensitivity 

is required for docetaxel monitoring in a clinical trial where the drug is given in low 

dosages in combination with the cytochrome P 450 3A4 inhibitor ritonavir. 

We have developed a method to analyze prenylated Ras proteins in complex 

biological matrices. Celllysates are fractionated on a CI8 reversed phase HPLC 

column. Upon tryptic digestion of the Ras protein containing fraction, prenylated 

C-terminal endpeptides are quantified with LC/MS/MS. Reference peptides, also 

stabIe isotopically labeled peptides to be used for calibration and as internal 

standards, have been synthesized for this project by Henk Hilkmann. First results 

show that this assay can be used and is sensitive enough for the detection of 

endogenous famesylated H-Ras in tumor tissue, buccal mucosa cells and white blood 

cells. Gradient HPLC-UV was used to determine enzyme kinetics of purified 

farnesyltransferase in vitro. IC so of the famesyl transferase inhibitor L788,I23 

was 40 nM. 

A novel field in our laboratory is the pharmacogenetic screening of Single 

Nucleotide Polymorphisms (SNPs) with re gard to phase I metabolizing enzymes 

(CYP2D6, CYP3A4, CYP3A5, CYP2C9 and CYP2CI9), phase Ir metabolizing 

enzymes (UGTIAI, DPD and GST) and drug transporters (MDRI, MRP2 and BCRP) 

employing PCR-RFLP and sequencing assays. One hundred healthy volunteers have 

been extensively screened for these SNPs to determine their frequencies and to 

discover new ones. The assays are also used in clinical pharmacological research with 

docetaxel and irinotecan, to correlate the pharmacokinetics and pharmacodynamics 

of these drugs with the genotype for these enzymes. 

Our laboratory equipment has been extended with two new mass spectrometers 

(SELDI-TOF-MS and ICP-MS). SELDI-TOF-MS, Surface Enhanced Laser 

Desorption/lonization Time-Of-Flight Mass Spectrometry is a novel, fully integrated 

platform combining ProteinChip array-technology for protein capture coupled with a 

sensitive linear TOF MS analyzer and bioinformatics software for complex protein 

profile recognition. So far we have analyzed serum samples from 140 breast cancer 

patients (stage I-lIl) and found distinguishable protein profiles in comparison with 

serum from healthy controls. In Figure X.I a typical example is depicted from a 

serum sample of a healthy volunteer and a patient with breast cancer showing the 

differences in protein profiles. Identification of proteins that emerge in patient 

samples is currently ongoing. One of our major goals in this project is to find protein 

biomarkers that could predict (chemo-) therapy outcome and could be used as a 

sensitive pharmacodynamic parameter. 

Î 

8939.9+H 

§ 

n 

t 

e 

n 

s 

i 

t 

Y § 

3971.6+H 

patient 

healthy 

~------------------------------------------~ 

M/Z~ 4000 6000 8000 10000 

Figure X.l: Proteins with mjz 8935 Da, 3972 Da and 3086 Da are present in serum ofbreast cancer 

patients but not in serum from healthy individuals.

IMMUNOTHERAPY 

Bert De Gast, John Haanen, Martijn Kerst, Willem Boogerd, Henk Mallo 

The aim of this program is to contribute to the development of effective 

immunotherapy in two immunogenic tumors: malignant melanoma and renal cell 

carcinoma. 

Metastatic melanoma. A phase 111 study of Temozolomide with or without 

immunotherapy in stage IV melanoma was stopped after inclusion of 80 patients 

because of non-availability of GM-CSF. Preliminary results do not show a difference 

in response between the two arms. A phase 11 study of neoadjuvant and adjuvant 

chemo-immunotherapy around surgery in stage 111 B/C melanoma was stopped for 

the same reason af ter the inclusion of 26 patients, but continued with chemotherapy 

only. The results of the 26 patients af ter a median follow-up of 26 months (range 

12-38 months) show a low relapse rate (8/26 = 31%) in the first year (versus about 

50% in 2 similar studies). Moreover, the biomarker SrooB, which was elevated in 

13/26 patients, predicted early relapse and correlated with decreased overall survival. 

As there was no relation of SrooB with tumor load, the marker may reflect the 

presence ofhematogenous dissemination. 

Renal ce" carcinoma (ReC). The phase 11 study ofpegylated Interferon-a 

(PEG-Intron®) in progressive metastatic RCC did not show a higher response rate 

(3/22; I CR, 2 PR) nor lower toxicity than standard IFNa. Temozolomide in IFNa 

resistant patients did not show a response (0/12). Continuous low dose IL-2 

(I MIU /m 2 for 6 weeks) with Thalidomide and concomitant radiotherapy of soft 

tissue or bone lesions as primary treatment also did not show a promising response 

rate (I PR, IS SD, 6 PD) and only short-term stabilization. Three patients who did not 

respond to cytokine-based biotherapy were included in a study of non-myeloablative 

allogeneic peripheral blood stem cell transplantation. Patients pres enting with an 

RCC and metastases (synchronie RCC metastases) were treated with IFNa to 

evaluate the response to IFNa prior to a possible nephrectomy: In case of progressive 

metastatic disease, the nephrectomy was cancelled. 

Selected publicatlons (continued) 

Immunology 

De Gast GC, Batchelor D, Kersten MJ, 

Vyth Dreese FA, Sein J, 

Van de Kasteele WF, Nooijen WJ, 

Nieweg OE , De Waal MA, Boogerd W. 

Temzolomide followed by combined 

immunotherapy with CM-CSF, low dos 

IL-2 and IFNa in patients with metastatic 

melanoma. BrJ Cancer 2003; 88:175-80. 

Verra N, Jansen R, Groenewegen G, 

Mallo H, Kersten MJ, Bex A, 

Vyth-Dreese FA, Sein j, 

Van de Kasteele WF, Nooijen WJ, 

De Waal M, Horenbias S, De Gast Gc. 

Combined immunotherapy with 

subcutaneous CM-CSF, low dose IL-2 and 

IFNa in patients with progresive metastatic 

renal cell carcinoma. Brit] Cancer 200]: 

9:1346-51. 

Schrama JG, Rodenhuis S, De Gast Gc. 

Prolonged survival associated with early 

lymphocyte recovery after autologous 

hematopoietic stem celt transplantation for 

patients with metastatic breast cancer. 

Bone Marrow Transpl 2003; 31:141-2. 

Immunotherapy around nephrectomy. The study ofperi-operative 

immunotherapy was completed following inclusion of 25 patients. A maximum 

tolerated dose was determined and immunologie studies showed a strong increase of 

CD8+ T cells in the tumor with infiltration between tumor cells and an increased 

infiltration of dendritic cells (DC) . A subsequent study of pre-operative 

immunotherapy (d -10 through d -2) showed a stronger infiltration of CD8+ T cells 

that were TNFa positive, but alesser increase of De. One of the 5 patients, who had 

extensive lymph node metastases in mediastinum and supraclavicular, reached a 

complete remis sion that has now been maintained for more than 10 months. Studies 

are ongoing to detect anti-tumor T ceU immunity in the tumor infiltrating 

lymphocytes and in peripheral blood. 

Peptide vaccination in melanoma patients A phase I study of peptide vaccination 

(gpIOO, tyrosinase and MART-I) together with GM-CSF, tetanus toxoid as helper 

factor and Montanide ISA SI as adjuvant was started in HLA-A2.1 positive patients 

with stage IV melanoma, progressive af ter prior chemotherapy. Weekly 

subcutaneous vaccinations with peptide doses of 250 and 500 Ilg for 4 weeks we re 

non-toxie. No clinical responses were observed. Immunologie responses are being 

analyzed. 

AUTOLOGOUS HEMATOPOIETIC PROGENITOR CELL 

TRANSPLANTATION PROGRAM 

Sjoerd Rodenhuis, Joke Baars, Jos Beijnen, jan Paul De Boer, Milly De jonge, Marjo Holtkamp, 

Martijn Kerst, Jan Schel lens, Jan Schornagel, Jolanda Schrama 

The main objective of this program is to offer potentially curative therapy to patients 

with high-risk primary breast cancer and to patients with so-called oligometastatic


Autologous hematopoietic progenitor celt 

transplantation program 

Rodenhuis S, Bontenbal M, Beex LVAM, 

Wagstaff J, Richel DJ, Nooij MA, Voest EE, 

Hupperets P, Van Tinteren H, Peterse HL, 

TenVergert EM , De Vries EGE. High-dose 

chemotherapy with hematopoietic stem celt 

rescue in high-risk breast cance r. N EnglJ 

Med 2003; 349:7-16. 

Weigeit B, Bosma AJ , Hart AAM, 

Rodenhuis S, Van 't Veer LJ. Marker genes 

for circulating tumour celts predict survival 

in metastasized breast cancer patients. 

BrJ Cancer 2003; 88:1091-4. 

Schrama JG , Holtkamp MJ, Baars JW, 

Schornagel J H, Rodenhuis S. Toxicity of 

the high-dose chemotherapy CTC regimen 

(cyclophosphamide, thiotepa, carboplatin): 

the Netherlands Cancer Institute 

experience. BrJ Cancer. 2003; 88:1831-8. 

Faneyte IF, Schrama JG , Peterse JL, 

Remijnse PL, Rodenhuis S, 

Van de Vijver MJ . Breast cancer response to 

neoadjuvant chemotherapy: predictive 

markers and relation with outcome. Br ] 

Cancer 2003; 88:406-12. 

disease. Oligometastatic disease is defined as stage IV disease, in which all tumor 

localizations could, in principle, be managed by local therapy such as surgery or 

radiation therapy. As in adjuvant chemotherapy, the high-dose chemotherapy serves 

to eradicate microscopie disease. 

High-risk primary breast cancer In 2003, the analysis ofthe Dutch national trial 

ofhigh-dose chemotherapy in the adjuvant treatment of high-risk breast cancer was 

analyzed and published. The trial shows a modest relapse-free survival advantage for 

patients who received high-dose chemotherapy. This advantage is, however, 

important and statistically significant in patients with HER-2/neu negative disease 

(Figure X.2). Other (smaller) randomized studies do not always show a relapse-free 

survival advantage for patients receiving high-dose chemotherapy, possibly because 

of high toxic death rates following transplantation. As a re sult, the role ofhigh-dose 

chemotherapy in breast cancer continues to be a controversial subject. Based on the 

Dutch findings, the indication for high-dose therapy for a small and highly selected 

group of patients with high-risk breast cancer has been recognized by both the Dutch 

Health Council and the Dutch Health Insurance Council, who had sponsored the 

trial. 

HO 

Subgroup 

events/N 

Age 

< 40 yrs 38/113 

40-50 yrs 85/234 

50+ yrs 26/95 

HER-2Jneu 

0 851300 

1+ 0/5 

2+ 7/ 10 

3+ 39/85 

Grade 

Grade I 10/78 

Grade 11 42/138 

Grade lil 85/184 

MAl 

= 15 72/150 

Overall result 149/442 

Conv 

events/N 

58/ 11 2 

84/23 1 

28/ 100 

113/282 

5/10 

5113 

37/96 

29176 

56/154 

77/182 

57/149 

42/1 19 

63/141 

170/443 

Subgroup estimates 99%, Overall 95% confidence intervals 

E 

• 

- 

• 

- 

• - 

- 

• 

- 

THORACIC ONCOLOGY 

Nico Van Zandwijk, Paul Baas, Sjaak Burgers, Ank Sonnenberg, Sabine Linn, Jan Schellens, 


Non-Small Cell lung Caneer (NSCLC) The dose intensification studies with the 

gemcitabinejcisplatin combination in patients with advanced and metastatic NSCLC 

were repeated using areverse schedule with cisplatin preceding gemcitabine every 

two weeks. As previously found with gemcitabine preceding cisplatin, the interval of 

two weeks allowed a doubling of the standard dose intensity of cisplatin suggesting 

that a 2-weekly schedule is to be preferred over a weekly or 3-weekly schedule. A 

phase IIA study combining gemcitabinejcisplatin with CYC202, an inhibitor of the 

CDK2jCyclin E complex, was initiated. A randomized study on the role of induction 

chemotherapy for operabie NSCLC patients was activated and a translational project 

on pre-operative therapy for NSCLC stage lIlA patients was accepted by the EORTC. 

This project entails micro-array analyses of tumor biopsies before and af ter 

sequential therapy with gemcitabinejcisplatin and ZD1839 (gefitinib, Iressa) and 

attempts to find reliable intermediate markers of response to gemcitabinejcisplatin 

andjor gefitinib. 

More than 160 patients with recurrent NSCLC received gefitinib in a daily dose of 

250 mg p.o. day as part of an expanded drug access program. Gefitinib is welltolerated 

and safe and is able to induce long-Iasting (> 2 years) responses in a 

subgroup of patients. Predictors of response and survival were histology 

(adenocarcinomajbronchioloalveolar carcinoma) and a non-smoking history. 

Small Cell Lung Ca neer (SCLC) The phase II study in patients with limited disease 

SCLC with carboplatin, etoposide, paclitaxel and concurrent radiotherapy was 

evaluated. The objective response rate was confirmed to be 95% and a median 

survival of more than 22 months was documented. A national study combining 

platinum containing chemotherapy with early radiotherapy has been activated as a 

phase I study on the combination of irinotecan and cisplatin given concurrently with 

radiotherapy in patients with extensive disease. 


Thoracic oneology 

Zimmermann A, Walt H, Haller U, 

Baas P, Klein S. Effects ofehlorin-mediated 

photodynamie therapy eombined with 

jluoropyrimidines in vitro and in apatient. 

Caneer Chemother Pharmaeol2003; 

51:147-154. 

Baas P, Ardizzoni A, Grossi F, 

Nackaerts K, Numico N, Van Marck E, 

Van de Vijver M, Monetti F, 

Smid-Geinaerdt, MJA, Van Zandwijk N, 

Debruynr C, Legrand C, Giaccone G. 

The aetivity of raltitrexed (Tomudee®) in 

malignant pleural mesothelioma: on 

EORTC phase IJ study (08992). EurJ 

Ca neer 2003; 39:353-357. 

Van Zandwijk N, Pastorino U. 

Chemoprevention oflung eaneer: soon daily 

practis? Expert Rev. Antieaneer Therapy 

2003; 3:91-8. 

Chemoprevention The randomized (phase IIB) chemoprevention study with 

inhalational fluticasone versus placebo in 'healthy' smokers was significantly 

expanded. Thirty-four volunteers with over 25 packjyears smoking histories were 

included. PS3 and hTERT expressions in bronchial biopsies taken before and at the 

end of the intervention will serve as intermediate markers to determine the 

chemopreventive effect of fluticasone. 

The first images with Optical Coherence Tomography were obtained and this project, 

which is conducted in conjunction with the Laser Centre of the Academie Medical 

Center of the Amsterdam University, is now weIl on its way. 

Malignant Mesothelioma To increase the diagnostic accuracy of thoracoscopical 

diagnosis of malignant pleur al mesothelioma (MPM) we have tested a photodynamic 

diagnosis system using s-Aminoleavulinic Acid (SAA) . Fourteen patients received 

SAA before thoracoscopic examination and a majority showed marked fluorescence 

of the pathological pleura (figure X.3 and X.4). Sensitivity and specificity analyses are 

currently in progress. 

Based on previous experience with Thalidomide in MPM a randomized phase III 

study was activated to evaluate the effect of Thalidomide in the maintenance setting. 

In this study, patients receive a platinumj Alimta induction regimen followed by 

Thalidomide. An institutional phase 11 study with imatinib was completed and the 

results will be available in 2004. Alimtajplatinum before pleuropneumonectomy 

followed by chest irradiation is being planned as a multi-institutional effort for 2004.


Gastroenterology 

ZuetenhorstJM, Taal BG . Carcinoid he art 

disease. N EnglJ Med 2003; 348:2359-61. 

Zuetenhorst JM, Bonfrer JM, Korse CM , 

Bakker R, Van Tinteren H, Taal BG. 

Ca rcinoid heart disease: the role of urinary 

5-hydroxyindoleacetic acid excretion and 

plasma levels of atrial natriuretic peptide, 

traniforming growth factor-beta and 

fibroblast growth factor. Cancer 2003; 

97:16°9-15. 

Kruijtzer CM, Boot H, Beijnen JH, 

Lochs HL, Parnis FX, Planting AS , 

Pelgrims JM, Williams R, Mathot RA, 

Rosing H, Schot ME , Van Tinteren H , 

Schellens JH. Weekly oral paclitaxel as 

first-line treatment in patients with 

advanced gastric cancer. Ann Onco12003; 

14:197-2°4. 

Figure K3: Image of diaphragm on the leJt site with adjacent lung (leJt lower lobe). 

Figure X+ Same image in PDD mode, the diaphragm shows severaljluorescence spots. Biopsies 

confirmed the diagnosis malignant mesothelioma. 

GASTROENTEROLOGY 

Annemieke Cats, Henk Boot, Babs Taal 

Gastric ca neer Surgieal reseetion of gastric and esophageal eaneer remains the 

eomerstone of eurative treatment of these malignancies. However, the long-term 

prognosis of these malignaneies remains po or in loeally advaneed disease. Reeently, 

MaeDonald et al. performed a randomized phase III study eomparing surgery alone 

with surgery and postoperative adjuvant therapy, eombining radiotherapy (45 Gy) 

with 5FU-Ieukovorin. A redueed risk of relapse and improved survival were observed. 

In order to improve the ehemotherapeutie regimen, we started a dose-finding 

phase I-Il study, using a fIXed radiotherapy regimen with esealating doses of cisplatin 

and eapecitabine, further exploring the feasibility and toxicity of ehemo-radiotherapy 

af ter gastric andjor esophageal reseetion. Beeause the influenee of radiotherapy on

the absorption of the orally administered drug capecitabine is not known, 

pharmacokinetics of capecitabine and metabolites are being studied as weIl. Up till 

now IO patients have been included in the study. Dose limiting toxicity has not been 

encountered yet. 

Co-administration of cyclosporin A (CsA), which acts as a P-glycoprotein and CYP-3A 

blocker, results in an 8-fold increase in the systemic exposure of oral paclitaxel. In a 

phase 11 study, chemonaive patients with advanced gastric cancer received oral 

paclitaxel and CsA once weekly. In 25 patients, toxicity was mainly gastrointestinal 

and manageable. CTC neutropenia grade 3-4 occurred in 5 patients (20%). In 24 

evaluable patients, 8 partial responses were observed, II patients had stabie disease 

and 5 patients showed progressive disease. The median time to progression was I6 

weeks. Therefore, oral paclitaxel in combination with CsA is both active and safe in 

chemonaive patients with advanced gastric cancer. 

Carci noid tu mou rs Heart failure, due to right-sided heart valve fibrosis, is an 

important cause of death in carcinoid patients. As expected, in 37 patients with 

carcinoid heart disease, the median level of urinary 5-hydroxyindoleacetic acid 

(5-HIAA) excretion between carcinoid diagnosis and cardiac ultrasonography 

correlated with the presence of carcinoid heart disease. However, a much stronger 

relation with the serotonin load over time (i.e., the area under the curve for urinary 

5-HIAA excretion during the interval) was observed. Transforming Growth Factor-~ 

(TGF-~) and Fibroblast Growth Factor (FGF) are both associated with pulmonary and 

renal fibrosis development. These factors, however, we re not associated with 

carcinoid heart disease in our patients. 

Hereditary non-polyposis colorectal cancer The adenoma-carcinoma sequence 

in hereditary non-polyposis colorectal cancer (HNPCC) is accelerated, but it remains 

unknown whether the mismatch-repair (MMR) defect also promotes the 

development of adenomas. Therefore, in a national multicenter study, the risk of 

developing colorectal adenoma and carcinoma between carriers and non-carriers 

(controls) was determined during colonoscopies performed for the purpose of 

surveillance. In 86 families with a known MMR-gene mutation (249 carriers, 

247 controls) the proportion of subjects free of an adenoma at the age of 60 years 

was 29.7% for carriers and 70.8% for controls (P

XI DIVISION OF 

SURGICAL ONCOLOGY 

Division head, Group leader Bin Kroon 

Board 

Bin BR Kroon MD PhD FRCS (Head) 

Fons JM Balm MD PhD FRCS FACS 

Omgo E Nieweg MD PhD 

General Surgical Oncology 

Bin BR Kroon MD PhD FRCS, Academic staff, 

Head Division 

Houke M Klomp MD Academic staff 

Omgo E Nieweg MD PhD Academic staff 

Hester SA Oldenburg MD PhD Academic staff 

Emiel JTh Rutgers MD PhD FRCS Academic staff 

Frits Van Coevorden MD PhD Academic staff 

Vic J Verwaai MD Academic staff 

Frans AN Zoetmulder MD PhD Academic staff 

Susanne H Estourgie MD Academic staff 

lan Faneyte MD Academic staff 

Reinie Kaas MD Academic staff 

Marten R Kapma Academic staff 

Eduard LAR Mutsaerts MD Academic staff 

Philip Meijnen MD Academic Staff 

Eva M Noorda MD Academic staff 

Marjan Piek-den Hartog MD Academic staff 

Fatih Polat MD Academic staff 

Leonie Smit MD Academic staff 

Johan C Van Mourik MD Academic staff 

Serge Van Ruth MD Academic staff 

Pieter S Verduijn MD Academic staff 

Bart C Vrouenraets MD PhD Fellow 

Jan Wijsman MD PhD Fellow 

Arjen J Witkamp MD Academic staff 

Yaïr Acherman Undergraduate student 

Lysette N Broekhuizen Undergraduate student 

Bart Takkenberg Undergraduate student 

Maart je C Van Rijk Undergraduate student 

Head and Neck Oncology and Surgery 

Frans JM Hilgers MD PhD Academic staff, Head 

Fons JM Balm MD PhD FRCS FACS 


Marcel P Copper MD PhD Academic staff 

Peter JFM Lohuis MD PhD Academic staff 

Ludi E Smeele MD DMD PhD Academic staff 

I Bing Tan MD PhD Academic staff 

Michiel MW Van den Brekel MD PhD 


Annemieke H AckerstaffPhD Academic staff 

Lucille Dorresteyn MD Academic staff 

Tom W Geurts MD Academic staff 

Steven Gonggrijp DOS Academic staff 

Pet ra Jongmans Academic staff 

Frans HM Kroon DOS PhD Academic staff 

HEAO ANO NECK ONCOLOGY ANO SURGERY 

Fons JM Balm, Michiel WM Van den Brekel, Marcel P (opper, Frans JM Hilgers, Peter JFM Lohuis, 

Ludi E Smeele, I Bing Tan 

The head and neck oncology group was expanded with three part-time head and neck 

surgeons enlarging clinical research capacity. Collaboration with the Ear, Nose and 

Throat department of the Academic Medical Center and the Institute of Phonetic 

Sciences of the University of Amsterdam has been intensified, so that new research 

activities on ototoxicity af ter chemoradiation, combined modality treatment in 

childhood rhabdomyosarcomas, pulmonary physiology after totallaryngectomy and 

postlaryngectomy voice characteristics could be implemented. Research on 

rehabilitation after totallaryngectomy in close collaboration with Division XII and on 

organ sparing treatment in collaboration with Divisions IX and X is ongoing. The 

research collaboration with Divisions VIII and VII is continued on the development 

ofhypoxic markers and development of a conditional knock-out mouse model using 

the Cre-lox system respectively. 

Rehabilitation Avoice prosthesis has been developed with a new valve mechanism, 

applying Candida-resistant fluoroplastic (Teflon-like) material for the valve and valve 

seat, and magnets to generate an active closing force, preventing inadvertent opening 

of the valve during swallowing or de ep inhalation (Figure XLI). Several prototypes 

were tested in 13 laryngectomized patients and, subsequently, the final design was 

assessed in a prospective clinical trial in a cohort of 18 patients with a short device 

lifetime of their standard indwelling voice prosthesis (mean 30 days). Prototype 

testing and the long-term clinical trial confirmed that the new valve material 

remained free of Candida growth and that the use of magnets can prevent 

inadvertent opening of the valve during swallowing andjor deep inhalation. This 

resulted in a highly significant increase in device lifetime in the 18 laryngectomized 

patients in the prospective trial (I4-fo1d increase on average, range 3-39-fold; 

p

valve se at and valve of fluoroplastic 

(teflon-like material) 

Heike J Nyst MD Academic staff 

Adriaan P Timmers DDS Academic staff 

Corina J Van As PhD Academic staff 

Guido B Van den Broek MD Academic staff 

Vincent LM Vander Poorten MD PhD 

Academie staff 

Charlotte L Zuur MD Academic staff 

J Karel Zuur MD Academic staff 

magnets 

Fig. XI.l: Provox ActiValve voice prosthesis for postlaryngectomy vocal rehabilitation. This device solves the 

problem of early leakage through the valve caused by excessive Candida growth and/or inadvertent 

opening of the valve by swallowing and inhalation-related underpressure in the esophagus. 

lesions). LOH analysis showed metastases in II cases, probable metastases in 3 cases, 

second primaries in 21 cases and a probable second primary in I case. In 2 cases 

conclusions could not be drawn. In 19 patients LOH supported the clinical criteria 

and in 17 cases it did not. Although LOH profiling cannot always offer certainty due 

to genetic imbalance, it seems a useful tooI to differentiate between metastases and 

second primaries. A great number of clinically assumed metastases appear to be 

second primaries rather than metastases on molecular biological grounds. 

Research on prognostic markers The work on prognostic factors in 92 patients 

treated with targeted chemoradiation for unresectable head and neck squamous cell 

carcinoma was continued. Local control and overall survival at five years for the 

whole group were 64% and 38%, respectively. Tumor volume (P= 0.01) and 

unilateral intra-arterial infusion (P=0.03) were predictive for local control. In a 

multivariate analysis, tumor volume (P= 0.001), co-morbidity (P=0.008), lowest 

involved neck node level (P=O.OI), male gender (P=0.02) and more than 10% weight 

loss (P=0.04) were significantly predictive for overall survival. Using tumor volume 

as a continuous variabie it was found that Icm3 increase of volume was correlated to 

2.8% decrease oflocal control (Figure XI.2). 

Urological Oncolog}' 

Simon Horenbias MD PhD FEBU Academie staff, 

Head 

Axel Bex MD PhD Academic staff 

Wim Meinhardt MD PhD Academic staff 

Henk G Van der Poel MD PhD Academie staff 

Anoesjka Claessen MD Fellow 

Bin K Kroon MD Academic staff 

Anne P Lont MD Academie staff 

Nader Naderi MD Fellow 

Jacco A Nieuwenhuijzen MD Academie staff 

Gynecological Oncolog}' 

Matthé PM Burger MD PhD Academie staff, Head 

Marc Van Beurden MD PhD Academie staff 

Guus Fons MD Academie staff 

Lottie AC Lubsen·Brandsma MD Academie staff 

Marianne AC Slot MD PhD Aeademic staff 

Arjanneke C Van Hof MD Aeademic staff 

Roelien Olivier Aeademic staff 

Plastic and Reconstructive Surgery 

J Joris Hage MD PhD Academie staff, Head 

Leonie AE Woerdeman MD Aeademic staff 

Brigit Drost MD Academie staff 

David Jairath MD Fellow 

Petra Koster MD PhD Fellow 

Anne Van Schijndel Undergraduate student 

1.0r-c::--


Chen B, Van den Brekel MWM, 

Buschers W, Balm AJM, 

Van Velthuysen MLF. Validation of tissue 

array technology in head and neck 

squamous celt carcinoma. Head Neck 2003; 

25:922-3°. 

Copper MP, Tan IB, Oppelaar H, 

Ruevekamp MC, Stewart FA. Meta-tetra 

(hydroxyphenyl) chlorin photodynamic 

therapy in early-stage squamous celt 

carcinoma of the head and neck. Arch 

Otolaryngol Head Neck Surg 2003, 

129:7°9-11 . 

De Braud F, Khayat 0, Kroon BBR, 

Valdagni R, Bruzzi P, Cascinelli N. 

Malignant melanoma. Crit Rev Oncol 

Hematol2003; 4T35-63· 

Deurloo EE, Tanis PJ, Gilhuijs KG, 

Muller SH, Kroger R, Peterse JL, 

Rutgers EJ, Valdes Olmos R, 

Schultze Kool LJ. Reduction in the number 

of sentinellymph node procedures by 

preoperative ultrasonography of the axilla 

in breast cancer. EurJ Cancer. 2003; 

39:1068-7]. 

Estourgie SH, Nieweg OE, 

Valdés Olmos RA, Kroon BBR. A review 

and evaluation of sentinel node procedures 

in 250 patients with cutaneous melanoma 

with a median follow up of six years. Ann 

Surg Oncol2003; 10:681-8. 

Estourgie SH, Tanis PJ, Nieweg OE, 

Valdés Olmos RA, Rutgers EJTh, 

Kroon BBR. Should the hunt for intemal 

mammary chain sentinel nodes begin? Ann 

Surg Onco12003; 10:935-41. 

Hilgers FJM, Ackerstaff AH, Balm AJM, 

Van den Brekel MWM, BingTan IB, 

Persson JO. A new problem-solving 

indwelling voice prosthesis, eliminating the 

need for frequent Candida- and 

'underpressure'-related replacements: 

Provox ActiValve. Acta Otolaryngol2003; 

12Y972-79· 

Hilgers FJM, Ackerstaff AH, Van As CJ, 

Balm AJM, Van den Brekel MWM, Tan IB. 

Development and clinical assessment of a 

heat and moisture exchanger with a multimagnet 

automatic tracheostoma valve 

(Provox FreeHands HME) for vocal and 

pulmonary rehabilitation after total 

laryngectomy. Acta Otolaryngol2003; 

123:91-9. 

HYPERTH ERM Ie INTRAPERITON EAL eH EMOTH ERAPY 

(H I PEe) STU DY G ROU P 

Frans AN Zoetmulder, Vic J Verwaai, Gooike W Van Slooten, Serge Van Ruth, Arjan J Witkamp 

Both hyperthermic intraperitoneal chemotherapy (HIPEC) and hyperthermie 

intrathoracic chemotherapy (HITHOC) have the advantage of a high local dose of 

cytostatic drugs in the cavity with limited systemic uptake. Hyperthermia is used to 

potentate the cytotoxicity of the used chemotherapy. Because the penetration depth of 

the cytostatic drugs is limited, preceding cytoreductive surgery is mandatory. 

Feasibility and toxicity studies of cytoreductive surgery and HITHOC for malignant 

pleural mesothelioma and pleural thymoma were performed. Cisplatin and 

doxorubicin we re perfused during 90 minutes under mild hyperthermic conditions 

(40-4I°C). Doxorubicin was chosen because of its enhanced activity under 

hyperthermic conditions. Radiotherapy (3x8Gy) on the thora cic scar and drainage 

tracts was given to mesothelioma patients to prevent scar recurrences. This 

combined treatrnent modality appears feasible but is accompanied by considerable 

toxicity. The longest mesothelioma survival time without disease was eight months, 

the longe st thymoma survival time without disease was 31 months af ter radiotherapy 

for alocal recurrence. 

The promising results of this feasibility study prompted us to extend the inclusion of 

patients to a proper phase 11 study. The results of this study of 20 patients, nineteen 

males and one female with a median age of 57 years (range 38-67), showed a median 

survival of eleven months with a one-year survival of 42% after a median follow-up of 

fourteen months. There was no operative mortality, but significant morbidity was 

seen in thirteen patients (65%). Complications included broncho-pleural fistuia, 

diaphragm rupture, wound dehiscence, persistent air-leakage and chylus effusion. 

Most recurrences of mesothelioma occurred in the ipsilateral hemithorax. 

Based on the high morbidity rate and the discouraging survival figures, cytoreductive 

surgery and HITHOC in the present form cannot be recommended for 

mesothelioma patients. Increasing the dose of the cytostatic drugs, adding systemic 

chemotherapy or hemithorax radiotherapy could be considered in an attempt to 

improve the results. 

Pharmacokinetics of doxorubicin and cisplatin in this approach were also studied. 

Between 1998 and 2001,24 perfusions were performed. The dose was initially based 

on square meters body surf ace area, whereas in later studies a fixed concentration 

per liter perfusion fluid was used. Samples of blood and perfusion fluid were 

collected for doxorubicin measurements. The penetration characteristics of 

doxorubicin in intercostal muscle specimens were determined by fluorescence 

microscopy. The mean AUCperfusate/AUCplasma ratios for doxorubicin and cisplatin 

were 99 and 59 respectively. During the perfusion, the concentration in the perfusate 

declined essentially according to first-order elimination kinetics for both doxorubicin 

and cisplatin with half-lives of 74 and 138 minutes respectively. At the end of the 

perfusion, about 35% and 52% of the dose of doxorubicin and cisplatin was recovered 

in the perfusion fluid. One patient developed a nephrotoxicity grade 11. This 

complication was not longer observed after improving intra-operative hydration. 

Doxorubicin penetrated into the intercostal muscle specimen albeit that there was 

considerable variation in distribution throughout the specimen. 

The pharmacokinetics of mitomycin C was studied during HIPEC. Comparative 

analysis of reported mitomycin C pharmacokinetics is not possible because the 

diversity of treatrnent techniques. Basing the dosage on milligram per square meter 

body surf ace area appears to be a good method. Three drug additions over time are 

preferabie compared to a single adrninistration. The population pharmacokinetics 

and pharmacodynamics of rnitomycin C were analyzed. The observed concentrationtime 

profiles in the 47 patients included were used to develop a population 

pharmacokinetic model using nonlinear mixed-effect modeling. The area under the 

plasma concentration versus time curve was related to the hematological toxicity. 

Concentration-time profiles of mitomycin C in perfusate and plasma were adequately 

described with a one- and two-compartment model, respectively. The relationship 

between the area under the curve in plasma and the degree of leucopenia was 

described with a sigmoidal Emax model. In this series, 28% grade lIl/IV leucopenia 

was observed. The pharmacokinetics of mitomycin C during HIPEC could be fitted

.,......... ~ •• L.. ___-I 

successfully to a multi-compartment model. The developed pharmacokineticpharmacodynamic 

model can be used to simulate different do sage schemes in order 

to optimize mitomycin C dosing during HIPEC. 

Histology and the relation between pathological features and survival of patients 

with pseudomyxoma peritonei were studied as weIl. pathology was classified as 

disseminated peritoneal adenomucinosis or peritoneal mucinous carcinomatosis 

with intermediate or discordant features. Sixty-two patients with pseudomyxoma 

peritonei (24 males, 38 females) were included in this study. The appendix was 

normal in one patient (2%), adenomatous mucosal changes were present in 

31 patients (50%) and the appendiceal status was unknown in 30 patients (48%). 

In females, the ovaries we re normal in five patients (13%), ovarian pseudomyxoma 

was present in 20 patients (53%) and the ovarian status was unknown in thirteen 

patients (34%). Ofthe patients with minimal atypia (n=p), those with 1% focal 

proliferation or less (n=38) had a better survival (p=0.0008) than those with more 

focal proliferation (n=14). Patients with disseminated peritoneal adenomucinosis 

(n=38) had a better survival (p=0.0002) than patients with mucinous carcinomatosis 

with intermediate or discordant features (n=24). 

LYM PHATIC MAPPI NG 

Omgo E Nieweg, Susanne H Estourgie, Anne PLont, Emiel JTh Rutgers, Simon Horenbias, 

Bin BR Kroon 

Lymphatic mapping Lymphatic mapping with sentinel node biopsy is a staging 

technique devised to de te ct non-palpable tumor involvement of the regional 

lymph node fields. This approach identifies patients who may benefit from early 

regional treatment and from adjuvant systemic therapy. The lymphatic mapping 

research program was initiated ten years ago and the technique has evolved into a 

clinically weIl accepted procedure. The general aims of this branch of the research 

program are to refine the technique oflymphoscintigraphy and surgery, and to 

determine its implications in terms of more accurate treatment, survival and 

regional control. 

The tumor types that are subjects of investigation within the lymphatic mapping 

research program are melanoma, breast cancer, carcinoma of the penis, testicular 

cancer and carcinoma of the larynx. An essential element in the program is the 

multidisciplinary approach. The team involves surgeons, urologists, head and neck 

surgeons, nuclear medicine physicians, pathologists, radiologists, a physicist and a 

statistician. Studies are done in cooperation with the Medisch Spectrum Twente, the 

WHO Melanoma Programme and The J ohn Wayne Cancer Center. A selection of the 

work is presented here. 

Breast cancer The Netherlands Cancer Institute pioneered lymphatic mapping in 

breast cancer. The technique that was developed involves administration of the 

tracers into the primary breast cancer. Small volumes are used to avoid disturbing 

the normal physiology oflymph drainage. This approach is technically more 

challenging than the superficial tracer administration technique that is used by some 

other investigators but has a number of distinctive advantages. Excision of 

radioactivity remaining at the injection site in the breast cancer removes the gamma 

ray scatter that may hamper probe-guided retrieval of a sentinel node. The 

intralesional injection technique avoids potential injection of tracer fluid across a 

lymphatic watershed and allows probe-guided excision of non-palpable tumors. 

These benefits outweigh the disadvantage of the technically more demanding 

injection. 

The visualization of sentinel nodes on the lymphoscintigraphy images proved highly 

reproducible using the intratumoral injection technique. This was concluded from a 

prospective study in 25 patients who underwent the procedure twice. Two observers 

evaluated the paired images independently and count rates were calculated. A 

sentinel node was visualized in all patients. Drainage to the axilla was observed in 

seventeen patients, drainage to both the axilla and extra-axillary basins in seven 

patients, and drainage exclusively to extra-axillary sentinel nodes in one patient. The 

second scintigraphic study revealed the same drainage pattern in all 25 patients 


Hurkmans CW, Borger JH, Rutgers Ej, 

Van Tienhoven G; EORTC Breast Cancer 

Cooperative Group; Radiotherapy 

Cooperative Group. QuaLity assurance of 

axillary radiotherapy in the EORTC 

AMAROS trial 1°981/22023: the dummy 

run. Radiother Oncol. 2003; 68:233-40. 

Kaas R, Peterse JL, Hart AA, Voogd AC, 

Rutgers Ej, Van Leeuwen FE. The injluence 

of tamoxifen treatment on the oestrogen 

receptor in metachronous contralateral 

breast cancer. BrJ Cancer. 2003; 88:7°7-10. 

Lont AP, Horenbias S, Tanis PJ, 

Gallee MPW, Van Tinteren H, Nieweg OE. 

Management of clinically node negative 

penile carcinoma; improved survival after 

the introduction of dynamic sentinelnode 

biopsy. ] Uro12003; 170783-6. 

Mutsaerts EL, Van Coevorden F, 

Krause R, Bore! Rinkes IH, Strobbe, Lj, 

Prevoo W, Tollenaar RA, Van Gulik TM. 

Initial experience with radiofrequency 

ablation for hepatic tumours in the 

Netherlands. EurJ Onco12003; 29731-4. 

Nieweg OE, Bartelink H. ImpLications of 

lymphatic mappingfor staging and 

adjuvant treatment of patients with breast 

cancer. EurJ Cancer (in press). 

Nieweg OE, Estourgie SH, 

Valdés Olmos RA, Rutgers EJTh, 

Hoefnagel CA, Kroon BBR. Sentinel node 

biopsy with tracer administration into the 

primary breast cancer. EurJ Surg Oncol 

2003; 29:95-7· 

Noorda EM, Vrouenraets BC, Nieweg OE, 

Klaase JM, Van der Zee j, Kroon BBR. 

Long-term results of a double perfusion 

schedule using high-dose hyperthermia and 

melphalan sequentially in extensive 

melanoma of the lower limbo Melanoma 

Res 2003; 13:395-9. 

Noorda EM, Vrouenraets BC, Nieweg OE, 

Van Coevorden F, Van Slooten GW, 

Kroon BBR. Isolated Limb perfusion with 

tumor necrosis factor-alpha and melphalan 

for patients with unresectable soft tissue 

sarcoma ofthe extremities. Cancer 2003; 

98:1483-9°.


Roozendaal KJ, De Valk B, Ten Velden JJ, 

Van der Woude HJ, Kroon BBR. Alveolar 

soft-part sarcoma responding to interferon 

alpha-2b. BrJ Cancer 2003; 89:243-5. 

Van As CJ, Koopmans-Van Beinum FJ, 

Pols LC, Hilgers FJM . Perceptual 

evaluation of tracheoesophageal speech by 

naive and experienced j udges through the 

use of semantic differential scales. ] Speech 

Lang Hear Res 2003; 46:947-59. 

Vander Poorten VLM, Hart AAM, 

Van der Laan BFAM, 

Baatenburg De Jong RJ, Manni JJ, 

Marres HAM, Meeuwis CA, Lubsen H, 

Terhaard CHJ, Balm AJM. Prognostic index 

for patients with parotid eareinoma: 

extemal validation using the nationwide 

1985-1994 Duteh Head and Neek Oneology 

Cooperative Group database. Cancer 2003; 

97:1453-63. 

(Figure XI.3) (reproducibility IOO%; 95% confidence interval 86% - IOO%). 

An additional advantage of the intralesional injection technique is that it enables the 

pursuit of sentinel nodes in the intemal mammary chain. Such nodes we re present 

in 150 of a series of 691 patients (incidence 22%). The nodes were surgically 

retrieved in 130 of these patients (87%) and contained metastasis in 21 (16%). Stage 

migration was seen in all patients with a tumor-positive intern al mammary sentinel 

node. As a re sult, treatment was better adjusted to the individual needs of 37 patients 

(28%). 

Melanoma Lymphatic mapping was studied in a total of 250 melanoma patients 

who were followed for a median of six years. Lymphoscintigraphic visualization was 

IOO% and surgical identification was 99.6%. Occult metastatic disease was 

discovered in 60 patients (24%). Postoperative complications were seen in 

23 patients (9.2%). All resolved without long-term sequelae. Late complications af ter 

sentinel node biopsy were seen in 35 patients (18%). The false-negative rate was 9%. 

In-transit metastases we re seen in 7% of the sentinel node-negative patients and 23% 

of the sentinel node-positive patients. The estimated five-year overall survival rates 

were 89% and 64%, respectively (P

BREAST CANCER 

Emiel JTh Rutgers, Hester SA Oldenburg 

I ncidence of micro-metastases in Iymph nodes from patients with ductal 

carcinoma in situ (DeiS) and small invasive breast cancer From our database, 

we identified 242 patients diagnosed with a pure DCIS (n= 179), invasive 

ductaljlobulair carcinoma (IDCjILC) ~ S mm (n= 40) or tubular carcinoma ~ 10 mm 

(n= 23), treated between 1989 and 1998. An axillary lymph node dissection was 

performed in 72, 30 and 17 patients, respectively. All archived nodes were reevaluated 

using immunohistochemistry staining (IHC) at deeper levels. 

More metastases were found with the use ofIHC (tabie). All patients who had IHC 

positive lymph nodes remained free of disease after a mean follow-up of74 months. 

In conclusion, there is no indication for a complete axillary lymph node dissection 

when a micro-metastasis is detected in a lymph node of a patient with a Tis or a Tla 

carcinoma. 


Van Ruth S, Van Tellingen 0, Korse CM, 

Verwaai VJ, Zoetmulder FAN . 

Pharmaeokineties of doxorubicin and 

cisplatin used in intra·operative 

hyperthermie intra-thoraeie ehemotherapy 

after cytoreductive surgery for malignant 

pleural mesothelioma and pleural 

thymoma. Antieaneer Drugs 2003; 14:57-65. 

The influence of previous tamoxifen use on the receptor status of contralateral 

breast cancers Thirty-five tarnoxifen treated patients with contra-Iateral 

breast cancer (CBC) and IlS patients without previous hormonal treatment were 

Tabl 

Incidence of metastases on routine staining in 278 patients and a review with IHC 

on IlO patients who were node negative. 

routine 

staining 

micrometa- IHC I solitary\micrOstasis 

\macro- 

metastasis\ 

metastasis 

macrometastasis 

DCI S 

1% 

1\72 

0\1 n% 6 \ I \ 0 

7\66 

DCI S + micrometastasl 

's~2mm 

IDC \ILC 2-5 mm 

0% 

- 

6% 

0\12 

1\18 

0\0 27% 2 \ 0 \ I 

3\Il 

-- -- 

0\1 12% 0\2\0 

2\17 

IDC \ILC 6-10 mm 

15% 

23\159 

6 \ 17 No rtWision No revision 

tubul ar carcinoma 

1-10 mm 

0% 

0\17 

0\0 13% I \ 1\0 

2\16 

studied to investigate the hypothesis that adjuvant tamoxifen reduces the occurrenee 

of estrogen receptor (ER) positive CBC, but not the growth of ER-negative CBCs and 

to examine survival af ter the diagnosis of CBC. The ER status was 

immunohistochemically assessed by revision in the contra-Iateral breast cancers. The 

cases were retrieved from a series of patients treated from 1984-1995 at nine 

hospitals. The interval between ipsi- and CBC was at least one year. 

The proportion of patients with an ER-negative CBC was significantly higher among 

those with prior tarnoxifen treatment: 37% versus 18% (P=0.047) No difference in 

overall and disease specific survival following CBC was found between the two 

groups. However, the tumor stage differed in both groups: tamoxifen-users had more 

often node-positive contra-lateral disease (P=0.045). 

Conclusion: Metachronous CBC developing after one to three years of tarnoxifen 

treatment are more often ER-negative than without such treatment. So far, this does 

not seem to have a major impact on survival.

• 

Trends in diagnosis and treatment, and outcome of ductal carcinoma in situ 

of the breast in 403 patients over 1986 - 2002 Four hundred and three patients 

with DCIS underwent surgery at the NKIjAvL between January 1986 and December 

2002. All patients with 'pure' DCIS and no prior history ofbreast cancer was 

included. 

One hundred and sixty-five patients (41%) we re treated with breast-conserving 

therapy (BCT) , 97 (24%) with excision alone (BCT+E) , and 68 (17%) with excision 

plus radiotherapy (BCT+E+RT) and 238 (59%) with mastectomy. The median age was 

51.0 years (range: 24-81 years). 

Mammographically detected DCIS increased from 50% in the late nineteen-eighties 

to 8}6% in the nineteen-nineties. Ten years af ter its introduction, more than 70% of 

all DCIS are currently diagnosed by stereotactic co re biopsy. As aresult, the number 

of surgical multi-step procedures necessary for definitive diagnosis and treatrnent 

declined from 77% in 1995-1997 to 41% in 2001-2002. Today, 59% of DCIS patients 

are being treated surgically in one step. The BCT j mastectomy ratio did not change 

over time with about 60% of patients treated ultimately by mastectomy (either 

simple mastectomy or skin-sparing masrectomy with reconstruction). 

At a median follow-up of 5.3 years, 20 tumor-related events occurred. Eight patients 

(8.2%) developed alocal recurrence in the BCT+E group, seven patients (10.3%) in 

the BCT+E+RT group and five patients (2.1%) in the mastectomy group (four local 

and one distant). The median time to recurrence was 2.9 years for all groups. Four 

(1%) patients died of invasive breast carcinoma af ter recurrence af ter a mean followup 

of 4-4 years. 

Histological differentiation grade of the primary tumor and mar gin status are not 

equally distributed. The poorly differentiated and mar gin positive tumors are more 

of ten present in the BCT+E+RT than in the BCT+E or mastectomy group, 62(25%) 

vs. 27(18%), and 51(4%) respectively. 

Contralateral breast cancer developed in seven (7.2%) , two (2·9%) and twelve (5%) 

cases in the BCT+E, BCT+E+RT and mastectomy group respectively. 

ISOLATED LI M B PERFUSION 

Bin BR Kroon, Omgo E Nieweg, Gooike W Van Slooten, Eva M Noorda, Bart C Vrouenraets 

Isolated limb perfusion for unresectable melanoma ofthe extremities From 

our computer-assisted database, containing all characteristics and results of ILP for 

extremity melanoma, 130 patients could be selected who had undergone an isolated 

limb perfusion (ILP) for truly unresectable lesions. This selection was made on the 

basis ofnumber (>10) or size (>3 cm) ofthe lesions or their location on the extremity. 

A complete tumor response was obtained in more than half of the patients with a 

tendency for a better response rate af ter ILP with Tumor Necrosis Factor a (TNFa) 

and melphalan compared to ILP with melphalan alone. The limb salvage rate was 

96%. Overall five-year survival was 29% (95% Cl 20-38%). The absence oflymph 

node metastasis increased the chance of a complete response and was associated with 

longer tumor-free survival. Patients with a complete response and relatively small 

tumor nodules «3 cm) had a significantly better overall survival in multivariate 

analysis. 

It is concluded that ILP is a valuable treatrnent option in patients with locoregional 

unresectable extremity melanoma, for whom there are no effective alternatives. 

Repeat I LP with tumor necrosis factor a and melphalan for recurrent limb 

melanoma af ter failure of previous perfusion Between 1978 and 2001, 

21 patients underwent repeat ILP using TNFa for recurrent melanoma or persisting 

disease af ter a previous ILP. First ILPs had been performed with melphalan alone in 

13 patients and with TNFa and melphalan in eight, for a median of nine lesions. 

Repeat ILP was performed with TNFa and melphalan in all 21 patients for a median 

of nine lesions. Mean follow-up af ter repeat ILP was 48 months (range 2-II5 

months). Thirteen patients attained a complete response (CR) af ter repeat ILP 

compared to II of 17 with measurable lesions at the first ILP. Nine patients relapsed 

again af ter CR. Median limb recurrence-free survival was 13 months (95% Cl 5-22 

months). Fourteen patients had mild acute regional toxicity af ter repeat ILP

compared to 18 af ter the first ILP. One patient underwent amputation for critical 

limb ischaemia 10 months following repeat ILP, leading to a limb salvage rate of 

95%. Overall median survival was 62 months (95% Cl 36-89 months) after CR 

compared to 13 months (95% Cl 0-4-26 months) for those without CR (P=0.006). 

Repeat ILP with TNF? and melphalan appears to be well feasible in patients with 

recurrent disease after previous ILP with mild regional toxicity. The complete 

response rate is relatively high and comparable to that of the fust procedure with a 

considerable limb recurrence-free survival and high limb salvage rate. 

Long-term results of a double perfusion sehedule using high-dose 

hyperthermia and melphalan sequentially in extensive melanoma of the 

lower limb The efficacy of a sequential ILP schedule was studied in search ofbetter 

tumor response rates in patients with extensively recurrent or bulky extremity 

melanoma. This schedule consists of an ILP with high-dose hyperthermia (42-43°q, 

without cystostatic drug, followed a week later by ILP with melphalan under 

normothermic conditions. It is known that hyperthermia has a cell-killing effect on 

tumor cells, but its simultaneous application with cytostatics had proven to be too 

toxie. This sequential schedule was developed to have the cytotoxic effect ofboth 

high-do se hyperthermia and melphalan, without the increase in locoregional toxicity 

caused by their synergy. Seventeen patients were treated with this schedule between 

1989 and 199+ Eleven patients had a complete response (65%). Three patients had 

limb recurrences (27%) after five, six and eighteen months respectively. The five-year 

limb recurrence-fee interval for patients with CR was 63%. Limb toxicity was mild 

with only pressure-related blistering and transient sensory disturbances after the 

hyperthermic ILP. Af ter the second ILP, 88% of the patients had the desired grade 11 

reaction (mild erythema and ederna). 

Concluding, this sequential ILP schedule results in a high complete response rate 

and a low limb-recurrence rate in patients with extensive, recurrent melanoma of the 

limbs at the co st of only mild toxicity. This regimen could be an alternative to ILP 

with TNFa and melphalan, which seems indicated in extensive or bulky lesions. 

AN ESTH ESIOLOGY A 0 I TENSIVE AR 

Dirk R Buitelaar, Johannes M Huitink, May Ronday, Peter FE Schutte, Julia ten Cate 

Anesthesiologieal management of patients with earcinoid disease Surgery in 

patients with carcinoid disease is often complicated by hemodynamic instability due 

to release of vaso-active agents of which somatostatin is the most important one. 

Since the introduction of octreotide (Sandostatine®), these symptoms of carcinoid 

syndrome can be attenuated. 

Since 2000 all patients with carcinoid disease who are scheduled for an invasive 

procedure are treated with intravenous octreotide, until48 hours after the procedure. 

In review articles, epidural anesthesia is advised against because of the possible 

aggravation of the hemodynamic instability. We reviewed records of all carcinoid 

patients (n = 65) undergoing invasive procedures at our institution. These procedures 

were performed under either epidural or a combination of general and epidural 

anesthesia. There were no serious haemodynamic events. We conclude that epidural 

anesthesia can be safely performed in patients with carcinoid disease who are treated 

with octreotide. 

Anesthesiologieal aspects of head en neek surgery The incidence and the time 

course of cardiovascular and respiratory complications were studied in 469 patients 

after major head and neck surgery. The incidence of these complications was 12% 

and n% (heart failure - on day I, and pneumonia - on days 2 and 6). Guidelines 

were developped to prevent these complications in the future. 

Airway management in emergency tracheotomies was studied prospectively in 

26 patients. Clinical criteria were studied to select the best intubation strategy: 

laryngoscopy, fiberoptic intubation or direct tracheotomy after local anesthesia. The 

common finding in the patients with difficult or impossible fiberoptic intubation was 

CO 2 

narcosis.

" ' 

".' ~ . . 

) ", 

'; : :J';' 

• • 

Epidural analgesia In I997 we started a prospective database of postoperative 

epidural analgesia in cancer patients. In 2003 the first data (complications and sideeffects 

in cancer patients) ofthe NKI epidural database (> 4700 patients) were 

reported during the annual meeting of the Dutch Anesthesiology Society. This is the 

largest database of epidural analgesia in cancer patients in the Netherlands. 

Mobile anesthesiology The expertise of one of the staffmembers with mobile 

intensive care unit transportation resulted in the publication of a book.

XII DIVISION OF PSYCHOSOCIAL 

RESEARCH AND EPIDEMIOLOGY 

PSYCHOSOCIAL ONCOLOGY 

The subdivision of psychosocial oncology is pursuing three major research lines: 

(1) quality oflife assessment in clinical research and clinical practice (Group 

Aaronson); (2) psychosocial issues in cancer clinical genetics (Group Aaronson); 

and (3) symptom perception and management (Group van Dam). 

Health-related quality of life (H RQl) in long-term survivors of isolated limb 

perfusion for melanoma of the limbs This cross-sectional study investigated the 

HRQL of 51 long-term survivors of extremity melanoma who had been treated with 

isolated limb perfusion between 1978 and 2001. QL was assessed with the SF-36 

Health Survey, and a condition-specific questionnaire. SF-36 scores were compared 

with those of an age- and gender-matched sample drawn from the general Dutch 

population. The patient sample scored, on average, better than the comparison group 

on all SF-36 scales, with statistically significant differences observed for general 

health perceptions, bodily pain, and the physical and mental health component 

scores. Prevalent condition-specific symptoms included stiffness or edema of the 

affected limb (49-55%), skin discoloration (31%) and visible skin lesions (38%). Fear 

oflocal and distant disease recurrence was reported by 77% and 63% of the patients, 

respectively. Fewer than 10% of patients reported health-related problems in 

obtaining health or life insurance or a home mortgage. Nearly all patients indicated 

that they would undergo perfusion again or would recommend it to others in a 

similar situation. 

Division Head, Group leader Neil Aaronson 

Neil Aaronson PhD Group leader 

Joanna Madalinska MA, MSc Academic staff 

Babs Taal MD, PhD Academic staff 

Heiddis Valdimarsdottir PhD Academic staff 

Marc Van Beurden MD, PhD Academic staff 

Sen no VerhoefMD Academie staff 

Eveline Bleiker PhD Post-doc 

Karin Gehring MSc Graduate student 

Doranne Hilarius MSc Graduate student 

Rianne Hoopman MSc Graduate student 

Ruud Knols MSc Graduate student 

Martin Muller MSc Statistica I analyst 

Fatima Bouali Research assistant 

Miranda Gerritsma Research assistant 

Masjda Idrissi Research assistant 

Esther Janssen Research assistant 

Ruben Van Kreij Undergraduate student 

The prognostic value of cognitive functioning in the survival of high-grade 

glioma patients This study investigated cognitive functioning as a potential 

predictor of survival among 68 newly diagnosed high-grade glioma patients. In a 

combined Cox proportional hazards model, ol der age and higher tumor grade were 

associated with poorer survival. Although cognitive functioning was not found to be 

an independent prognostic factor for the total sample, it was associated significantly 

with poorer survival among older patients with WHO grade IV gliomas. 

H RQl assessment among ethnic minority cancer patients In 2003, data 

collection was completed for a study whose primary aims are: (1) to translate, adapt 

and validate two widely used generic QL questionnaires (the SF-36 and the 

COOP jWONCA charts) and two cancer-specific questionnaires (the EORTC 

QLQ-C30 and the Rotterdam Symptom Checklist) for use among Turkish and 

Moroccan cancer patients in the Netherlands; and (2) to investigate the accuracy of 

proxy (family member) ratings of these patients HRQL. In total, I05 Turkish and 

87 Moroccan patients and 40 Turkish and 25 Moroccan proxies have been included 

in the study. Quantitative and qualitative data analyses are on-going. 

The psychosocial and behavioral impact of genetic counseling for colorectal 

cancer (eRC): a prospective, multicenter study In 2003, the (follow-up) data 

collection was completed for this multicenter (AvL, VUMC, AMC, RUG, and LUMC) 

prospective study, whose aims are to: (1) assess the effect of genetic 

counselingjtesting on risk perception, distress, family relationships, work, and 

family and financial planning; (2) identify risk factors for poor psychological 

adjustrnent to and early withdrawal from the genetic counseling process; and 

(3) establish rates of short-term compliance with recommended screening practices. 

Initial questionnaires we re completed by 347 (87% of eligible) individuals following 

the first counseling session. Follow-up questionnaires (3 weeks and 6 months after 

the final counseling session) were completed by 240 (69%) and 178 (51%) 

individuals, respectively. The most important reasons for undergoing genetic 

counseling were to determine personal cancer risk or risk for off-spring, and to take 

preventive actions. Fewer than IO% of counselees reported clinically significant levels 

of psychological distress at the time of initial counseling, and this figure remained


Aaronson N K. Cross-cultural use ofhealthrelated 

quality oflife assessments in clinical 

oncology. In: Lipscomb J, Gotay CC, 

Snyder CF (Eds.) Outcomes Assessment 

in Cancer. Cambridge: Cambridge 

University Press (in press). 

Bleiker EMA, Hahn DEE, Aaronson NK. 

Psychosocial issues in cancer genetics: 

Current status and future directions. Acta 

Oncol42:276-286, 2003. 

Bleiker EMA, Menko FH, Kluijt I, 

Wever LDV, Gerritsma MA, Vasen HF, 

Aaronson N K. E;lCperience of discharge from 

colonoscopy of mutation-negative HNPCC 

family members. ] Med Genet 40:e55, 2003. 

De Haes JCJM, Curran D, Aaronson NK, 

Fentiman IS. Quality oflift in brast cancer 

patients aged over 70 years participating in 

the EORTC 10850 randomised clinical 

trial. EurJ Cancer 39:945-951, 2003. 

Fearon KCH, Von Meyenfeldt MF, 

Moses AGW, Van Geenen R, Roy A, 

Gouma DJ, Giacosa A, Van Gossum A, 

Bauer J, Marber MD, Aaronson NK, 

Voss AC, Tisdale MJ. The effect ofa protein 

and energy dense, n-3fatty acid enriched 

oral supplement on loss of weight and 

lean body tissue in cancer cachexia. A 

randomised double blind trial. Gut 52:1479- 

1486,20°3, 

fairly stable through to,the 6 month post-counseling assessment. Distress levels are 

not related significantly to genetic test results. Twenty percent of the counselees 

indicated that their family relationships had changed as a result of the genetic 

counseling process, almost always for the better. Although approximately 20% 

experienced the counseling as burdensome, fewer than 5% of counselees expressed 

any regrets about having undergone genetic counseling. Data analysis is on-going. 

The impact of prophylactic oophorectomy in women from hereditary 

breast/ovarian cancer families on psychosocial health and symptom 

experience This multicenter, cross-sectional and longitudinal (AvL, AZVU, AMC, 

LUMC, AZN, AZG, AZM, UMCU) study is investigating: (1) the decision-making 

process surrounding the choice of preventive health options among wo men at 

increased risk of developing ovarian cancer; (2) the impact of screening versus 

prophylactic surgery on psychosocial well-being; (3) complianee with screening 

advice; and (4) the prevalenee and severity of menopausal symptoms among women 

who opt for surgery, and the use and perceived benefit ofhormone-replacement 

therapy (HRT). In total, 856 women have completed the self-report questionnaire for 

the cross-sectional part of the study. Preliminary results indicate that women who 

have undergone preventive oophorectomy have significantly fewer cancer worries 

and perceive their cancer risk as lower than women who have opted for gynecological 

screening. Women who use HRT following prophylactic oophorectomy experienee 

very few endocrine symptoms, and do not differ significantly in this re gard from 

premenopausal women in the screening group. The levels of self-reported physical 

and mental health are within normallimits for both the surgical and screening 

groups. Final data analyses of the retrospective data are planned for 2004. 

To date, 257 of 302 eligible women (87%) have agreed to participate in the 

longitudinal part of the study. Preliminary analyses of the baseline questionnaires of 

146 women indicate that 4r% intend to undergo prophylactic oophorectomy, 44% 

gynecologic screening and rs% have not yet decided on which course to pursue. 

Women who intend to undergo prophylactic oophorectomy report significantly more 

cancer worries and anxiety, and perceive their risk of developing ovarian cancer as 

significantly higher than do women from the screening group. The choice of 

preventive oophorectomy is associated with mutation status (BrCar/2 carrier), parity 

(having at least one child), education (lower) and an active coping style. Sample 

accrual, data collection, and statistical analyses are on-going. 

Fossa SD, De Wit R, Roberts JT, 

Wilkonson PM, De Mulder PHM, 

Mead GM, Cook P, De Prijck L, 

Stenning S, Bottomley A, Aaronson NK, 

Collette L. Quality oflife in good prognosis 

patients with metastatic gum cell cancer: A 

prospecdtive study ofthe EORTC GU 

Group/MRC Testicular Cancer Study 

Group. ] Clin OnCOl21:1107-1l18, 2003. 

Klein M, Engelberts NHJ, 

Van der Ploeg HM, Kasteieijn-Noist DGA, 

Aaronson NK, Taphoorn MJB, Baaijen H, 

Vandertop WP, Muller M, Postma TJ, 

Heimans J). Epileptic seizures in low-grade 

gliomas: the impact on cognition and 

health-related quality oflife. Ann of Neurol 

54:514-520, 2°°3· 

Non-participation in genetic counseling for cancer: concerns and family 

issues Approximately one-third of individuals who apply for genetic counseling for 

cancer decide not to undergo such counseling. The aim of this study is to investigate 

reasons for non-attendance af ter a first request for genetic counseling. Self-report 

questionnaires were sent to 73 women who had requested but did not attend genetic 

counseling for breast/ovarian cancer. A comparison group was formed of ros women 

who completed the genetic counseling process. Response of the eligible nonattendees 

and attendees was 66% and 8r%, respectively. The most important reasons 

expressed for withdrawing from counseling were: (1) difficulties in anticipating the 

consequences of genetic counseling (28%); (2) worries about not being able to cope 

weIl with an unfavorable test-re sult (20%); and (3) a preferenee to postpone the 

process (20%). The best predictors of non-participation were having no or only few 

worries about cancer, and being the first and only member in the family to request 

genetic counseling. None of the non-attendees reported problems with the initial 

contact with the clinic service as a reason for not pursuing the counseling. 

Penson DF, Litwin MS, Aaronson NK. 

Health-related quality oflife in men with 

prostate cancer: current status and future 

research. ] Uro1169: 1653-1661, 2003.

SYMPTOM PERCEPTION AND MANAGEMENT 

Neuropsychological and neurophysiological studies The impact of cytostatic 

drugs on the patients cognitive functioning is evaluated with neuropsychological and 

neurophysiological techniques. We have investigated cognitive functioning following 

chemotherapy in (high-risk) breast cancer patients, lymphoma patients, patients with 

testicular cancer and patients who have been treated during childhood for 

osteosarcoma and Ewing sarcoma. In all of these studies the same standard battery 

of neuropsychological tests is used, making possible a comparison between tumor 

types and chemotherapy regimens. To date, over 1000 test assessments have been 

carried out. Data analysis is in progress, and first results are expected at the end of 

this year. 

Furthermore, we started a study into the neuropsychological sequelae of 

postmenopausal breast cancer patients receiving endocrine therapy in an adjuvant 

setting. In this study, two groups of patients receiving endocrine therapy 

(tamoxifen versus exemestane) will be assessed twice: before the start of the 

endocrine therapy and af ter 12 months of treatrnent. Healthy controls will be 

assessed with the same time interval. Patients will be recruited from the TEAM 

trial; an open label, randomized multicenter study of 5 years adjuvant exemestane 

versus tamoxifen. 

A four-year neurophysiological study was designed to gain a better understanding 

of the effects of chemotherapy on specific stages of information processing in various 

groups ofbreast cancer patients. A task based on the Additive Factors Method (AFM) 

was used to obtain information on which specific stages of information processes in 

the brain have been affected by chemotherapy. Electrocortical activity was registered 

during task administration. The neurophysiological assessment also included 

standard EEG and an auditory task. So far 48 breast cancer patients participated in 

this study: 12 patients treated with a high-do se CTC regimen, 17 patients treated with 

a standard-dose FEC regimen and 19 stage 1 breast cancer patients not treated with 

chemotherapy. All patients also participated in the prospective neuropsychological 

study and were tested approximately 3 years af ter cessation of treatrnent. Currently 

the neurophysiological and behavioral data are being analyzed. 

Quality of care studies 

Alternative treatments We determined the use of alternative diets and other 

alternative treatments in 2002 compared to 1999. During the period 13-26 May 2002 

a survey was held among all patients visiting the outpatient clinic of the N etherlands 

Cancer I nstitute I Antoni van Leeuwenhoek hospital. Patients were asked about their 

current and past use of alternative therapies, their reasons for using these therapies, 

the way they we re informed about these therapies and the expenses involved. The 

data were compared with a similar study during the period 15-19 March 1999. Of 

the 729 patients who fulfilled the inclusion criteria, 66 (9%) declined to participate 

in the study. Ofthe remaining 663 patients (average age 58.5 years; 28% male) 

131 (20%) used an alternative therapy. Of these, 43 patients (7%) used an alternative 

diet, mainly the Houtsmuller diet, and 88 patients (13%) used a mixture of alternative 

diets such as homeopathy, vitamins and herbs. In 1999,121 patients (30%) used an 

alternative form of treatrnent, 51 (13%) of whom used a diet. Of the 43 us ers of diets 

in 2002, II (26%) believed that the di et would slow down the disease process; in 

1999 this was 53% (27/5 1). Of the 131 users of alternative therapies in 2002, 55% had 

been made aware of the possibilities of alternative treatments via family and friends. 

Internet and TV played a minor role as a source of information. 33 (79%) of the diet 

us ers informed their physician or nurse about the use. The diet us ers spent an 

average of 170 euro per month on their diets. It can be concluded that both the 

percentage of cancer patients who used an alternative diet and the percentage of diet 

users who believed that a diet could affect the course of the disease were reduced by 

half compared to three years earlier. 

In order to determine if their should be smoking facilities for patients and whether 

there would be interest in a stop smoking course, we asked 347 patients at the 

outpatient department, the day care facility and the hospital ab out their smoking 

behavior. 49 patients (14%) declined participation in the study. 72 patients (24%) 

Group leader Frits Van Dam 

Frits Van Dam PhD Group leader 

Willem Boogerd MD, PhD Academic staff 

Frans Hilgers MD, PhD Academic staff 

Sjoerd Rodenhuis MD, PhD Academic staff 

Corinne Van As PhD Academic staff 

Aafke Donker Registered nurse 

Hilda Hauer Registered nurse 

Suzy Oppeneer Registered nurse 

Karin Van Rooijen Registered nurse 

Marjan Hummel PhD Post-doc 

Sanne Schagen PhD Post-doc 

Baudewijntje Kreukels MSc Graduate student 

Christien Schilder MSc Graduate student 

Martin Muller MSc Statistica I analyst 

Anky Cas pers Research assistant 

Tinneke Denhof Research assistant 

Walter Oomen Research assistant 

Marion Weevers Research assistant 

Angelique Biervliet Undergraduate student 

Kirsten Douma Undergraduate student/ 

Research assistant 

Marthe Ford Undergraduate student 

Kim Karsenberg Undergraduate student 

Sanne Koemans Undergraduate student 

Suzanne Leering Undergraduate student 

Lisette Mennen Undergraduate student 

Sietske Sikkes Undergraduate student/ 

Research assistant 

Simone Verwer Undergraduate student 

Miriam Zaagsma Undergraduate student


Polak R, Van As C, Van Dam F, Hilgers F. 

Reuk revalidatie voor gelaryngectomeerden, 

handleiding voor logopedisten. Swets «[ 

Zeitlinger, 2002. ISBN 90 265 17238. 

were smokers (hospital 21%; outpatient department 26%, day care 18%, radiotherapy 

28%) and would appreciate a place to smoke in the hospita!. On average patients 

smoked 10 cigarettes a day. Of the smokers, 21 (30%) were interested in a stopsmoking 

course when organized by the hospita!. 

Puts M, Versloot ). Muller M, Van Dam F. 

Tussen wal en schip: een onderzoek naar de 

visie op de zorgverlening van 

kankerpatiënten die op de dagverpleging een 

palliatieve behandeling ondergaan. Ned 

Tijdschr Geneeskd (in press) . 

Schagen SS, Muller M). Soogerd W, 

Van Dam FS. Cognitive dyifunction and 

chemotherapy: neuropsychologicalfindings 

in perspective. Clin Breast Cancer. 2002; 

3 Suppl ]:S100-8. Review. 

Van Dam FSAM, Goudsmit M, Jonker Th, 

Eeltink CJT, Muller MJ. Less use of 

alternative treatments by cancer patients in 

2002 than in 1999. [article in Dutch: 

Minder gebruik van alternatieve 

behandelingen door kankerpatiënten in 

2002 dan in 19991 Ned Tijdschr Geneeskd 

2003; 147(36):1731-1734.

EPIDEMIOLOGY 

The ca neer epidemiology group is currently concentrating on two principal research 

lines: (1) the etiology ofhormone-related cancers; (2) the long-term health 

consequences of cancer treatrnent, particularly in terms of the risk of developing a 

second cancer. 

Risk factors for hormone-related cancer In our nationwide cohort study in breast 

and/or ovarian ca neer families (GEO-HEBON study), we are studying (1) whether 

hormonai/life-style factors modify cancer risk in BRCA1/2 families, (2) the agespecific 

cumulative risks ofbreast, ovarian and other cancers based on full pedigree 

information. In 2003 we evaluated cancer risks for sites other than breast and ovary 

in 517 BRCA1 and 139 BRCA2 mutation families (6,585 and 1,821 individuals, 

respectively). In a selected cohort of 50% presumed carriers the observed cancer 

incidence was compared with age- and sex-specific cancer incidence rates in the 

Dutch population. Among BRCA1 carriers cancer risks we re significantly elevated for 

pharynx (relative risk (RR)=).6), stomach (RR=2.5), colon (RR=2.5), liver (RR=8.2), 

cervix (RR=4-4), endometrium (RR=2.9), and bra in (RR=5.0). 

For BRCA2, cancer risks were increased for the digestive tract (RR=I.4), liver 

(RR=9.6), pancreas (RR=5.8), bone (RR=1).6), pro state (RR=2.5), and among women 

liver (RR=13.8) . To examine whether metastasis ofbreast or ovarian cancer or 

treatrnent for these cancers affected the results, we excluded all breast and ovarian 

cancers prior to a second cancer diagnosis at another site. Only for BRCA1 did the 

increased risk of endometrial cancer not hold. In conclusion, these results confirm 

earlier findings of increased risks for eancers of the stomaeh, colon, liver, brain, 

cervix and endometrium in BRCA1 mutation carriers. In contrast, earlier reported 

increased risks of pro state and pancreatic cancer were not found. This studyalso 

provides confirmation of an elevated risk of pro state cancer among BRCA2 carriers, 

which may have important implications for counseling and screening advice for male 

BRCA2 carriers. 

Early childhood risk factors have been hypothesized to play a role in breast cancer 

etiology. We investigated the association between body weight and height at age ro 

and breast cancer risk as an adult in our population-based case-control study 

(469 cases with invasive breast cancer, 435 cases with ductal carcinoma in situ and 

424 controls). Preliminary data suggest that women who were taller than their peers 

at age ro had a 30-50% increased risk of in situ as weIl as invasive breast cancer, 

whereas being shorter seemed to be protective (3°-40%). Women who were heavy as 

a child seemed to have a slightly lower breast cancer risk during adulthood than 

those who we re thin at age ro. 

We continued in 2003 with the data collection of our cohort study on hormonerelated 

cancer risk in women who were exposed to diethylstilbestrol (DES) in utero. 

We started with the medical verification of DES-exposure and of all disorders 

reported in 7,779 risk factor questionnaires from DES daughters registered at the 

Netherlands DES Center. For the part of the cohort traeed in the historical archives 

we searched through a total of 126.233 medical records to identify 624 DESdaughters 

and 696 controls. 

Since 1993 we participate in the Oxford meta-analysis on Horrnonal Risk Factors for 

Breast Cancer (Collaborative Group on Hormonal Factors in Breast Cancer, Imperial 

Cancer Research Fund, Oxford, UK). The analysis on the association between alcohol 

and tobacco use with risk ofbreast cancer showed an increasing risk ofbreast cancer 

with increasing intake of alcohol by 7.3% per rog (- per glass). An average of 4 drinks 

a day was associated with a 32% increased risk. Tobacco consumption was not 

associated with breast cancer risk. 

We are also closely involved in an intervention study focusing on the insulin-like 

growth factor system in relation to breast caneer and colorectal carcinogenesis, a 

study on gynaecologieal screening of women with a high genetic risk of ovarian 

caneer and a study on the efficaey of contralateral prophylaetie mastectomy among 

women with a BRCA1/2 mutation (see Division of Diagnostic Oneology I Familial 

Caneer Clinic). 

In our nationwide historical cohort study of caneer risk in 25,152 women treated 

for subfertility in the Netherlands between 1980 and 1995, we examined 

Group leader Flora Van Leeuwen 

Group leader Matti Rookus 

Flora Van Leeuwen PhD Group leader 

Matti Rookus PhD Group leader 

Berthe Aleman MD Academic staff 

Pet ra NederlofPhD Academic staff 

Nicola RusselI PhD Academic staff 

Emiel Rutgers MD, PhD Academic staff 

Laura Van 't Veer PhD Academic staff 

Senno VerhoefPhD Academic staff 

Lisette Hoogendoorn PhD Post-doc 

Ina Mulder PhD Post-doc 

Olga Van der Hel PhD Post doc 

Dorien Voskuil PhD Post doc 

Richard Brohet MSc Graduate student 

Mathilde Cardous-Ubbink MSc Graduate student 

Evelien De Boer MSc Graduate student 

Maart je Hooning MD Graduate student 

jojanneke Van de Swaluw MD Graduate student 

Agnes Van Rosmalen MSc Graduate student 

janneke Verloop MSc Graduate student 

Willem Klokman MD, MSc Statistica I analyst 

Thea Mooij MSc Statistical analyst 

Annemarieke Bruinsma Research assistant 

Geri De Leeuw-Mantel Research assistant 

Shula Grivell Research assistant 

Vivianne Hartog Research assistant 

Nandy Hofland Research assistant 

Esther janssen Research assistant 

Heidi Kruizenga Research assistant 

Marianne Kuenen Research assistant 

Monica Legdeur Research assistant 

Bart Maertzdorf Research assistant 

Gabey Ouwens Research assistant 

Sandra Van den Belt-Dusebout Research assistant 

Annewil Van der Meij Research assistant 

Mirjam Zwaai Research assistant 

Rebecca Bareman Undergraduate student 

Akke Botman Undergraduate student 

Miranda Kramer Undergraduate student 

Vivien Marasigan Undergraduate student 

Sabien Olsthoorn Undergraduate student


Aleman BMP, Van den Belt-Dusebout AW, 

Klokman Wj, Van 't Veer MB, Bartelink H, 

Van Leeuwen FE. Long-tenn causespecific 

mortality of patients treated for 

Hodgkin's disease. J Gin On col 2003; 

21 (18}:]431-3439. 

Barneveld TA, Sasco Aj, Van Leeuwen FE. 

A cohort study of cancer mortality among 

biology research laboratory workers in the 

Netherlands. Cancer Causes Control [in 

pressJ. 

Gilbert ES, Stovall M, Gospodarowicz M, 

Van Leeuwen FE, Andersson M , 

Glimelius B et al. Lung cancer after 

treatment for Hodgkin 's disease:focus on 

radiation effects. Radiat Res 2003; 

159(2):161-173· 

Klaren HM, Van 't Veer Lj, 

Van Leeuwen FE, Rookus MA. Potential 

for bias in studies on efficacy of prophylactic 

surgery for BRCAI and BRCA2 mutation. 

J Natl Cancer Inst. 2003; 95(13):941-947. 

Klip H, Van Leeuwen FE, Schats R, 

Burger CW, For The OMEGA Project 

Group. Risk ofbenign. gynaecological 

diseases and honnonal disorders according 

to responsiveness to ovarian stimulation in 

IVF: a follow-up study of 8714 women. 

Hum Reprod. 2003; 18(9):1951-1958. 

Moil AC, ImhofSM, Cruysberg JRM , 

Schouten-van Meeteren AYN, Boers M, 

Van Leeuwen FE. Incidence of 

retinoblastoma in children bom after 

in-vitro fertilisation. Lancet 2003; 

361 (9354):]09-]1 0. 

Travis LB, Hili 0, Dores GM, 

Gospodarowicz M, Van Leeuwen FE, 

Holowaty E, Glimelius B, Andersson M, 

Wiklund T, Lynch CF, Van 't Veer MB, 

Glimelius I, Storm H, Pukkala E, 

Stovall M, Curtis R, Boice JO, Gilbert E. 

Breast Cancer Following Radiotherapy and 

Chemotherapy Among Young Women With 

Hodgkin 's Disease. JAMA 2003; 

29°(4):465-475. 

Van Leeuwen FE, Klokman Wj, Stovall M , 

Dahler EC, Van 't Veer MB, Noordijk EM, 

Crommelin MA, Aleman BMP, Broeks A, 

Gospodarowicz M, Travis LB, RusselI NS . 

Roles of Radiation Dose, Chemotherapy, 

and Honnonal Factors in Breast Cancer 

Following Hodgkin 's Disease. J Natl Cancer 

Inst 2003; 95(13) :971-929. 

whether responsiveness to ovarian stimulation in IVF is related to the risk of 

benign gynecological disorders af ter IVF. Contrary to our expectation, low 

responders (wo men with less than 4 retrieved oocytes per IVF cycle) tended to have 

higher risks of developing ovarian cysts and uterine leiomyoma than high 

responders. 

Late effects of cancer treatment Now that curative treatrnent is available for a 

substantial group of cancer patients, it is increasingly important to evaluate how the 

occurrenee oflate complications of treatrnent affe cts their long-term survival. We aim 

to evaluate the risk of second cancers and cardiovascular disease (CVD) after radioand 

chemotherapy (RT and CT) for Hodgkin's disease (HD) (n=2,800), testicular 

cancer (n=2,750) and breast cancer (n=8,ooo) over a period of up to 30 years after 

primary treatrnent. 

In 2003, we completed collection of treatrnent and follow-up data for 8,000 survivors 

ofbreast ca neer treated between 1970 and 1985 in The Netherlands Cancer Institute 

(NKI) or the Dr. Daniel den Hoed Cancer CenterjEUR (DDHK). We studied 

mortality from CVD in a group of 3900 patients who were treated for early stage 

breast ca neer between 1970 and 1981. CVD mortality was not only compared 

between irradiated and non-irradiated patients, but also between the study population 

and the general female population. For 92% of the patients medical status was 

complete up to at least January 1998 and for 34% of the patients follow-up time was 

longer than 20 years. Compared to the general female population, the number of 

CVD deaths in the study population was close to expected. However, when we 

examined CVD risk by treatment, we found a significant 2.2 fold increase in risk for 

irradiated patients compared to non-irradiated patients. Surprisingly, for nonirradiated 

patients, CVD mortality was significantly decreased (by 50%) in 

comparison with the general population, indicating that the risk factor profile of 

breast cancer patients may be protective against CVD. A healthier life style af ter 

breast cancer mayalso play a role. The radiation-related risk increased especially 

after more than 10 years of follow-up, and even more so for patients treated before 

age 45 (RR=2.6). Af ter radiation to the internal mammary chain (IMC), CVD 

mortality was similarly increased for left- and right-sided breast cancer; for chest 

wall radiation, irradiation to the left side revealed significantly increased CVD 

mortality against no radiation (RR=2.5). 

In conclusion, there is a large breast cancer survivor population at increased risk for 

CVD. As for currently treated patients, RT techniques and indication (especially for 

the IMC-field) have changed substantially since the patients in our study were 

treated. Furthermore, the present results are not applicable to the tangential fields 

used in breast conserving therapy (BCT); in the near future we will evaluate patients 

treated by BCT from 1981 to 1986. 

Our study on late effects of treatrnent for testicular cancer focuses on the long-term 

risk of second malignancies following radiation (with an emphasis on 

gastrointestinal and urogenital tract cancers) and the risk of CVD after CT. In 2003, 

we continued collection of treatrnent and follow-up data on 2,750 5-year survivors of 

testicular cancer treated between 1965 and 1995 in the NKI, the DDHK, Groningen 

University Hospital, and hospitals participating in the Eindhoven Cancer Registry 

(IKZ). In total lAoO medical records have been reviewed and 2,100 letters have been 

sent to general practitioners to obtain follow-up information. Up till now, we have 

received complete follow-up data for 79% of patients. 

In a previous study on tamoxifen's late effects, we showed that the risk of poorpro 

gnosis endometrial cancers increased with duration of tamoxifen treatrnent for 

breast cancer. In 2002, we started a new nationwide study to confirm our previous 

findings and to also obtain insight into the mechanism of tamoxifen-induced 

carcinogenesis. In 600 cases of uterine malignancies af ter breast cancer we will 

investigate whether the clinicopathologic characteristics and prognosis of uterine 

malignancies differ between women with and without long-term tamoxifen 

treatment for breast cancer. We will also search for tamoxifen-specific genomic 

aberrations (using comparative genomic hybridization) and differences in geneexpres 

sion (using micro-array techniques). A nationwide signaling system was set up 

to obtain fresh tumor samples of uterine malignancies af ter breast cancer; so far 

20 samples have been collected.

Female survivors of HD have a strongly elevated risk ofbreast cancer, but factors 

responsible for this excess risk are not weil known. We investigated the effects of 

radiation dose, CT, and reproductive factors in a nested case-control study in a 

cohort of 770 female patients who had been diagnosed with HD before age 41. 

Detailed treatrnent information was collected for 48 case patients who developed 

breast cancer S or more years af ter diagnosis of HD and 17S matched control 

subjects. The radiation dose was estimated to the area of the breast where the case 

patient's tumor had developed and to a comparable location in matched control 

subjects. 

The risk ofbreast cancer increased significantly with radiation dose; patients who 

received 38.S Gy or more had a RR of 4.S times that of patients who received less than 

4 Gy. Patients who received both CT and RT had significantly lower risk than those 

treated with RT alone (RR=0.4S). Among patients who received RT only, the 

association with radiation dose was much stronger than among patients treated with 

CT and RT. Sixty-nine percent of control subjects treated with RT and more than six 

cycles of CT, but only 9% of those who received RT alone, reached menopause before 

age 4I. Reaching menopause before age 36 was associated with a 90% reduced risk 

of breast cancer. 

We concluded that breast cancer risk rises with increasing radiation dose up to at 

least 40 Gy. The substantial risk reduction associated with CT appears to reflect its 

effect on menopausal age, suggesting that ovarian horrnones promote tumorigenesis 

af ter radiation has produced an initiating event. These results were confirmed in a 

internationally pooled analysis to which the above data were contributed. 

In collaboration with Division VIn (Annegien Broeks, Laura Van 't Veer) we are 

examining the contribution of cancer susceptibility genes to the development of 

radiation-induced breast cancer. To examine the separate and joint effects of 

radiation exposures and ATM germline mutations on breast cancer risk, we will 

perform a case-control study of contralateral breast cancer. 

In close collaboration with the department of Paediatric Oncology of the 

Emma Children's HospitaljAMC and the Department of Medical Oncology ofthe 

AMC we are involved in several studies oflate effects of childhood cancer treatrnent. 

In 2003 we assessed long-term cause-specific mortality in 1,378 s-year survivors who 

were treated for childhood cancer between 1966 and 1996; follow-up was complete 

for 99% of survivors. After a median follow-up of 16 years, 120 patients had died. 

The overall RR was 17-fold increased compared to the general population, mainly due 

to excess mortality from the primary childhood cancer. Our cohort experienced an 

excess risk of 7 deaths per 1,000 person-years. The RR appeared to stabilize at an 

about 4 to S-fold increased risk of death after 20 years of follow-up. OnIy af ter 

20 years of follow-up excess mortality from other causes exceeded excess mortality 

from primary childhood cancer. After 2S years of follow-up, the RR of death due to 

other causes was stillS-fold increased.

XIII DIVISION OF 

DIAGNOSTIC ONCOLOGY 

DEPARTM ENT OF CLI N ICAL CH EM ISTRY 

D Bakker, JH Beumer, T Buckle, W Douwenga, N Franke, M Kemper, N Proost, 0 Van Tellingen 

Division Head, Group leader Marc Van de Vijver 

Marc Van de Vijver MD PhD Head Division XIII 

DEPARTMENT OF CLINICAL CHEMISTRY 

Willem Nooijen PhD Academic staff 

Hans Bonfrer PhD Academic staff 

OlafVan Tellingen PhD Academic staff 

Heleen Bardelmeijer G raduate student 

Marleen Kemper Graduate student 

Jan Hendrik Beumer Graduate student 

Niels Franke Undergraduate student 

Mariët Ouwehand Technical staff 

Wanda Douwenga Technical staff 

Tessa Buckle Technical staff 

Tiny Korse Technical staff 

Dorothé Linders Technical staff 

DEPARTMENT OF NUCLEAR MEDICINE 

Cornelis A Hoefnagel MD PhD Academic staff 

Philippe C Baars MD Academic staff 

Saar H Muller PhD Academic staff 

Michiel Sinaasappel PhD Academic staff 

Ferida Sivro-Prndelj MD Academic staff 

Renato A Valdés Olmos MD PhD Academic staff 

Marina S Kartachova MD Clinical fellow 

Jules lavalaye MD PhD Registrar 

Saskia Baank Technical staff 

Martine C Bakker Technical staff 

Carolien M Bauhuis Technical staff 

Petra A Doodeman Technical staff 

Hester Frentzen Technical staff 

Iris J Van den Heuvel Technical staff 

Bert A Pool Technical staff 

Gerrie van Steeg Technical staff 

DEPARTMENT OF PATHOLOGY 

Frans Hogervorst PhD Academic staff 

Daphne De Jong MD PhD Academic staff 

Wolter Mooi MD PhD Academic staff 

Petra Nederlof PhD Academic staff 

Hans Peterse MD Academic staff 

Hester Van Boven MD PhD Academic staff 

Marc Van de Vijver MD PhD Academic staff, 

Head Division XIII 

loes Van Velthuysen MD PhD Academic staff 

laura Van 't Veer PhD Academic staff 

Annuska Glas PhD Post-doc 

Douwe Atsma Technical staff 

lucie Boerrigter-Barendsen Technical staff 

Marie-Christine Hermus Technical staff 

leonie Delahaye Technical staff 

Jeroen Poodt Technical staff 

Carla Schippers-Gillissen Technical staff 

Pharmacological studies in mice. We had previously established that the brain 

penetration of paclitaxel is very low due to the presence of P-glycoprotein (P-gp) in 

the blood-brain barrier (BBB). P-glycoprotein inhibitors may help to increase the 

brain penetration, but in most cases the inhibition ofP-gp was not complete. We 

have now investigated the activity of a number of novel so-called 3rd generation P-gp 

blockers, including zosuiquidar, laniquidar and ONT-093. However, the results with 

these compounds that we re selected for their potency and selectivity in drug 

developments programs, were also very modest. These results highlight how difficult 

it is to completely annul the activity of P-gp in the blood-brain barrier. To investigate 

the role of P-gp in protecting intracranial tumors from systemic chemotherapy, we 

have developed a set of experimental intracranial tumor models showing infiltrative, 

invasive or expansive growth pattem with intact andjor locally disrupted BBB 

properties, similar as observed in primary and secondary brain tumors. To all ow for 

non-invasive in vivo bioluminescent monitoring of tumor burden during these 

chemotherapy intervention studies, tumor celllines we re stably transfected with 

luciferase. The expansively growing U87MG xenografts were highly vascularized 

with leaky BBB properties as determined by contrast-enhanced MRI. By contrast, the 

murine melanoma cellline KI73S displays a highly infiltrative pattem using existing 

vasculature with (almost) completely intact BBB properties. Other celllines 

(MDA MB 435, MeIS7) gave rise to tumors of intermediate phenotype. Chemotherapy 

intervention studies show that paclitaxel is more active against intracranial MDA 

MB 43S xenografts in P-gp knockout mice than in wild-type mice. MelS7 brain 

xenografts were not responsive to paclitaxel chemotherapy, whereas KI73S 

xenografts were also more sensitive to paclitaxel chemotherapy when growing in 

P-gp knockout mice; however, only when given in combination with the potent P-gp 

inhibitor elacridar. These KI735 cellline intrinsically express P-gp, however, at 

much lower levels than present in the tumor vessels as determined by western 

blotting. Overall, these preliminary results are in agreement with the idea that P-gp 

in the BBB can protect intracranial tumors from systemic chemotherapy. More 

research to confirm these results is ongoing. In addition to brain tumor modeis, we 

are also developing other luciferase tagged tumor models for therapeutic intervention 

studies. 

We are also investigating the role of P-gp in the disposition and 

pharmacodynamics ofET-743, a marine-derived anticancer agent. We have shown 

that this compound is a P-gp substrate by in vitro transport studies. Interestingly, 

P-gp appears to confer drug resistance in tumor cells only when present at very high 

levels. Apparently the membrane permeability of ET-743 into tumor cells is high and 

P-gp activity in unselected P-gp expressing celllines to low to reduce drug 

accumulation. P-gp knockout mice are by a factor 1.S to 2-fold more sensitive than 

wild-type mice. Pharmacokinetic studies indicate that metabolism is the main route 

of elimination. 

DEP RTMENT OF NUCLEAR MEDICINE 

APE Besnard, E Deurloo, R Haas, D De Jong, PC Baars, CA Hoefnagel, MS Kartachova, SH Muller, 

F Sivro, RA Valdés Olmos 

Reproducibility of Iymphoscintigraphy after excisional breast lesion biopsy 

We have previously shown a 100% reproducibility for preoperative 

lymphoscintigraphy using intralesional tracer injection in patients with T 1 

-T 2 

No 

breast cancer. To establish the reproducibility oflymphoscintigraphy before and af ter 

breast lesion excisional biopsy, intratumoral administration of 99 m Tc-nanocolloid 

guided by palpation, ultrasound or stereotaxis followed by lymphoscintigraphy was 

performed in 20 patients the day before surgery. At least two weeks af ter surgery a

second lymphoscintigraphy was perforrned following tracer injection around the 

biopsy cavity. Similar drainage pattern and sentinel nodes af ter excisional biopsy 

we re found only in 6 patients (reproducibility 30%). Reproducibility for the axilla was 

50% and for the internal mammary chain 20%. These findings imply that sentinel 

node biopsy should be perforrned prior to excisional biopsy to prevent false negative 

sentinel node results. 

Radio-guided surgery for therapeutic excision in non-palpable invasive 

breast eaneer The value ofradio-guided tumor excision (RTE) in addition to a guide 

wire was studied in 65 patients with clinically occult breast cancer. Intratumoral 

injection of 99 m Tc-nanocolloid was guided by ultra sound or stereotaxis and patients 

received a localization wire after scintigraphy was performed. The data were 

compared with retrospective data from 67 consecutive patients who underwent wiredirected 

excision only. Complete excision was obtained in 54/65 patients (83%) who 

underwent combined RTE/wire-Iocalisation and in 43/67 patients (64%) who 

underwent wire-directed excision only (P=0.014). RTE was found to be an 

independent predictor of complete excision of the invasive component (p=O.012) 

following alogistic regres sion model. 

Internal mammary ehain measurements A high agreement between two 

operators was found in the gamma camera measurement of depth, lateralization 

(later al distance to the midsternalline) and height (distance to the sternal noteh) on 

the internal mammary (IM) lymph nodes. The study included 64 patients referred to 

for lymphoscintigraphy before radiotherapy to the IM chain and was performed 

using an automated computer software application. Correlations of 0.95 were found 

for depth and lateralization, and 0.99 for height. 

High resolution PET of head and neek in unknown primary tumors An 

accuracy of77% (sensitivity 77%, specificity 80%) was found for high resolution 

r8p_PDG PET in detecting unknown primary tumors in he ad and neck in 18 

consecutive patients presented with squamous cell carcinoma neck metastases. 

Histological confirmation of an occult primary tumor during panendoscopy under 

general anesthesia was used as the gold standard. Resolution of he ad and neck PET 

was first improved using a 128 x 128 matrix with halved transverse POV and 

correction (attenuation and scatter). In a nuther optimization, the protocol 

incorporated the use of a 256 x 256 matrix with full POV in order to obtain an 

optimal visualization of lower structures of the neck such as the larynx and 

hypopharynx. 

Imaging of tumor apoptosis Planar and SPECT images with 99 m Tc Hynic-rh 

Annexin V were performed in 8 patients with non-small celIlung cancer in the 

context of an ongoing phase H/lIl multicenter study (M02APO) aimed to establish 

the value of this tracer in predicting response to platinum-containing chemotherapy. 

Gamma camera tumor uptake measurements, performed before and within 72 hours 

after first course of chemotherapy, are being evaluated in relation to the tumor 

response as assessed by CT. A second multicenter study in patients with small cell 

lung cancer scheduled to receive platinum-based chemotherapy (M03APS) was 

recently activated. 

In II patients with follicular lymphoma, increase in tumor uptake as assessed by 

99 m Tc Annexin V scintigraphy was concordant with the appearance of apoptotic 

morphology as determined by fine needle aspiration cytology in the measurement 

of tumour apoptosis before and after localized low-dose radiotherapy (HORA-I). 

Increase in apoptosis was associated with a favorable therapy response. 

Co-registration and matching of SPECT and CT is being used to optimize the 

quantification of 99 m Tc Annexin V tumor uptake and the variations in apoptosis after 

therapy. Tumour delineation is firstly effectuated on CT and subsequently uptake 

quantification is perforrned on SPECT. 

Anke Witteveen Technical staff 

Guido Brink Quality staff 

THE NETH ERLANDS CA NCER 

INSTITUTE FAM ILY CANCER CLINIC 

Frans Hogervorst PhD Academ ic staff 

laura Van 't Veer PhD Academic staff 

Sen no VerhoefMD PhD Academic staff 

Irma Kluijt MD Academic staff 

Marjanka Schmidt PhD Post-doc 

Marielle Ruijs MD Cl inical fellow 

Marieke Bronk Genetic consultant 

Anja Van Rens Genetic consultant 

Gea Wigbout Genetic consultant 

Mohamed Achahchah Technical staff 

Rob Plug Technical staff 

RoelofPruntel Technical staff 

Paul Van der Voort Technical staff 

Majella Boutmy-de lange Technical staff 

Bart Maertzdorff Research assistant 

Renate Udo Research assistant 

Rob Van der Spruit Research assistant 

Guido Brink Quality staff 

DEPARTMENT OF RADIOLOGY 

Jelle Teertstra MD Academic staff 

Peter Besnard MD Academic staff 

Kenneth Gilhuijs PhD Academic staff 

Wim Koops MD Academic staff 

Robert Kröger MD Academic staff 

Saar Muller PhD Academic staff 

Frank Pameijer MD PhD Academic staff 

Warner Prevoo MD Academic staff 

Claudette loo MD Clinical fellow 

Eline Deurloo MD Graduate student 

William Klein Zeggelink Graduate student 

Angelique SchliefTechnical staff 

Marja Van Vliet Technical staff


Chen B, Van den Brekel MW, Buschers W, 

Balm Al. Van Velthuysen ML. Validation of 

tissue array technology in head and neck 

squamous cell carcinoma. Head Neck. 2003 

Nov; 25(11):922-3°. 

De Jong 0, Glas AM, Boerrigter L, 

Hermus MC, Dalesio 0, Willemse E, 

Nederlof PM, Kersten MJ. Very late relapse 

in diffuse large B-celilymphoma represents 

clonally related disease and is marked by 

germinal center cell features. Blood. 2003 

JUlI; 102(1):]24-7· 

Deurloo EE, Tanis Pl. Gilhuijs KG, 

Muller SH, Kroger R, Peterse JL, 

Rutgers El. Valdés Olmos RA, 

Schultze Hooi LJ. Reduction in the number 

of sentinellymph node procedures by 

preoperative ultrasonography of the axilla 

in breast cancer. Eur J Cancer 2003; 

39:1068-1073. 

Estourgie SH, Nieweg OE, 

Valdés Olmos RA, Hoefnagel CA, 

Kroon BBR. Review and Evaluation of 

Sentinel Node Procedures in 250 Melanoma 

Patients With a Median Follow-Up of6 

Years. Ann Surg Onco12003; 10:681-688. 

Haas RL, Valdés Olmos RA, 

Hoefnagel CA, Verheij M, De Jong 0, 

Hart AA, Bartelink H. Thallium-201- 

chloride scintigraphy in staging and 

monitoring radiotherapy response in 

follicular lymphoma patients. Radiother 

Onco12003; 69:]23-]28. 

Haas RLM, Poortmans Ph, De Jong 0, 

Aleman BMP, De Wit LGH, Verheij M, 

Hart AAM, Van Oers MHJ, 

Van der Hulst M, Baars JW, Bartelink H. 

High Response rates and lasting remissions 

by low dose involved field radiotherapy up to 

4 Gy in indolent lymphomas. J. Clin. 

Oncol. 2003; 21:2474-2480. 

Hogervorst FB, Nederlof PM, Gille )J, 

McElgunn CJ, Grippeling M, Pruntel R, 

Regnerus R, Van Welsem T, 

Van Spaendonk R, Menko FH, Kluijt I, 

Dommering C, VerhoefS, Schouten JP, 

Van 't Veer Lj, Pais G. Large genomic 

deletions and duplications in the BRCAI 

gene identijied by a novel quantitative 

method. Cancer Res. 2003 Apr 1; 

63(7):1449-53· 

DEPARTM ENT OF PATHOLOGY 

DAtsma, L Boerrigter-Barendsen, L Delahaye, A Glas, M-C Hermus, B Maertzdorf, R Olivier, J Poodt, 

A Witteveen, H Van Boven, F Hogervorst, D de Jong, P Nederlof, H Peterse, L Van 't Veer, 

L Van Velthuysen, M van de Vijver 

M LPA for the detection of H ER2 amplification in breast carcinomas The 

HER2 status of a breast carcinoma is an important parameter in guiding optimal 

therapy. Current techniques, such as immunohistochemistry (IHC) and fluorescence 

in situ hybridization (FISH), have several drawbacks. We have developed a new, easy 

to perform, high-throughput quantitative PCR-based method, Multiplex Ligationdependent 

Probe Amplification (MLPA), which reliably detects HER2 gene copy 

numbers in tumors. We included a probe for topoisomerase-2 since clinical 

implications of its overexpression has been suggested. Comparison of HER2 MLPA 

test and Southern blot analysis on 46 fresh-frozen breast carcinomas revealed a 

IOO% concordance. On 80 formalin-fixed paraffin embedded tissues MLPA and 

FISH showed very similar results; 40 out of 42 tumors with HER2 amplification by 

FISH also showed amplification by MLPA. There was also good concordance 

between MLPA and IHC 0,1+ and 3+ cases: 31 IHC negative (0 or 1+) tumors showed 

no amplification by MLP A, and 32 out of 33 cases with IHC 3+ showed amplification 

by MLPA. In contrast, the IHC 2+ group contains both tumors with and without 

36.00 

34.00 

32.00 

30.00 

28.00 

~ 26.00 

Q) 

.c 24.00 

E 22.00 

::l 

r:: 20.00 

~ 18.00 

8 16 . 00 

Q) 14.00 

; 12.00 

C) 10.00 

8.00 

6.00 

4.00 

2.00 

0.00 

co 0 N 

N .,. 

tumor sample 

Chromosome 17 probes, HER2 amplicon 

21 .00 ,.------------------------------__. 

20.00 

19.00 

18.00 

17.00 

16.00 

15.00 

14.00 - 

13.00 . 

12.00 I-----~--'----. 

11.00 

10.00 

9.00 

8.00 

7.00 

6.00 

5.00 

4.00 

3.00~ ............... -~ _____ _ 

1.00 - 

2.00 [:!~2;S~~~~r!~~~~§!::~§:~~:!~==~~~~=~=J 

0.00 

.., 

N N N 

CL 

________________________________________________________________________ ~----------~~~{~~(.~,.[~r..~[~~li~~~~~r.~ft------J 

amplification by FISH and MLPA. An additional 157 tumors we re analyzed with a 

different probe set with 7 additional probes on chromosome 17 including genes 

directly flanking the HER2 gene (less than I Mb up and downstream) and 

topoisomerase-2, to obtain information in the amplicon size. Topoisomerase-2 was 

amplified in 17% of the tumors that showed amplification of HER2, tOPO-2 

amplification without co-amplification of HER2 was not observed. 

In conclusion, MLP A has been demonstrated to be an efficient and effective test for 

the molecular diagnosis of HER2 amplification in breast carcinomas. 

Very late relapse in diffuse large B-cel1 Iymphoma represents c1onal1y related 

disease and is marked by germinal center cel1 features Patients with diffuse 

large B-celllymphoma (DLBCL) rarely show relapse af ter 4 years of complete 

remis sion (CR). In this study, we addressed the following questions: (I) Does laterelapsing 

DLBCL represent clonally related disease or a second malignancy; and 

(2) is there a characteristic biologic background? In 10 of 13 DLBCL patients with 

relapse after 4 to 17 years, a clonal relationship was established based on identical 

IgH-sequences andjor identical bc12-IgH translocation. Most tumors (77%) showed 

features of germinal center (GC) cells, as defined by expression of CDIO, bcl-2, and 

bcl-6 protein and ongoing immunoglobulin heavy chain variabie region (VH) 

hypermutation. A GC phenotype was seen in 8 (20%) of 38 control patients matched 

for age, stage, and (extra)nodallocalization with relapse within 2.5 years (P = .005). 

In conclusion, we have found evidence that late-relapsing DLBCL represents truly 

clonally related disease episodes in most cases and that this clinical behavior may be 

related to the biologic features of GC cells. (Blood. 2°°3;102:324-327) 

Gene expression profiling in Fol1icular Lymphoma to predict clinical 

aggressiveness and to gu ide the choice of treatment Follicularlymphoma (FL) 

is a disease characterized by a long clinical course marked by frequent relapses that 

vary in clinical aggressiveness over time. Therefore, the main dilemma at each 

relapse is the choice for the most effective treatment for optimal disease control and 

failure-free survival while, avoiding over-treatment and harmful side effects. The 

selection for more aggressive treatment is currently based on histological grading 

and clinical criteria; however, in up to 30% of all cases these methods prove to be 

insufficient. Using supervised classification on a training set of paired samples from 

the indolent and aggressive phases of the disease, a gene-expres sion profIle of 

81 genes was established that could, with an accuracy of 100%, distinguish 

indolent from aggressive disease. This profIle accurately classified 93% of the FL 

samples in an independent validation set. Most importantly, in a third series of FL, 

cases where histological grading was ambiguous, precluding meaningful 

morphological guidance, the 8I-gene profIle shows a classification accuracy of 94%. 

The FL-stratification profile is a more reliable predictor of clinical behavior than the 

currently used histological grading and clinical criteria, and may provide an 

important alternative to guide the choice of therapy in FL patients both at 

presentation and at relapse. Currently, we are analyzing if also the capacity to 

transform is a intrinsic feature of FL and may be predicted on the basis of geneexpres 

sion characteristics in the primary presentation. 

Gene expression profiling in ovarian cancer Approximately 25% of patients with 

high stage ovarian cancer will survive for more than 5 years af ter diagnosis. 

Chemotherapy improves progression free survival and overall survival; the impact is 

more prominent in those patients optimally debulked. 

To predict more precisely which patients will respond to chemotherapy based on 

their gene expression profIle we collected 38 tumor samples of patients with known 

long follow-up from the tissue banks of AVLjNKI, AMC and LUMC. To perform the 

gene expression profiling related to clinical outcome, i.e. overall survival, two series 

of ovarian cancers were identified: a series with a favourable outcome (overall 

survival of at least 3 years) versus a series with poor outcome (dead of disease within 

2 years). Optimally debulked ovarian cancer patients were matched for stage, 

histology, and subsequent completed platin-based chemotherapy. Gene expression 

profIling using microarray analysis has been performed in a test series using RNA of 

19 ovarian carcinomas. A gene expression profIle of 25 genes was associated with 

Selected publications (contlnued) 

Bilous M, Dowsett M, Hanna W, Isola J, 

Lebeau A, Moreno A, Penault-Llorca F, 

Ruschoff J, Tomasic G, Van de Vijver M. 

Current perspectives on HER2 testing: a 

review of national testing guidelines. 

Mod Pathol. 2003 Feb; 16(2):173-82. 

Kaas R, Peterse JL, Hart AA, Voogd AC, 

Rutgers EJ, Van Leeuwen FE. The influence 

of tamoxifen treatment on the oestrogen 

receptor in metachronous contralateral 

breast cancer. Br] Cancer. 2003 Mar 10; 

88 (5) :7°7-10. 

Kemper EM, Van Zandbergen AE, 

Cleypool C, Mos HA, Boogerd W, 

Beijnen JH, Van Tellingen O. Increased 

penetration of paditaxel into the brain by 

inhibition of P-Glycoprotein. Gin Cancer 

Res 2003; 9:2849-2855. 

Kok M, Bonfrer JM, Korse CM, De Jong D, 

Kersten MJ. Serum soluble CD27, but not 

thymidine kinase, is an independent 

prognostic factor for outcome in indolent 

non-Hodgkin's lymphoma. Tumour Biol. 

2003 Jan-Feb; 24(1):53-60. 

Klein Zeggelink WFA, Deurloo EE, 

Bartelink H, Rutgers EJTh, Gilhuijs KGA. 

'Reproducibility ofthe assessment of 

tumor extent in the breast using multiple 

image modalities ', Med. Phys. 2003, 

3°:2919-2926. 

Rodenhuis S, Bontenbal M, Beex LV, 

Wagstaff J, Richel DJ, Nooij MA, Voest EE, 

Hupperets P, Van Tinteren H, Peterse HL, 

TenVergert EM, De Vries EG, Netherlands 

Working Party on Autologous 

Transplantation in Solid Tumors. Highdose 

chemotherapy with hematopoietic 

stem-cel! rescue for high-risk breast cancer. 

N Engl] Med. 2oo3]ul 3; 349(1)7-16. 

Ruskoné-Fourmestaux A, Dragisics B, 

Morgner A, Wotherspoon A, De Jong D. 

Paris staging system for primary 

gastrointestinallymphomas. Gut 2003; 

52 :912-913. 

Tanis PJ, Valdes Olmos RA, Muller SH, 

Nieweg OE, Rutgers EJTh, Hoefnagel CA, 

Kroon BBR. Reproducibility of 

lymphoscintigraphy for lymphatic mapping 

in patients with breast cancer. Radiolog}' 

2003; 228:546-551.


Van de Vijver MJ , Peterse H. The diagnosis 

and management of pre-invasive breast 

disease: pathological diagnosis-problems 

with existing classifications. Breast Cancer 

Res. 2003; 5(5):269. 

Van Tellingen 0 , Buckle T, Jonker JW, 

Van der Valk MA, Beijnen JH. 

P-glycoprotein and Mrp1 collectively 

protect the bone marrow from vincristineinduced 

toxicity in vivo. Br ] Cancer 2003; 

89:1776-1782. 

Verkooijen HM, Peterse JL, Schipper ME, 

Buskens E, Hendriks JH, Pijnappel RM , 

Peeters PH, Borel Rinkes IH, Mali WP, 

Holland R; COBRA Study Group. 

Interobserver variability between general 

and expert pathologists during the 

histopathological assessment oflarge-core 

needle and open biopsies of non-palpable 

breast lesions. EurJ Cancer. 2003 Gct; 

39(15):2187-91. 

outcome. At present, we are investigating the predictive value of this profile in a 

validation series of 19 independent tumour samples. In addition, we are exploring 

whether gene expression profiles can be identified related to histological features of 

the tumours (type and grade) . 

Screening for ovarian cancer in high-risk women Women with a genetic risk to 

develop ovarian cancer are offered screening byevaluation of serum CA125 and 

transvaginal ultra sound (TVUS). At present, the data are collected for all women, 

who visited our clinic between 1996 and 2002. The cohort to be evaluated will 

include approximately 550 women. The prevalence of BRCA1j2 mutation carriers in 

this group is expected to be around 25-30%. The purpose of the study is to evaluate 

the outcome and to address the efficacy of the screening tests. Since the number of 

wo men for evaluation of screening is rather low in one institution, a national study is 

aimed to incorporate the data of eight institutions (HEBON study). 

An alternative to screening is to surgically remove the organs at risk by (salpingo) 

oophorectomy. We have evaluated the histopathologic findings of 142 prophylactic 

(salpingo)-oophorectomy. Between 1990 and 2001,142 women had undergone 

this risk-reducing surgery. Of these 142 wo men, 84 women were known BRCA1 

mutation carriers. In tota!, five occult fallopian tubejovarian carcinomas were 

diagnosed; in the follow up after surgery three primary peritoneal carcinomas 

occurred. All eight patients were BRCA1 mutation carriers. The findings support 

that BRCA carriers are at risk and that the risk-reducing surgery consisting of 

salpingo-oophorectomy is recommended for BRCA carriers after completion of 

child bearing or at age 35 years. 

Vrieling C, Col lette L, Fourquet A, 

Hoogenraad WJ, Horiot JC, Jager JJ, 

Bing Oei S, Peterse JL, Pierart M, 

Poortmans PM, Struikmans H, 

Van den Bogaert W, Bartelink H; 

EORTC Radiotherapy, Breast Cancer 

Groups. Can patient-, treatment- and 

pathology-related characteristics explain the 

high local recurrence rate following breastconserving 

therapy in young patients? EurJ 

Cancer. 2003 MaY;39(7):932-44. 

THE NETHERLANDS CANCER INSTITUTE FAMILY CANCER 

CLI N IC 

M Achahchah, M Boutmy-de Lange, G Brink, M Bronk, C Dommering, D Hahn, FBL Hogervorst, 

I Kluijt, R Plug, R Pruntel, A Van Rens, LJ Van't Veer, S Verhoef, P Van der Voort, G Wigbout. 

Since 1995, over 1300 families have sought genetic advice in our hospital. In many 

families, at least two affected relatives were tested by DNA-analysis. DNA-testing 

revealed a germline mutation for breastjovarian cancer syndrome in the BRCA1 

gene in no families, and in BRCA2 in 32 families. This includes hereditary breast 

cancer families counseled at the Academic Medical Hospital, Amsterdam. In 

about 40 HNPCC families, mutations were demonstrated in one of the mismatch 

repair genes hMLH1, hMSH2 or hMSH6. In mutation screening, the 

implementation of MLPA (multiple ligand-dependent probe amplification) analysis 

has proven useful for the detection ofboth mutations in MMR genes as well as in 

the BRCA1 gene. 

In selected colon cancer families mutation analysis for the mutY(e.coli)homolog 

MYH gene has been initiated, as these mutations have been found to be 

associated with some forms of polyposis-like autosomal recessive hereditary colon 

cancer. 

The family cancer clinic contributes data to several multi-center national and 

international research projects, e.g. GEO-HEBON (gene environment interactions 

in hereditary breast and ovarian cancer, see Division XII), DNA-profiling by 

classic cGH and array cGH ofbreast and ovarian cancer patients (see Division VIII), 

a national study of families with Li Fraumeni and variant Li Fraumeni(-like) 

syndrome, studies to elucidate the role of unclassified DNA-variants of the BRCA1 

and 2 genes in hereditary breast cancer, the Breast Cancer Linkage Consortium, 

and others. 

A second clinical geneticist (IK) who had already made longstanding contributions to 

the family cancer clinic as guest consultant, joined our clinical staff. New incentives 

have been given to both the psychosocial studies, in collaboration with the 

department of psychosocial research and epidemiology (Division XII) and clinical 

and genetic research in families with familial stomach cancer and other gastrointestinal 

malignancies (in collaboration with DI. A. Cats, Division X).

DEPARTM ENT OF RADIOLOGY 

H Bartelink, APE Besnard, EE Deurloo, C Loo, W Klein Zeggelink, 5 Muller, JL Peterse, EJTh Rutgers, 

A Schlieff, HJ Teertstra, MJ Van de Vijver, M Van Vliet, KGA Gilhuijs 

Computer-aided diagnosis of lesions in contrast-enhanced M RI of the breast 

The aim of this ongoing study is to investigate whether computerized analysis of 

clinically and mammographically occult breast lesions at magnetic resonance (MR) 

imaging complements clinical reading, how it complements clinical reading, and to 

assess the potential impact of the system. 

A previously developed computerized analysis system was enhanced by training on 

100 breast lesions and validating on 136 independent lesions. 

The performance of reading in clinical setting (Az=0.86) was similar to that of the 

computerized analysis (Az=0.8S; P=0.99). A significant improvement was obtained 

by the combined model (Az=0.9I; P=0.03). Improvement was mostly accomplished 

for lesions graded indeterminate and suspicious by the radiologists. In the combined 

model, an increase in specificity of approximately 20% was observed without 

reduction of sensitivity. Computerized analysis thus complements clinical reading, 

making computer-aided diagnosis feasible. 

Additional breast lesions detected by M R imaging only in patients with 

breast ca neer Obtaining histopathological proof ofbreast lesions only visible at 

MR imaging can be a difficult task. Clinical management of additional findings 

would be greatly facilitated if subgroups of benign lesions can be identified during 

reading with high accuracy, thus obviating the need for further clinical workup. 

One hundred sixteen patients with lI8 proven breast cancers underwent MRI of 

both breasts prior to breast-conserving therapy. The frequency of occurrence of 

additionallesions, the additional clinical workup that was required to manage these 

findings, and the number of patients in which treatment was changed due to more 

extensive disease detected by MRI were established. Performance of clinical reading, 

and performance of the combination of clinical reading and a computerized analysis 

system were obtained for the task of identifying the benign lesions among all 

additionallesions. 

MRI showed a larger extent of the index lesion in 10% of the patients (n=I2). 

Furthermore, fifty additionallesions in 40 patients (3S%) were detected. Twenty 

lesions were proven to be malignant, 30 were benign (7 pathology-proven, and 23 by 

mean follow up of 21 months). Additional clinical workup before surgery was 

performed in 78% of the additionallesions (39/So). In almost half of the cases 

(49%), the les ion was visible at clinical workup and fine-needle aspiration was 

performed to obtain the diagnosis. Treatment was changed to a more extensive 

approach due to larger extent of the index lesion (n=I3, lI%) and due to additional 

malignant lesions (n=I2, 10%). 

Multi-modality analysis and radiological guidance in breast conserving 

therapy This year, our MARGINS database which is aimed at providing preoperative 

estimation of tumor margins, and guidance to surgery has increased to 

include the data of 697 patients with breast cancer. As of yet, the database includes 

mammograms (679), ultrasound images (6S9), MR images (299), and x-ray 

specimen photographs (481). 

U sing our MARG INS database measurements of the largest tumor diameter in 

mammography, ultrasonography, contrast-enhanced magnetic resonance imaging, 

and at pathology were retrieved retrospectively for lOS patients eligible for BCT. 

The overall random variations in the assessment of tumor extent were on the order 

of 3 mm (I SD) with only little differences of about 0.3 mm between the four 

techniques. The dependence of the random variations on tumor size was found 

significant (p

(P15% eumulative lifetime risk for breast caneer were 

screened by six monthly clinical breast examination (CBE) and yearly 

mammography and MRI with independent readings. Tumor characteristics of 

detected cancers were compared to those of two different age-matched control 

groups. 

The aims of our study were to assess the value of regular surveillance and 

the efficacy of Magnetic Resonance Imaging (MRI) as compared with 

mammography. 

In this prospective multicenter study 1874 women including 322 gene 

mutation carriers were screened. The results of this study will soon be published, 

the conclusions are that MRI is twice as sensitive than mammography, and 

intensive surveillance including MRI facilitates early diagnosis ofhereditary 

breast cancer. 

Clinical predictors of outcome after targeted chemoradiation; impact of tumor volume: 

The purpose of this study was to determine prognostic factors for outcome after 

chemoradiation in advanced head and neck cancer patients. The radiologie part of 

this study was to determine pre- and post-tumor volume in ninety-two inoperable 

patients with stage IV squamous cell carcinoma of the eral cavity, oro/hypopharynx 

and supraglottic larynx who were treated with selective-targeted chemoradiation 

(RADPLAT intra-arterial). 

The significance of circumscribed mammographic masses in the surveillance of BRCA 1/2 

gene mutation carriers: In this study, in twenty nine BRCAI/2 mutation carriers 

under surveillance who developed 31 breast cancers between 1994 and 2001 at a 

mean age of 44.2 years and 63 women with 67 breast cancers as controls in the 

same period at a mean age of 53.8 years, a review of mammographies in the two 

groups was do ne by seven radiologists, to establish technical assessment, with 

special attention for circumscribed lesions and estimated probability of malignancy. 

In the mutation carriers seven (23%) circumscribed non-calcified mammographic 

masses were found and in the controls three (4.5%) p = o.O!. These masses were 

proven to be malignant. In both groups around 70% of these lesions we re detected 

by the patient. The masses were situated in breasts with a good interpretable breast 

pattern. The conclusion is that BRCAI/2 gene mutation carriers have a higher 

percentage of circumscribed non-calcified mammographic masses that proved to be 

malignant, compared to women from families with a high risk for breast cancer but 

inconclusive DNA. 

Monitoring breast cancer response to neoadjuvant chemotherapy with dynamic Contrast 

Enhanced MRI mammgraphy (CE-MRM ): This pilot study was designed to 

determine if CE-MRM is useful to evaluate tumor response during chemotherapy in 

locally advanced breast cancer ( LACB) and to demonstrate with confidence residual 

tumor af ter therapy. In a group of 42 women with LABC, CE-MRM was performed, 

the first time at the beginning of treatment, the second after 2 courses of 

chemotherapy and the third after completion, before operation was planned.

Criteria for evaluation were:tumor size ( RECIST-criteria ), earlyenhancement, 

pattem of enhancement and wash-out. The tumor size measured on the third MR 

was correlated with pathology (tumor specimen af ter breast-sparing therapy or 

mastectomy), that was regarded as the gold standard. Preliminary results show good 

correlation between fmding of CE-MRM and pathology. 

Figure XIII.2: Uptake of contrast agent in the tumor at contrast-enhanced magnetic resonance imaging 

before neoadjuvant chemotherapy (left), after two courses (middle), and after the last course (right). The 

extent and intensity of the area of contrast uptake diminishes during the chemotherapy, signaling that 

chemotherapy has effect.

BIOMETRICS DEPARTMENT 

CLINICAL STUDIES AND OVERVIEWS 

Division Head, Group leader Otilia Dalesio 

Otilia Dalesio MSc Head 

Ninja Antonini MSc Academic staff 

Ellen Peter PhD Academic staff 

Harm Van Tinteren MSc Academic staff 

Danny Baars Technical staff 

Roman Bohoslavsky MSc Technical staff 

Wilma Deurloo Technical staff 

Marjolijn De Waal MSc Technical staff 

Jitske Dijkstra Technical staff 

Annelies Hiemstra Technical staff 

Lea Kooij Technical staff 

Jan Lieverst MSc Technical staff 

Jolanda Maaskant Technical staff 

Marianne Mahn-Schaefers MSc Technical staff 

Ingrid Mandjes MSc Technical staff 

Carla Modder Technical staff 

Gemi Moppi Technical staff 

Anneke Reinders-Som Technical staff 

Suzanne Reitsma Technical staff 

Jolanda Remmeizwaai Technical staff 

Danny Roberts Technical staff 

Dea Storm Technical staff 

Gijsbert Suren Technical staff 

Jane Tyrrell Technical staff 

Heleen Vaessen MSc Technical staff 

Renato Valdes Olmos Technical staff 

Ludy Valkenet MD Technical staff 

Emile Van der Donk Technical staff 

Tony Van der Velde Technical staff 

Els Van Oosterwijk Technical staff 

Wil Van Waardenberg Technical staff 

Anneke Wals Technical staff 

Lidwina Wever Technical staff 

Els Willemse Technical staff 

Lonny Ziblat Technical staff 

Hans Oosting PhD Guest 

Tineke Meindertsma Secretary 

Overview of randomized trials in prostate ca neer Since 1989, we coordinate 

an overview of all randomized trials of the treatment of pro state cancer. The 

Prostate Cancer Trialists' Collaborative Group (PCTCG) was created to bring 

together these randomized trials and currently counts more than 4°° investigators. 

The statistical analyses are made centrally in collaboration with R Peto and his 

group at the University of Oxford. The initial cycles of this collaboration assessed the 

use of maximum androgen blockade (MAB). For the current cycle of the overview 

(2002-20°7) we have obtained funding from the EC Quality of Life program. To 

identify all relevant randomized trials, a wide variety of sources is used, including 

literature databases and data nets, the Cochrane Controlled Trials Register, abstract 

books from relevant meetings and contact with individuals, trial groups and 

pharrnaceutical companies. We have identified 345 randomized trials that included in 

excess of 70,000 patients. For each trial data on each of the patients randomized is 

sought from the different trialists, institutes and cooperative groups. These data are 

analyzed centrally and the results of the different trials are combined appropriately to 

pro duce an overall estimate of the differences between the treatments assessed. 

Trials identified are grouped according to the general question they are addressing. 

The most important questions identified are: 

Immediate vs. deferred hormonal treatment 

Neoadjuvant treatment vs. not 

Local treatment vs. not 

Chemotherapy vs. not 

Maximum Androgen Blockade (MAB) 

(23 trials) 

(14 trials) 

(9 trials) 

(30 trials) 

(31 trials) 

The trials studying the effect of immediate endocrine therapy as compared to 

deferred endocrine treatment in patients with no recorded metastases (M -) are 

currently sufficiently mature to combine into an overview. Already 91% of the 

14.000 patients entered in these trials have been obtained and analyzed. A 

preliminary report of the results of the effects on prostate cancer mortality and nonpro 

state cancer mortality has been discussed with the PCTCG and a final report is 

being prepared for publication. Results are still subject to embargo. 

Collaboration with national and international trial groups We have developed 

collaborations with several cooperative groups; industry and clinical research 

organizations, and we function as partner for clinical trials, being involved from the 

generation of the idea, protocol setting, the planning, and providing randomization 

services, quality assurance, data handling and statistical expertise. 

The collaboration with the Dutch Chest Physician Association (NVALT) already 

resulted in four randomized studies in which in excess of 30 hospitals participate. 

In the first study treatment with docetaxel + carboplatin in a three weekly schedule 

was compared to docetaxel in a weekly schedule in patients with stage IIIb and 

IV NSCLC. Af ter accruing 297 patients in 3 years, the study was stopped following 

the advice of the Independent Data Monitoring Committee based on the results of 

an interim analysis. Weekly docetaxel was found to have insufficient activity in this 

category of patients. Results have been presented during ASCO meeting last ApriL 

The NVALT2 study, performed in collaboration with the British Medical Research 

Council, is aimed at evaluating the bene fit of neo-adjuvant chemotherapy in operable 

NSCLC. More than twenty percent of the 4°0 patients randomized have been 

entered by the NVALT. NVALT3 is a study of chemotherapy comparing Carboplatin 

and gemcitabin versus carboplatin and paclitaxel in elderly patients with non-small 

celllung cancer, with main endpoints toxicity and quality oflife. The study is carried 

out in selected centers where special nurses are trained to support the study and 

29 patients have already been included. NVALT4 study is a randomized placebocontrolled 

study of docetaxeljcarboplatin with celecoxib or placebo in patients with

locally advanced or metastatic non-small celllung cancer. Accrual to the study began 

end of200} 

Collaboration with the Dutch Colorectal Cancer Group (DCCG) has resulted in a 

randomized study of sequential versus combination chemotherapy in patients with 

previously untreated advanced colorectal carcinoma (CAIRO study). Seventy 

institutes participate in this study randomizing and average of 32 patients per month. 

Currently more than half of the planned 640 patients have already been entered in 

the study. We have developed an information system and have made it available to an 

Extranet of investigators involved. 

The departrnent has also been involved in 2 trials that could have a major impact on 

clinical practice and that were published this year. The study of the Dutch Breast 

Cancer Study group on breast cancer patients with at least four tumor-positive 

axillary lyrnph nodes but no distant metastases, in which 885 patients were 

randomized by ten Dutch centers was extensively analyzed and the results were 

published in a prestigious medical journal. The second study is the HIPEC trial, in 

which patients with peritoneal carcinomatosis of colorectal cancer were randomized 

between standard adjuvant chemotherapy (SFU jLeucovorin) on outpatient basis and 

experimental therapy consisting of cytoreduction with Hyperthermic Intra PEritoneal 

Chemotherapy (HIPEC), followed by the same systemic chemotherapy. Patients in 

the HIPEC arm had an increased survival as can be seen in the graph (Figure I). 

Subgroup analyses did not suggest interactions between treatrnent and the factors 

studied (Figure 2). 


Rodenhuis S, Bontenbal M, Beex LV, 

Wagstaff J, Richel DJ, Nooij MA, et al. 

High-dose ehemotherapy with 

hematopoietie stem-cell reseue for high-risk 

breast eaneer. N Engl J Med 2003; 

349(1)7-16. 

Verwaai VJ, Van Ruth S, De Bree E, 

Van Sloothen GW, Van Tinteren H, 

Boot H, et al. Randomized trial of 

cytoreduction and hyperthermie 

intraperitoneal ehemotherapy versus 

systemie ehemotherapy and palliative 

surgery in patients with peritoneal 

earcinomatosis of eolorectal eaneer. J Clin 

Oneo12003; 21 (20):3737-3743. 

Van Tinteren H, Hoekstra OS, Boers M. 

The need for Health Teehnology 

Assessments of PET. Eur J Nucl Med 

MolImaging 2003; 30(10):1438. 

1.0 

0.8 

0.6 

0.4 

0.2 

51 

0.0 - 54 

overall survival by treatment 

-.. -, 

~, 

t, 

" 

L __ .. ___ ", 

19 

26 

L. _____ ..... ____ , 

p= 0.032, logrank test, two-sided 

-l·l.._..., 

\ "1 

~----------·---·---1 

5 

11 

~------------- 

HlPEC 

control 

4 HIPEC 

Herder GJ, Van Tinteren H, Comans EF, 

Hoekstra OS, Teule GJ, Postmus PE, et al. 

Prospeetive use of serial questionnaires 

to evaluate the therapeutic effieacy of 

18F:fluorodeoxyglueose (FDG) positron 

emission tomography (PET) in suspected 

lung ca neer. Thorax 2003; 58(1):47-51. 

Lont AP, Besnard AP, Gallee MP, 

Van Tinteren H, Horenbias S. 

A eomparison of physieal examination 

and imaging in determining the extent of 

primary penile eareinoma. BJU Int 2003; 

91 (6):493-495. 

0 

12 

24 

months from randomization 

36 

Figure 1 

Overall survival of Hyperthermie Intra Peritoneal Chemotherapy 

in peritonea I eareinomatosis of eoloreetal origin 

HIPEC Control 

Subgroup 

events/N events/N 

Sex 

male 17/34 18124 

female 

- 

7120 13127 

Age 

BOyrs 5/16 10/15 

Site of tumor 

appendix 2n 7111 

colon 20141 21/34 

rectum 

- 

216 3/6 

Origin 

primary 14/30 16/28 

10/24 15123 

Subgroup estimates 99%, Overall 95% confidence intervals 

Overall result 24/54 31/51 ~ 

0.55 ( 0.321 , 0.95 1 ) 

0.5 

HIPEC better ~ ~ no HIPEC better 

Figure 2

In addition, several studies from the pharmaceutical industry in colorectal, ovarian 

and breast cancer have been handled and analyzed by the Department and reports 

have been presented at international meetings. 

With support from the Dutch Cancer Society, we collaborate with the radiotherapy 

department in carrying out a N ational study in he ad and neck cancer comparing 

radiotherapy with intra-arterial cisplatin to radiotherapy to cisplatin IV. In 2003, af ter 

160 patients have been included, a confidential interim analysis was prepared for the 

Independent Data Monitoring Committee. Furthermore, support for 3 new 

randomized radiotherapy studies in lung and breast cancer has been obtained. 

In Europe and many other countries, children with renal tumours (nephroblastoma 

or Wilms' turnours) are treated according to a protocol developed by a specialized 

group within the SlOP collaboration. SlOP is an internationalorganization for the 

treatment of cancer in children (Société International d'Oncology Pediatrique). 

Survival in these patients has improved dramatically over the past 30 years and 

current protocols in general aim at a reduction of post-operative treatment, in 

particular subgroups. In 2001 a new protocol was developed including a study 

question with the design of a randomized trial. Patients with stage 11 and 111 cancer 

are randomized between the current standard of docetaxel versus a reduced schedule 

without docetaxel. At the NKljAvL a randomization facility has been set up to allow 

24 hoursj7 days per week randomization of patients. This construction facilitates 

central randomization also for Brazil (BWTG) and the UK (UKCCSG). Other groups 

participating are the French group (SFOP) and the German group (GPOH). Patients 

from the Netherlands and other European countries are randomized in our 

randomization servers by the lKA. Central data collection and processing is done at 

the Biometrics Department. Twice yearly an overview report is produced and 

discussed by the group. Various questions of the previous study (including a 

randomization for stage 1 intermediate risk patients) are currently analyzed and 

reported. 

The functionality of our randomization system (ALEA) is fully deployed as an 

independent service for in-house and external groups and is now being extended for 

the registration and data collection of a pilot study introducing micro-arrays in 

clinical practice. Recentlya protocol has been approved for 'guided introduction of 

micro-arrays in breast cancer diagnosis and treatment'. In this pilot study, known as 

RASTER, surgeons from five institutes, including the NKljAvL, will register woman 

under 61 years of age with breast cancer into the ALEA system. ALEA serves as a 

central data collection facility and notifications will be sent automatically, bye-mail 

andjor fax, to the relevant investigators, e.g. the local pathologist, the pathology 

department ofthe NKljAvL, Agendia and specialists. This notification system will 

guarantee 'real-time' processing of information thereby preventing unknown or 

missing information at essential time-points in the process. The pilot study is setup 

to develop an efficient working infrastructure for national implementation of microarrays 

in breast cancer. The following logistics scheme has been developed. In this 

figure, the ovals indicate the ALEA-registration system. The straight lines indicate the 

communication flow through ALEA with the collaborators of the study. Different 

entities will have access only to pieces of information about a patient relevant for 

their activities. 

Positron emission tomography (PET) is a non-invasive imaging procedure, providing 

images ofbiological activity of cells. PET is different than other imaging techniques, 

such as magnetic resonance imaging (MRI) or computed tomography (CT) , which 

produce structural (anatomical) information. The clinical implementation of positron 

emission tomography is emerging in the Netherlands. In the lKA-region, the first 

PET facility was opened at the VUMC in 1997. Two multicenter randomized studies 

in non-small celllung cancer (NSCLC) we re designed and performed by the 

Comprehensive Cancer Center Amsterdam (IKA) in which the lung physicians of the 

AvLjNKI participated. The first study investigated the additional value of PET in the 

conventional work-up of a patient with suspected operabie and resectable NSCLC. 

Here PET showed to be able to reduce the number of unnecessary thoracotomies by 

50%. The second study positioned PET at the contact of patients with suspected 

NSCLC in order to investigate the potentialof substituting the conventional series of 

diagnostic tests by one image and subsequent confirmation. Data of this study are 

currently being analyzed. Several other studies with PET in NSCLC are performed or

ongoing: among others, the role of PET in the detection of solitary pulmonary 

nodules and the role of PET as an early diagnostic tooI in stage lIla lung cancer. The 

fusion of PET with CT-images is the most recent development and currently under 


ICT PROJECTS 

For more than a decade we have been involved in research and development in the 

area ofTelematics applied to healthcare. We have developed information and 

communication technology (ICT) projects and we have obtained funding under the 

various framework programs of the EC. 

An online randomization system for clinical trials is one of the services developed 

over these years of research. A major upgrade of the system was initiated in 2002 in 

the framework of the EU funded RTD project BePrO. The service was recognized by 

major European Cancer Clinical Trials Data centers as an efficient tooI. In 2003, the 

Biometrics department created a consortium of five N ational cancer centers in 

France, UK, Poland, Italy and Netherlands and two ICT service providers, and 

submitted a proposal to the EU requesting funding for the setup and operation of a 

N ational Randomization service in each of the participating countries. The proposal 

was submitted to the eTEN (Trans European Networks) programme under the 

acronym TENALEA and passed the evaluation phase in October 2003. Contract 

negotiations are expected to commence at the beginning of 2004- NKI win 

coordinate the project. In contrast with the various Research and Technology 

Development (RTD) programs, the focus of the eTEN program is a market validation 

and service initiation. 

A hospital actively involved in clinical trials may be entering patients in 100 active 

clinical trials, and be faced with over 30 different procedures for patient 

randomization. The main objective of the TENALEA project is to implement uniform 

European procedures for patient randomization in clinical trials and make these 

procedures available as a service on a Trans European Network of Clinical Trials Data 

centers. A uniform European procedure win improve accessibility and efficiency and 

consequently accelerate the numbers of patients accrued to the clinical trials. The 

current operational service in the Netherlands win be replicated in the partner 

countries sites. These sites will carry out a validation of the requirements for 

localization of the service. The output of this validation win provide the necessary 

information for the European procedures, and the initiation of a full deployment of 

the randomization service. 

The tech nology used includes state of the art standard products for general-purpose 

components, while niche functionality is provided by building blocks resulting from 

previous R&D projects (funded under BIOMED, FP3 and FP4 DG.xIlI Telematics 

Applications for Health). More specificaIly, a pilot service platform was set up in FP4 

Randomization Service - Building Blocks

project RUBIS, which provides a secure environment for validating telematics 

applications in clinical practice. The network layer of this platform consists of 

standard mechanisms for transport (lP) , encryption (SSL, IPSec) and authentication 

(PKI + X.509). On top of the network layer, application level interoperability is 

provided by the adoption of established XML standards in the domain ofhealthcare 

(CDISC ODM, HL7 RIM and CENTC251 prEnvI3606 EPR). At the application 

level , end us er services can be operated in clinical practice providing maximum 

interoperability with the clinical ICT environment while complying with strict rules 

of security, privacy legislation and local institutional security policies. 

The main objective of the new EC project is to conduct the tasks that have a specific 

European dimension: 

1. To replicate the service to four sites, mainly to stress the hypothesis of the 

business case to attract investors and to show commercial short term and long 

term benefits; 

2. To assess localization requirements for service replication to UK, Italy, France 

and Poland; 

3- To compile a standardized European procedure for patient randomization in 

clinical trials on the basis of the localization requirements and established 

standards; 

4. To implement a shortlist oflocalization requirements, such as translations to 

French, Polish, Dutch and Italian; 

5. To evaluate the market potential for a Trans-European Network for patient 

randomization in clinical trials; 

6. To produce a business plan for the fuH deployment of the randomization service. 

It is expected that the project will be completed in 2005.

RESEARCH FACILITIES 

The research divisions within the NKIjAvL are supported by a number of 

indispensable research service facilities. Each facility comprises a group of experts 

who provide information, instruction and service assistance and houses state of the 

art equipment. Besides the facilities described below, the institute has a central 

cryogenic storage facility, a glassware kitchen, a(n) (electro-) technical workshop and 

an audiovisual department. 

BIOMETRICS 

Otilia Dalesio MSc, Ninja Antonini MSc, Danny Baars, Roman Bohoslavsky MSc, Astrid Diemeer, 

Wilma Deurloo, Marjolein De Waal MSc, Jitske Dijkstra, Annelies Hiemstra, Lea Kooij, Jan Lieverst 

MSc, Jolanda Maaskant, Marianne Mahn-Schaefers MSc, Ingrid Mandjes MSc, Tineke Meindertsma, 

(aria Modder, Gemi Moppi, Hans Oosting PhD, Ellen Peter PhD, Anneke Reinders-Som, 

Suzanne Reitsma, Jolanda Remmeizwaai, Danny Roberts, Dea Storm, Gijsbert Suren, Jane Tyrrell, 

Heleen Vaessen MSc, Renato Valdes Olmos, Ludy Valkenet MD, Emile Van der Donk, 

Tony Van der Velde, Els Van Oosterwijk, Harm Van Tinteren MSc, Wil Van Waardenberg, 

Anneke Wals, Lidwina Wever, Els Willemse, Lonny Ziblat 

In addition to its own research activities (described separately), the Biometrics 

Department is the Data Center of the Institute and provides the infrastructure for 

clinical and fundamental research concerning bio-statistical support, centralized 

patient data collection and documentation, data processing and coordinated 

administration and monitoring of clinical trials. The statisticians and data managers 

collaborate in several clinical and research projects both within the institute and for 

national and international multicenter studies. 

The departrnent maintains a Tumor Register database, with information on patients 

with benign tumors, pre-malignant and malignant tumors seen in the institute since 

1977. This database is a valuable resource for research currently containing more 

than 100,000 registrations. Between I July 2002 and 30 June 2003 a total of 5891 

new tumors were seen in the hospita!, including 4794 malignancies, 78 premalignancies 

and 1019 non-malignancies. 

Approximately one quarter (1297) of the malignancies were seen only for a second 

opinion, while three quarters (3497) were actually treated in the Institute. Breast, 

gastro intestinal, lung and pleura, and genitourinary tract are the most frequently 

seen sites. 

In excess of 200 questions from investigators within the institute have been 

processed and data was extracted from the database to be used in specific projects.

Separate databases of several of these projects have also been developed. 

Support of the medical divisions for central administration, patient registration and 

data collection of clinical trials is provided through the department's Trial Office. 

Between July 2002 and June 2003, 593 patients were entered in one of78 trials open 

to accrual. Fifty percent of them we re included in studies sponsored by the institute, 

23 percent in studies supported by the NKB or by the Health Insurance, and 

37 percent in studies sponsored by the industry. Half of patients we re entered in 

early clinical trials (pilot, phase land 11). Two of the department data managers 

visited 31 institutes affiliated to the Dutch Chest Physician Organization (NVALT), 

all over the country to collect data on the patients entered to the 3 randomized trial 

of the group. Furthermore, central registration and randomization services for 

10 multi-center studies were provided. 

In addition to handling the evaluation routing of new clinical trials and the monthly 

production of a trial bulletin, the department maintained the web based information 

system TRION that gives access to trial summaries, nursing protocols, patient 

information and informed consent. A working group for quality assurance was 

created to study the last European Directive relating to the implementation of good 

clinical practice in clinical trials and to review the procedures (SOPs) within the 

department in the light of possible changes in the law associated with the N ational 

application of the directive. 

The course on Medical Statistics for researchers and physicians that has been so 

successful previous years was organized for the seventh time. 

COMPUTERS AND NETWORK SUPPORT 

Tjeerd Canrinus, Peter Bouman, Evert-Jan De Kruijf, Anne-Ruth Elffers, Luud Hagendoorn, 

Ferry Hoeben, Cecilia Hulscher, Fred Kasiander, Ton Luts, Saskia Nijk, Albert Pauw, Mia Pietersen, 

Jan Stoltz, Iija Van de Pavert, Elma Van der Sleen, Gert-Jan Van der Stroom, Franck Van Kerkwijk, 

Hein Van der Woord , Richard Van Schie, Anne Wierda 

The central ICT department provides general IT support for all research divisions as 

well as specific 'site wide' services (such as e-mail facilities) for all personnel. Tasks 

include: management of routers, Ethernet switches, file servers, job-and-database 

servers, configuration of network software on PCs and Apple Macintosh computers 

of end users and, if necessary, the development and maintenance of custom server 

andjor client side software. 

The I CT department provides IT services in line with the late st technology standards. 

The institute uses Gigabit Internet technology. A storage infrastructure for images 

will be introduced soon. Also, VPN (Virtual Private Network) to exchange data 

between protected zones of the NKI infrastructure and people outside the

organization in a secure way, has been introduced successfully, as well as Server 

Based Computing. 

DIGITAL MICROSCOPY 

Lauran Oomen PhD, Lenny Broeks PhD 

The Digital Microscopy Facility provides four imaging systems: two confocallaser 

scanning microscopes and two microscope setups with cooled CCD-cameras. Each of 

these systems has its own special functionality and together they form a 

complementary set of instruments. A large group of researchers from all Research 

Divisions make use of one or more of these central microscope systems. 

The confocals all ow high-resolution microscopie imaging ofliving cells. Most 

important herein are the studies of protein synthesis, assembly, transport and 

dynamics in living cells, making use of the green fluorescent protein (GFP) and its 

analogs (YFP, CFP). The increasing demand for this type of studies has led to 

unacceptable waiting times for timeslots on the confocals. Therefore the necessary 

steps have been undertaken to acquire a third confocal, which will be installed at the 

beginning of next year. 

The color microscopejCCD-camera system is primarily suited to directly capture 

digital color images from (immuno-)histochemically stained tissue sections, but has 

also been frequently used to capture bright field (phase-contrast or DIC-Nomarski) 

time-lapse image series from living cells. The two main subjects studied we re factors 

influencing cell migration and cell division. This year, a fully motorized microscope 

stand has been installed to replace the manually operated stand. This greatly 

enhances the system's capabilities to acquire complex image series from living cells: 

now multiple-channel fluorescence imaging can be easily combined with bright field 

imaging (PhC or DIC). In addition image sequences can now be recorded at multiple 

positions in the sample, hereby improving the efficiency of the system. 

This year we started to store all data recorded on the microscope systems on a central 

server provided by the Computers and N etwork support facility. All users now have 

direct access to their data from any computer system in the Institute. 

EL CTRO 

M IC OSCOPY 

Jero Calafat PhD, Hans Janssen 

The facility consists of an FEl CMIO and an FEl Tecnai 12 G 2 transmission electron 

microscope and Leica (cryo)-ultramicrotomes. In addition to its own research 

activities (see Division of Cell Biology), the facility assists researchers in their 

ultrastructural morphological analysis of macromolecules, viruses, cells and tissues. 

Immunoelectron microscopy on ultrathin cryosections reveals a great wealth of 

structural details in the 2 to 100 nm range providing information that can be 

obtained in no other way. This technique is used for the following studies: the exact 

localization of antigens jmolecules in the cell organelles; localization of two different 

antigens simultaneously using colloidal gold of different sizes; trafficking of 

molecules in the cell; localization of molecules in the cell during signal transduction 

events and cell-cell and celi-matrix interactions. 

F OW CYTOM TRY 

Anita Pfauth, Frank Van Diepen 

The Flow Cytometry facility provides instrumentation and technical assistance for 

flow cytometric analysis and sorting. Current instrumentation includes a multiple 

color Moflo high-speed sorter, a FACStar sorter for more fragile cells and sorting on 

DNA-content. Both sorters are equipped with three lasers and a device to perform 

sortings in 96-well plates. Operators carry out the sorting experiments; researchers 

can carry out the experiments on the analyzers independently after they have had 

training on one of the machines. The facility has four analyzers: two double-laser 

FACSCalibur, one single-laser FACScan and one FACScan suited to measure GFP

and YFP together. Measurements on cell surface markers, cell cycle progression and 

apoptosis are examples of experiments possible on these analyzers. Different 

software packages to analyze the data are available via the research computer 

network. 

LABORATORY AN I MAL DEPARTM ENT 

A large part of the oncology research in the institute is carried out using rodent 

models (mice). The Laboratory Animal Facility is located in a separate wing in order 

to maintain a good microbiological barrier. The department provides the 

infrastructure for animal experiments: breeding and housing, rendering of 

biotechnical assistance, sanitation of strains and production of gnotobiotic ani mals. 

The facility possesses a large isolator unit for gnotobiology, sanitation and 

quarantine, plus a separate microbiological diagnostic laboratory for monitoring 

animais, with special emphasis on sick animal screening. 

LABORATORY ANIMAL PATHOLOCY AND HISTOLOCY 

Marco Breuer PhD, Jurjen Bulthuis, Kees De Goeij, Elly Mesman, Ji-Ying Song, Marion Tjin-A-Koeng, 

Martin Van der Valk PhD, Evert Van Garderen PhD 

Due to the increasing use of animal models in tumor biology and clinical cancer 

research, it is expected that trans genie and knock-out mouse pathology will gain 

further interest, and in collaboration with clinical pathology, will play an important 

role in present day ca neer research. Experienced technicians perform a wide range of 

technical procedures, including all standard methods such as tissue processing, 

sectioning and staining, cryosectioning and immune histology. The pathologists 

perform histopathology on request, either on aregular, project-related basis, or 

incidentally, for most research groups in the institute. The staff also gives advice on 

subjects involving post-mortem examination, tissue sampling, handling and 

processing and different staining methods. 

LlBRARY 

Suzanne Bakker MSc, Irene Benne, Ina Goede, Meena Kanhai, Truud Kroeskamp 

The Central Cancer Library, serving the research, clinical, nursing and paramedical 

departments of the institute, is incorporating an ever increasing number of electronie 

publications and services. The electronic journal collection as weIl as all the library 

services (catalogue, current awareness bulletins, acquisitions, nursing literature 

databases, NKI-bibliography) are now accessible for all NKI-staff from home through 

the NKI Virtual private network connection (VPN). The print and electronic journal 

holdings of the NKI-library are integrated in the PubMed system for easy reference 

(available through the hypertext link to PubMed on the library webpages). The 

bibliography ofNKIjAvL publications, including hypertext linking to the electron ic 

full-text, will be fully integrated with the library catalogue. Digital formats of pictures 

and other archivalia will be catalogued for easy access in the ne ar future and further 

support ofthe activities ofthe 'NKIjAvL Historisch Genootschap' (The NKIjAvL 

Oncology History Interest and Study Group). Electronic books and applications on 

CD-ROM are now a substantial part ofthe collection; some will be made available on 

the Intranet, many more win be available for loan to NKIjAvL staff. Electronic 

discus sion lists are in use for communication with library us ers (e.g. CKB 

AANVRAG EN @nic.surfnet.nl is available for requests ofliterature retrieval or 

interlibrary loans). Nowadays most of our interlibrary loan requests are Hlled within 

2-3 working days and most of them are received as scanned images bye-mail. The 

library is responsible for the annual scientometric analyses (based on impact factors 

and citations). The library offers training opportunities and research projects for 

students in library and information science.

MICRO-ARRAY FACILITY 

Ron Kerkhoven PhD, Wim Brugman, Marcel Daemen, Mike Heimerikx, Daoud Sie, 

Arenda Schuurman, Arno Velds Msc 

The production of significant numbers ofhigh complexity human and mouse 

cDNA microarrays was a main aim this past year. We have maintained the high 

production levels of microarrays and we have equipped our printing robots with 

new pinheads with smaller print tips that enable us to print up to 25,000 spots per 

micro array. 

We acquired the non-overlapping NIA mouse cDNA extension set, making our 

mouse cDNA arrays 22K in complexity. We have focused on the production of 

the human I8K and the mouse I5K and 22K cDNA microarrays, of which 4,953 

have been delivered to users. We also acquired the Sanger Center BAC clone set 

(3K) of which 59I arrays were delivered to users. Oligonucleotide arrays were 

produced for siRNA bar code screens (collaboration with division of Molecular 

Carcinogenesis ). 

This year, the Central Microarray Facility fulfilled its role as a national resource of 

DNA microarray technology with continued support of the Dutch Cancer Society. 

As aresult, the facility produced and delivered I,739 cDNA arrays (24% increase) for 

Dutch Cancer Society-sponsored research projects nationwide. 

Protocols for RNA-amplification, labeling and post-processing are now wellestablished, 

which resulted in the production of top-quality microarrays. Initial 

steps have been set to adapt all the protocols to the MIAME standard. This will 

make the CMF-generated datasets eligible for comparative studies performed 

elsewhere. The installation of computer hardware and software was made 

possible thanks to a generous grant from the Josephine Nefkens Foundation. An 

automated backup system at the SARA computer center is used for the back-up of 

all CMF data. All the scanned microarray images are available for users of the facility 

through a new intranet website (http://res-store/microarrays), coupled to a data backup 

system. 

We have seen astrong increase in the use of the software tools for data analysis that 

is available through the website http://dexter.nki.nl. The software on this site executed 

I2,000 jobs and is visited by all our users in the Netherlands. The data quantification 

software Imagene has been upgraded with better spot finding routines and 

quantification algorithms. 

The combination of improved microarray production and analysis has facilitated the 

preparation of good quality datasets by users. Both the human and the mouse 

microarrays have proven their value in experimental research. 

To provide training and education, nine two-day micro array courses were given for 

77 us ers from a number of different research laboratories in the Netherlands. In total 

we have now introduced I92 people to the microarray-research field. 

The website (http://microarrays.nki.nl/) underwent significant revision and is 

adapted for the exchange of technical information and is used for ordering 

microarrays. 

The public genome databank ENSEMBL has been made available to the NKI 

(http://ensembl.nki.nl).Itis set up to gives researchers the opportunity to mine the 

database making use ofbatch processes and the option to insert local databases in the 

ENSEMBL display of the genome. The Celera database, which is hosted by the 

Microarray Facility, has been updated to the late st version. 

MONOCLONAL ANTIBODY UNIT 

Els Groeneveld 

The Monoclonal Antibody Unit provides support and advice concerning antibody 

production, development and characterization. Mice and rats are used for 

immunization. Immunochemical techniques, such as different immunoassays, cell 

staining, immunohistochemistry and Western blotting, are used to characterize the 

monoclonal antibodies and the antibody-antigen interaction.

PEPTIDf SYNTHESIS 

Henk Hilkmann 

The Pioneer synthesizer was installed last year, and after some start up problems, is 

running smoothly now. This synthesizer can monitor two separate synthesis-runs at 

the same time. This in contrast to the Syro 11 robot synthesizer, which can 

synthesize, without monitoring, 60 separate peptides. Peptides were mainly 

produced for researchers from our institute but also for other research institutions 

and universities collaborating within the framework of the Oncology Graduate 

School Amsterdam. As part of routine quality control each peptide is checked by 

HPLC. If needed, mass spectroscopy is performed in collaboration with the Free 

University of Amsterdam and the Department of Pharmacy and Pharmacology of the 

Slotervaartziekenhuis of Amsterdam. 

Most of the synthesized peptides are used for biological studies in tissue culture cells 

or for raising antisera, but also for in vitro binding experiments and for stimulating 

cells. Peptides used for the production of antibodies are now routinely synthesized 

onto a branching lysine core. The result of the multiple antigenic peptides (MAP's) is 

satisfactory. There is an increasing need for peptides with special features. Examples 

include the synthesis ofbiotinylated or acylated peptides, peptides containing 

phosphorylated amino acids or specific other groups and peptides existing of two 

chains of amino acids. It is also possible now to synthesize cyclic peptides or peptides 

with a deuterium containing amino acid. 

RADIONUCLlDF LABORATORY 

H Van Rooij, L janssen, TE Lamers 

In addition to departrnentallaboratories licensed for radioactive work, which are 

present on nearly each floor of the research building, there is a central 

radiochemistry facility (class D, C and B) available for specific and general 

experimental use. The staff of the Radionuclides Laboratory offers help and advice on 

all aspects of radioactive work. 

The department is equipped with up to date gamma and scintillation counters, 

gamma analysers, a tissue oxidiser and HPLC apparatus for on line radiodetection. 

There are also separate facilities for animal experiments with radioactive tracers for 

cancer research (e.g. multidrug-resistance and metabolic studies of radiopharrnaca). 

The departrnent provides regular courses on Radiation Protection, leve15B, for all 

new personnel (including students and guests) whose work entails the use of 

radioactive material. There are also practice courses level 4B for therapeutic 

technicians of the Inhollandcollege.

~EQUENCE FACllITY 

Roelof Pruntel, Frans Hogervorst PhD, Rob Plug 

The sequence Facility offers a service for DNA sequence and fragment analyses to 

the us ers in the research departrnents and the DNA-diagnostics laboratory of the 

Departrnent of Pathology. The Sequence Facility has an important role in the 

diagnostic analyses of patient samples. lts procedures and protocols are therefore 

qualified by RvA (formerly known as Sterlab). In total more than 250 researchers 

representing all divisions make use of the service provided by the facility. The facility 

is equipped with an ABI 3700 capillary sequence machine, which can handle up to 

96 samples simultaneously and a 377XL slab-gel sequencer. This year the sequence 

facility had a throughput of 4000 samples on average per month, an increase of I5% 

compared to last year. This is mainly due to some research projects producing 

thousands of samples for sequencing or fragment analysis. Furthermore, a novel 

application to rapidly screen for known SNPs, the SnaPshot method, has been 

assessed and is going to be implemented for the DNA-diagnostic laboratory of the 

F amily Cancer Clinic. 

TRANSGCNIC MOU 

ERVICE 

PJA Krimpenfort PhD, R Bin Ali, F Van de Ahe 

The production of genetically modified mouse strains has been reorganized last year. 

The result of this reorganization is that the Animal Departrnent supports the more 

routine procedures, such as super-ovulation, embryo isolation and transfer, and the 

Transgenic Mouse Service will focus on the development of new technologies. 

With respect to technology development the Transgenic Mouse Facility will be 

involved in a) the initial implementation of'in vitro' fertilization procedures critical 

for sperm cryopreservation, b) testing new ES celllines for the generation modified 

strains with a more suitable genetic background and c) the development of 

alternative methods gene transfer for transgenesis. New ES celllines include cell 

lines derived from FI hybrids that all ow a much faster production of mutant mice. 

STAFF GENERAL RESEARCH FACllITIES: 

Cryogenic storage: Minze Dijkstra, Erwin Kambey 

Glassware kitchen: Trees Holman-Van Doorn, Moustapha Aboutalib, Esther Holman, 

Erwin Kambey, Harry Kempff 

(Electro)-Technical workshop: Vanessa Rademaker, Wim Kraan 

Audiovisual departrnent: 5 Bakker, 5 Bos, AF Jans, ARM Jagt, evd Hagen, JM Lomecky

CLINICAL TRIALS IN THE 

NETHERLANDS CANCER INSTITUTE 

type of 

cancer study 

ALL SITES 

title 

study coordinator 

in AvL 

phase 

activated 

(closed) 

no pts. 

in AvL 

per 12. 

11.2003 

MOlOCC 

Dose-finding phase I clinical and pharmacokinetic 

study of orally administered irinotecan (CPT-l1) 

once daily for 14 days as single agent or in 

combination with capecitabine twice daily for 

14 days every three weeks in patients with 

advanced solid tumors. 

J H M Schellens 

12-7-2001 

1-8-2003 

22 

M02CCA 

An open label dose adjustment and phase II study 

of E7070 in combination with capecitabine: - Part 1: 

to determine the recommended dose of the 

combination - Part 2: to determine the efficacy of 

the combination in patients with metatstatic 

colorectal cancer (CRC) who are resistent to 

5-fluorouracil and irinotecan. 

JHM Schellens 

IIII 

28-1-2003 

11 

M02GFT 

A Phase I, Randomized, Open-Label, Parallel-Cohort, 

Dose-Finding Study of Elacridar (GF120918) in 

Combination with 2.0 mg Oral Topotecan in 

Cancer Patients. 


3-6-2002 

27 

M02GWO 

A ph ase I, open-label, pharmacokinetic study of the 

safety and tolerability of GW572016 in combination 

with Oxaliplatin/Fluorouracil/Leucovori n. 

JHM Schellens 

17 

M02LGC 

A phase I clinical and pharmacokinetic evaluation 

of oral LY317615 in combination with Gemcitabine 

and Cisplatin in patients with advanced cancer. 


16-5-2002 

8 

M02PPD 

Population pharmacokenetics and pharmacodynamics 

of Docetaxel in relation to genotype of CYP3Ä4 and 

P-glycoprotei n. 


28-1-2003 

14 

M02PPI 

Population pharmacokinetics and pharmacodynamics 

of irinotecan in relation to the genotype of UGT1A1, 

M RP2, CYP3Ä4 and P-glycoprotein. 

JHM Schellens 

28-1-2003 

7 

M02TTA 

A clinical trial on TopoTect (dexrazoxane) in the 

treatment of accidental extravasation of antracycline 

ant-cancer agents. 

J H Schornagel 

111 

15-8-2002 

o 

A phase 1 trial to determine the safety of AP5346 given 

as an IV infusion once a week for 3 consecutive weeks 

in patients with asolid progressive tumor. 

LCJ Van Warmerdam 

4 

Preventie catheter-gerelateerde centraal veneuze 

trombose met LMWH bij intensief chemotherapeutisch 

behandelde patiënten. 

JP De Boer 

111 

18-6-2003 

o

type of title study coordinator phase activated no pts. 

cancer study in AvL (closed) in AvL 

per 12. 

11.2003 

M03D24 Dose-finding and pharmacokinetic Trial of Orally J H M Schellens 2-4-2003 8 

Administered D-24851 to patients with Solid Tumors. 

M03MEN A ph ase I study ofintravenous MEN 4901/T-0128 JHM Schel lens 10-11-2003 2 

administered once every 6 weeks in patients with 

solid tumors. 

M99DIL Safety and tolerability oflong-term administration of APE Vielvoye-Kerkmeer III 7-9-2000 0 

Dilaudid SR (Hydromorphone HCI) in cancer pain. 14-1-2003 

NooRGC Ph ase I maximum tolerated dose (MTD) trial to JHM Schellens 7-9-2000 31 

determine the safety and pharmacokinetics of 7 days 7-11 -2003 

oral administration of farnesyl transferase 

inhibitor Rl15777. 

N010RH Evaluation of pharmacokinetics and safety after oral JHM Schellens 4-9-2001 19 

dose R115777, in at least 21 cancer patients with normal, 

mild and moderate impaired, hepatic function. 

NOlPER A clinical phase I study of combined treatment with M Verheij 9-1-2002 21 

the alkyl-Iysophospholipid Perifosine and ionizing 

radiation in patients with locally advanced solid 

tumors. 

N02MBB Pharmacokinetics and metabolism of {14C} BMS-247550 J H M Schel lens 31-12-2002 8 

in patients with advanced cancer. 

N02MET Mass balance study with ET-743 as an 3 or 24-hous J H M Schel lens 23-10-2002 8 

intravenous infusion to patients with advanced cancer. 10-9-2003 

N030GE Phase I dose escalation and pharmacokinetic JHM Schellens 24-9-2003 9 

evaluation of oral gemacitabine in patients with 

refractory tumors. 

N98CTO A ph ase I study to determine the maximum tolerated WW Ten Bokkei Huinink 19-4-1999 26 

doses ofCarboplatin and Topotecan administered 

intravenously every 28 days to patients with 

malignant tumors. 

N98NAM Phase land pharmacologic study with NAM I-A, J H M Schellens 20-9-1999 25 

a novel ruthenium anti-cancer agent. 11-6-2003 

BRAIN I CNS 

NooGLI Temozolomide and combined immunotherapy in W Boogerd 1/11 30-6-2000 0 

malignant glioma. 3- 6-2003 

BREAST 

E10021 Trial 10021: An IDBBC randomized, double blind, J H Schornagel 11/11 4-11 -2003 0 

placebo-controlled, multi-center phase II trial of 

anastrozole (Arimidex) in combination with Iressa 

(Zd1839) or placebo in patients with advanced 


E109 81 After Mapping of the Axilla: Radiotherapy Or Surgery? EJTh Rutgers III 21-12-2000 186

ire.ctJ1 !f.Rese.arch-Anton..Bems 


cancer study in AvL (closed) inAvL 

per 12. 

11.2003 

E16023 Phase I study of lonafarnib (SCH66336) in combination JHM Schellens 1-8-2003 4 

with herceptin plus paclitaxel in Her 2 neu 

overexpressing breast cancer. 

E22922 Phase 111 randomised trial investigating the role of GMM Bartelink 111 5-11-1996 62 

internal mammary and medial supraclavicular Iymph 31-12-2003 

node chain irradiation in stage 1-111 breast cancer. 

MooPCM Phase 11 study of weekly Paxoral (Oral Paclitaxel) with JHM Schellens 11 8-8-2000 23 

Cyclosporin A for advanced breast cancer. 18-2-2003 

MOlVIC Phase I study of oral Vinorelbine in combination with JHM Schellens 24-9-2001 11 

oral Cyclophosphamide in patients with metastatic 


M02HER HERA: A randomised th ree-arm multi-centre J H Schornagel 111 24-4-2003 

comparison of 1 year and 2 uears of Herceptin versus 

no Herceptin in women with H ER2-positive primary 

breast cancer who have completed adjuvant 

chemotherapy. 

Mo2TEA An open label, randomized multicenter comparative HSA Oldenburg 111 17-9-2002 7 

trial of 5 years adjuvant Exemestane treatment versus 

5 years adjuvant Tamoxifen treatment in postmenopausal 

women with early breast cancer. 

M03ARR The implementation of microarray~ in cancer diagnosis. SC Linn Pilot 21-10-2003 0 

M03HTI Open label, comparative, randomized, multicenter, J H Schornagel 11/11 22-8-2003 

study oftrastuzumab (Herceptin) given with 

docetaxel (Taxotere) versus sequential single agent 

therapy with trastuzumab followed by docetaxel as 

first-line treatment for metastatic breast cancer 

(MBC) patients with HER2-neu overexpression. 

M96ATLAS Reliable assesment of the efficay and safety of EJTh Rutgers 111 29-1-1997 35 

prolonging the use of adjuvant tamoxifen; a large 

simple randomized study. 

M99 BLU Lymphatic drainage analysis through preoperative OE Nieweg Pilot 4-5-2000 0 

intradermal injection of blue dye in patients undergoing 

a modified radical mastectomy. 

NooDCD Single-institution randomized study of S Rodenhuis 111 8-6-2000 63 

doxorubicin-cyclophosphamide versus 

doxorubicin-docetaxel in locally advanced 


NooRLY Reproducability of Iymphoscintigraphy for Iymphatic OE Nieweg Pilot 1-5-2000 0 

mapping in patients with breast carcinoma. 10-12-2003 

NOlNlG Nutrients and insulin-like growth factor (IGF) system. DW Voskuil Pilot 1-3-2002 0 

8-12-2003 

N02ANB Phase 11 study of anastrozole as neoadjuvant treatment ROosterkamp 11 5-2-2003 

in postmenopauzal women with ER+ locally advanced 

breast cancer.


ca neer study in AvL (closed) in AvL 

per 12. 

11.2003 

N02SNB Validation of the sentinel node procedure in patients OE Nieweg 13-8-2002 0 

with a breast les ion after prior excisional biopsy. 

N03BCC Proteomic profile and treatment response, in patients, HH Helgason 11 7-10-2003 4 

with advanced breast cancer, treated with Capecitabine 

chemotherapy. 

N99FCO Feasability and Phase 11 Study ofFEC-tCTC in S Rodenhuis 11 19-5-2000 23 

Oligometastatic Stage IV Breast Cancer. 

PooCOG Psychophysiological study of long-term cognitive W Boogerd 6-6-2002 0 

deficits as a consequence of high-dose chemotherapy: 

study in high-risk breast cancer patients. 

PooMRI M RI-scan bij patiënten met borstkanker ter bepaling KGA Gilhuijs 6-10-2000 2 

van de afmetingen van de afwijking. 

P02MAB Micro-array analysis of breast cancer as a diagnostic M Van de Vijver 27-3-2002 0 

tooi to guide optimal treatment. 

P99SHB Impact van regelmatige controle (screening) bij EJTh Rutgers 1-5-2000 0 

vrouwen met een verhoogd risico op borstkanker 

vanwege een familiaire predispositie. 

GASTRO INTESTINAL 

E40004 CLOCC trial (chemotherapy + local ablation versus VJ Verwaai 111 23-6-2003 

chemotherapy). Randomized ph ase 111 study oflocal 

treatment ofliver metastases by radiofrequency combined 

with chemotherapy versus chemotherapy alone in patients 

with unresectable colorectal liver metastase. 

M01BAX POCASTER trial (J-POuch- Colo-Anale anastomose FAN Zoetmulder 111 6-6-2002 3 

versus Side-To-End colo-rectale anastomose na 

preoperatieve radiotherapie en Totale Mesorectale 

Excisie (TME) bij Rectumcarcinoom; een multicenter, 

gerandomiseerd onderzoek. 

M02COL Een landelijke studie naar de waarde van periodiek A Cats Pilot 9-9-2003 

colonoscopisch onderzoek bij personen met een 

positieve familieanamnese voor colorectaal carcinoom. 

M02PCR Postoperative chemo-radiotherapy after surgical resection H Boot 11 16-1-2003 13 

of gastric and esophagal cancer. A multicenter ph ase 11 

study of a fixed radiotherapy regimen with concurrent 

chemotherapy with capecitabine. 

M02RFA Evaluatie van immuun- en stollingsactivatie na RFA F Van Coevorden Pilot 29-10-2002 14 

of resectie. 

M03CRE Chemoradiation for irresectable (T 4) esophageal BMPAleman 11 26-11-2003 0 

cancer - Phase 11 multi-center study. 

M99CCC CPT-11 in combination with Capecitabine as first line WW Ten Bokkei Huinink 11 6-7-2000 12 

chemotherapy for metastatic colorectal cancer. 2-1-2003 

NooSNC Lymphatic mapping and Sentinel Node Biopsy in F Van Coevorden Pilot 7-2-2001 0 

colonic cancer.

irector..ojResearch Anton Berns 


ca neer study in AvL (closed) inAvL 

per 12. 

11.2003 

N02ECC A si ngle i nstitution ph ase 11 study of ECC H Boot 11 29-8-2002 36 

(Epirubicin, Cisplatin, Capecitabin) in locally advanced 

or metastatic gastric cancer and adenocarcinoma of 

the oesophago-gastric junction and distal oesophagus. 

N02RCA Post operative chemo-radiotherapy after resection of H Boot 1/11 19-12-2002 11 

gastric and esophageal cancer. 

N03PCC Proteomic profile and treatment response, in patients, J H M Schel lens 23-6-2003 8 

with advanced colorectal cancer, treated with flrst-line 

Oxaliplatin and Capecitabine chemotherapy or 

second-line Irinotecan chemotherapy. 

N03PCE Proteomics profile and treatment response, in patients, J H M Schellens 23-6-2003 17 

with advanced gastric or distal esophageal cancer, 

treated with flrst-line Cisplatin, and Capecitabine (ECC) 

or 5FU/LV (ECF) chemotherapy. 

N98ECF A phase 11 feasability study of ECF (Epirubicin, Cisplatin BC Taal 11 10-8-1998 102 

and continuous 5-FU) in locally advanced meastatic 1-11-2002 

gastric cancer and adenocarcinoma at the 

oesophagogastric junction. 

N99SAN Conversion of short-acting octretide to the long-acting BC Taal 11 29-3-1999 38 

compound octreotide (SandostatinLAR): a phase 11 dose 8-12-2003 

fInding study in metastatic carcinoid tumors. 

GYNAECOLOG ICAL 

M02MCA Measurement of cisplatinum adduct in patients with M Verheij Pilot 1-5-2002 

cervical cancer treated with chemoradiotherapy. 

M02RTE PORTEC-2: Postoperative radiation therapy for BNFM Van Bunningen 111 4-3-2003 

endometrial carcinoma - a multicenter randomised 

ph ase 111 trial comparing external beam radiation and 

vaginal brachytherapy. 

HEAO ANO NECK 

E24001 Random ized phase 111 study on the selection of the C Rasch 111 28-11-2002 0 

target volume in postoperative radiotherapy for cervical 

node metastases of squamous cell carcinoma from an 

unknown primary (CUP). 

MooQPI Hypoxia in head and neck tumors: quantiflcation using AJM Balm 22-5-2001 17 

pimonidazole and IdUrd. 

M01AAP Multi-institutional pros[ective clinical trial on the effects FJM Hilgers 11 1-10-2002 0 

of automatic airway protection and occlusion on voice 1-12-2003 

quality, respiratory symptoms, and quality oflife in 

laryngectomized individuals. 

M02PDT A multicenter, single-group study of Foscan-Photodynamic IB Tan IV 22-1-2003 4 

therapy (PDT) in the palliative treatment of patients with 

advanced squamous cell carcinoma of the head and 

neck who have failed prior therapies and are unsuitable 

for curative therapy with radiotherapy, surgery or 

systemic chemotherapy.


cancer study inAvL (closed) inAvL 

per 12. 

11.2003 

M02STA The effect of a statin on the progression of the W Boogerd IV 28-1-2003 

inti ma-media thickness induced by radiotherapy. 

M99RAD Phase III multi-institutional trial oftargeted supradose C Rasch 111 23-11 -1999 143 

cisplatin chemoradiation versus systemic chemoradiation 

in locally advanced squamous cell carcinoma of the head 

and neck. 

NooVOX Development and clinical assessment of the Provox FJM Hilgers 11 29-6-2000 20 

FreeHands HME tracheostoma valve. 1-12-2003 

N02SNL Early detection of Iymph node metastasis of squamous PJFM Lohuis Pilot 3-4-2002 0 

cell carcinoma of the larynx with Iymphatic mapping 

and sentinel node biopsy. 

N98RTB Carotid arterial acclusive disease following radiation W Boogerd IJII 2-10-1998 12 

therpay; A prospective study in patients with a parotid 3-6-2003 

tumor. 

LEUKAEMIA I MOS 

M01H43 Randomized induction and post induction therpay in JW Baars 111 4-12-2001 0 

older patietns (>= 61 yrs of age) with acute 

myelocytic leukemoia (AML) and refractory anemia 

with excess ofblasts (RAEB, RAEB-t) . 

M01Hso A randomized phase 111 study on the effect of JW Baars 111 4-12-2001 2 

thalidomide combined with adriamycin, dexamethason 

(AD) and high dose melphalan in patients with 

multiple myeloma. 

M02H49 Randomised Phase 111 study in eldery patients with a M Kerst III 31-1-2003 0 

multiple myeloma on the value of Thalidomide added 

to Melphalan plus Prednison. 

M99H37 Early intensification by (un)related allogeneic autologous JW Baars 11 7-9-1999 

stem cell transplantation in adult acute Iymphoblastic 10-12-2003 

leukemia. HOVON 37 - CKVO 98-03. 

N98LPB De Lymfomen Plasma Bank. RLM Haas 22-11-2001 0 

LUNG 

E08941 Randomized trial of surgery versus radiotherapy in N Van Zandwijk 111 8-3-1996 31 

patients with stage lila NSCLC after a response to 20-12-2002 

induction chemotherapy. 

E08972 A randomized phase III study comparing induction JSA Beiderbos 111 11-1-1999 47 

chemotherapy to daily low dose cisplatin both combined 14-3-2003 

with high dose radiotherapy in patients with inoperable 

NSCLC stage I, II and low volume stage 111. 

M01ALI A randomized phase 3 trial comparing ALiMTA plus P Baas 111 20-3-2002 10 

best supportive care versus best suppotive care alone 

in previously treated patients with locally advanced or 

metastatic pleural mesothelioma.

're.ctm: oJ.ResearchÁn 

type of title study coordinator ph ase activated no pts. 

cancer study inAvL (closed) in AvL 

per 12. 

11.2003 

M01FLU The influence of fluticasone inhalation on intermediate N Van Zandwijk 11 6-11-2001 40 

markers of carcinogenesis in the bronchial epithelium 

of a high risk population: A double blind 

placebo-controlled randomised phase 11 study. 

MOlNLU Pre-operative chemotherapy in resectable NSCLC. P Baas 111 25-9-2001 3 

M02APO Phase 11/111 study of Technetium RA Valdes Olmos 11/11 28-11-2002 11 

99mTc-Hynic-rh-annexin V for imaging of 

apoptosis in patients with NSCLC. 

M02CCG An open label phase lIa study of CYC202 plus JHM Schellens 14-3-2003 2 

Gemcitabine/Cisplatin in patients with NSCLC. 

M02EPS A phase 11 trial of novel epothilone BMS-247550 in JHM Schellens 11 24-4-2003 0 

patients with Small Cell Lung Cancer refractory to 2-9-2003 

flrst-line chemotherapy. 

M03ACC Open-label Safety Study of ALiMTA (pemetrexed) P Baas 15-12-2003 8 

Single Agent or in Combination with Cisplatin or 

Carboplatin for Patients with Malignant Mesothelioma. 

M03APS Phase 11/111 Study of Technetium 99m Tc-rh-Annexin V RA Valdes Olmos 11/11 5-6-2003 

for Imaging of Apoptosis in Patients with Small-cell 

Lung Cancer. 

M031RS A double blind, placebo controlled, parallel group, JA Burgers 111 3-12-2003 

multicentre, randomised, phase 111 survival study 

comparing ZD1839 (Iressa) plus best supportive care 

versus placebo plus best supportive care in patients 

with advanced NSCLC who have received one or two. 

M031VC A randomized phase 111 study comparing induction JSA Beiderbos 111 22-8-2003 

chemotherapy to daily low dose cisplatin both combined 

with high dose radiotherapy in patients with inoperable 

non-sm all celilung cancer stage I, 11 and (Iow volume) 

stage 111. 

M98TDR A phase 1/11 dose escalation study using three JSA Beiderbos 1/11 11-6-1998 89 

dimensional conformal radiation therapy in patients 8-4-2003 

with inoperable non-small celilung cancer. 

NooALF Endoscopic detection op pre-neoplastic lesions and P Baas Pilot 30-6-2000 28 

carcinoma in the bronchial tree with delta-aminolevunic 

acid fluorescence. 

NooTHM Phase 11 trial ofthe antiangiogenic agent Thalidomidei P Baas 11 21-12-2000 46 

in patients with malignant pleural mesothelioma. 1-8-2003 

NOl PAL Feasibility study of targeting the plasma paclitaxel J H M Schellens 24-8-2001 25 

concentration above 0.1 umol/I during a defined 31-5-2003 

exposure-time in patients with Non-Small Cell 

Lung Cancer. 

N02GLM Phase 11 study of Glivec in malignant mesothelioma. N Van Zandwijk 11 19-4-2002 26 

13-11 -2003



per 12. 

11.2003 

N031CT Phase 1 study of irinotecan and eisplatin with concurrent P Baas 22-8-2003 

thoracic radiotherapy in patients with extensive stage 

small celilung cancer (ES-SCLC) . 

N98CIG Ph ase I study of dose-intensive Cisplatin + Gemcitabine J H M Schellens 8-9-1998 95 

in NSCLC. 

N99HIM Cytoreduetion and Hyperthermie Intra-Thoracic FAN Zoetmulder Pilot 26-1 -2000 24 

Chemotherapy (HITHOC) in patients with pleural 

mestastases opf thymoma or limited mesothelioma. 

X01IRS I ressa, patient named program. N Van Zandwijk IV 2-11-2001 165 

30-1-2003 

LYMPHOMA - HODGKIN'S DISEASE 

E20011 A randomized trial of BEAM plus PBSCT versus single JW Baars 111 23-1 -2002 0 

agent high dose therapy followed by BEAM plus PBSCT 

vpatietns with relapsed Hodgkin's disease. 

E20982 Prospective controlled trial in clinical stages 1-11 JW Baars III 10-12-1998 16 

supradiaphragma- tic Hodgkin's Disease. Evaluation 

of treatment effieacy, (long term) toxicity and quality 

of life in two different prognostie groups. Trial H9. 

NooMEG Ex vivo expanded megakaryocytes and their precursor JW Baars 11-9-2000 0 

cells to fill the platelet gap after high-dose chemotherapy. 10-12-2003 

N98LPB De Lymfomen Plasma Bank. RLM Haas 22-11-2001 0 

LYMPHOMA - NON-HODGKIN'S 

E20981 Chimerie anti-CD20 monoclonal anti body (Mabthera) JW Baars 111 20-1-1999 6 

in remission induetion and maintenance treatment of 

relapsed follieular non-Hodgkin's Iymphoma: a ph ase III 

randomised clinical trial. 

M01H46 A randomized phase III study of chimeric anti-CD20 JW Baars 111 4-12-2001 2 

monoclonal anti body (Rituximab) with 2-weekly 

CHOP chemotherapy (CHOP14) in elderly patients with 

intermediate- or high-risk non-Hodgkin's Iymphoma. 

M01H47 Chlorambueil versus 2X2 Gy involveld field radiotherapy RLM Haas III 22-5-2001 

in Stage lil/IV previously untreated follicular Iymphoma 31-10-2003 

patients. 

M02H44 A randomized phase III study on the effect of chimeric JPDeBoer 111 27-6-2002 4 

anti-CD20 monoclonal anti body (MabThera) during 

sequential chemotherapy followed by autologous stem 

cell transplantation in patients with relapsed B-cell 

non-Hodgkin's Iymphoma. 

M96FNL Treatment with BEAM and total nodal irradiation, both JW Baars Pilot 27-8-1996 

followed by PSCT support as consolidation after 10-12-2003 

remission induction for patients with high risk 

relapsed or refractory low grade (including certain 

subtypes intermediate grade) non-Hodgkins's 

Iymphoma. A feasibility study.

':reao cif~b.Anton--.Bmls 

type of title study coordinator phase activated no pts, 


per 12, 

11,2003 

M99H26 Intensified CHOP of 12-weeks duration plus G-CSF as JW Baars 111 21-4-1999 11 

compared with standard CHOP of 24-weeks duration for 

patients with intermediate prognosis non-Hodgkin's 

Iymphoma (HOVON26). 

NooMEG Ex vivo expanded megakaryocytes and thei r precursor JW Baars 11-9-2000 0 

cells to fill the platelet gap after high-dose chemotherapy. 10-12-2003 

N030FE A randomized phase IIjlll study of eradication therapy JP De Boer IIjll 17-6-2003 0 

with additional oral fludarabine versus eradication therapy 

alone in H.pylori positive gastric MALT Iymphoma. 

N030FP A phase II study of eradication therapy with additional JP De Boer 11 6-10-2003 0 

oral fludarabine in t(11 ;18) positive gastric 

MALT Iymphoma. 

N03RIM Radioimmunotherapy (1311-antiCD20 monoclonal JW Baars 25-6-2003 0 

anti body) as consolidation treatment after remission 

induction for patients with relapsed or refractory 

CD20+ B-cell NHL. 

N97HOR Low dose radiotherapy (2X2 Gy) in low grade RLM Haas 11 12-11-1997 35 

Non-Hodgkin Iymphomas. 

N98LPB De Lymfomen Plasma Bank. RLM Haas 22-11 -2001 0 

M ELANOMA I SKI N 

MooCIS Chemotherapy as neo-adjuvant and adjuvant therapy in CG De Gast 11 1-5-2000 48 

combination with surgery in in-transit or clinically 8-1-2004 

involved Iymph node metastases of malignant melanoma. 

MooTIM Temozolomide adjusted dose with or without combined CG De Gast 111 5-9-2000 79 

immunotherapy with GM-CSF, IFN-alpha and low 9-1-2003 

dose IL-2 in patients with metastatic melanoma. 

NooDER Specific immunotherapy bij intradermal vaccination with JBAG Haanen 24-4-2001 11 

tumor-(associated) peptides and GM-CSF in melanoma. 

A Phase Ijll study. 

NooPEM Aanvullende waarde van positron-emissie tomografie OE Nieweg Pilot 1-3-2001 0 

met FDG bij de opsporing van melanoom metastasen. 

N01TMM Protracted daily temozolomide patients with metastatic CG De Gast 11 28-11-2001 9 

melanoma and poor prognosis. 27-8-2003 

N02PVS Peptide vaccination after sentinel node biopsy and prior JBAG Haanen Pilot 26-6-2003 0 

to elective Iymph node dissection. 

N03CYL Pilot study of topical application of salicylate in ROosterkamp Pilot 24-9-2003 

familial cylindromas. 

N03LAM Longitudinal analysis of melanoma-specific immunity JBAG Haanen 22-8-2003 0 

in stage 111 and IV melanoma patients. 

N99TPC mTHPC mediated PDT and basal cell carcinoma: P Baas Ijll 8-3-2000 7 

a phase 1/11 study to determine optimal treatment 

parameters and its related pharmacological profile.


ca neer study in AvL (closed) in AvL 

per 12. 

11.2°°3 

M ISCELLAN EOUS 

M02BMC Bruikbaarheid van M R-angiografle (M RA) en JJ Hage I1 11 -10-2002 

color-flow-doppler voor de beoordeling van cruropedale 

arterieen voorafgaand aan de microchirurgische 

transplantatie van een fibula lap. 

N01RIT Identiflcations of molecular mechanisms involved in NS RusselI Pilot 7-5-2002 2 

radiation-induced telangiectasia. 

N021GF Nutrients and the Insuline-like Growth Factor (IGF) DW Voskuil I11 15-1-2003 0 

system in premeopausal women with previous 


N02NIG nutrients and the Insulin-like Growth Factor (IGF) DW Voskuil 111 28-1-2003 0 

system in men and women at increased risk of 

colorectal cancer. 

N03THY Therapeutic management of thymoma and thymic LGH Dewit Pilot 25- 11 -2003 0 

carcinoma: - a prospective registration study based on 

preoperative risk assessment oflocal failure. 

N99EOL Endoscopisch echo-onderzoek van de centrale P Baas Pilot 14-12-1999 0 

luchtwegen onder narcose: feasibility study. 

NIET WMO STUDIES 

NWMOO1 DELTA study: Eloxatin, 5FU and folinic acid in patients H Boot IV 12-7-2001 3 

not pretreated for metastatic colorectal cancer. 16-1-2004 

SOFT TISSUE IOSTEOSARCOMA 

E62011 Efficacy and safety of Brostallicin in patients with locally 5 Rodenhuis 11 12-8-2002 

advanced or metastatic soft tissue sarcoma failing one 

prior chemotherapy regimen. 

E62012 Randomised trial of single agent doxorubicin versus S Rodenhuis I11 9-7-2003 0 

doxorubicin plus ifosfamide in the flrst line treatment 

of advanced or metastatic soft tissue sarcoma. 

E62022 Phase 11 study oflressa (ZD1839) in locally advanced 5 Rodenhuis 11 31 -1-2003 

and/or metastatic synovial sarcoma expressing 

HER1/EGFRl. 

E62931 Adjuvant high dose Doxorubicin, Ifosfamide and 5 Rodenhuis 111 13-4-1994 26 

Lenograstim in patients with high grade soft tissue 15-11 -2003 

sarcoma. 

M01EUR EURO-E.W.I.N .G. 99. EUROpean Ewing tumor 5 Rodenhuis 111 18-9-2001 

Working Initiative of National Groups. 

M01ROS Phase 11 study of rosiglitazone in advanced liposarcoma. 5 Rodenhuis 11 24-8-2001 4 

X03GDL Gemcitabine and Docetaxel in patients with antracycline 5 Rodenhuis Pilot 8-9-2003 2 

refractory Leiomyosarcoma. 8-9-2003

~ 



per 12. 

11.2003 

URO-GENITAL 

E30983 Randomized ph ase lijlil study ofTaxol-BEP versus BEP J H Schornagel Iijll 7-9-1999 0 

in patients with intermediate prognosis germ cell cancer. 

E30986 Randomized phase lijlil study assessing J H Schornagel Iijll 7-5-2002 

GemcitabinejCarboplatin and 

MethotrexatejCarboplatinjVinblastine in 

previously untreated patients with advanced 

urothelial cancer ineligible for Cisplatinum based 

chemotherapy. 

E30987 Randomized phase 111 study comparing J H Schornagel 111 1-7-2002 9 

paclitaxeljcisplatinjgemcitabine and cisplatinjgemcitabine 

in patients with metastatic or Ically advanced urothelial 

cancer without systemic therapy. 

E30994 Randomized phase 111 trial comparing immediate versus M Kerst 111 28-1-2003 3 

deferred chemotherapy after radical cystectomy in 

patients with pT3-pT 4, andjor N+ Mo transitional cell 

carcinoma (TCC) of the bladder. 

MooLMT Identiflcation of occult Iymph node metastases in S Horenbias Pilot 14-11-2000 0 

testicular cancer to select patients for adjuvant treatment. 

Feasability of a laparoscopic selective retroperitoneal 

Iym phadenectomy. 

M01CHI Chimerism-inducing immunotherapy in patients with JBAG Haanen 11 5-9-2001 0 

advanced renal cell carcinoma. 

M01IGR Image-guided radiotherapy of the prostate. A study to JV Lebesque 9-10-2001 0 

determine the intra-fraction motion of the prostate and 8-12-2003 

normal tissues in patients receiving exernal beam 

radiation therapy for adenocarinoma of the prostate. 

M01MMI Improvement of Delineation quality in conformal C Rasch Pilot 5-9-2001 0 

radiotherapy of the prostate through multi-modality 8-12-2003 

imaging. 

M02EGF A single-arm multi-centre open lable phase 11 study of M Kerst 11 16-4-2003 0 

orally administered GW572016 as single agent second 18-9-2003 

line treatment of patients with locally advanced or 

metastatic transitional cell carcinoma of the urothelial 

tract. 

M94SAL Salvage regimen incorporating repeated ablative S Rodenhuis 11 4-7-1994 24 

chemotherapy with autologous PSCT, a phase 11 study. 

M97 RAD Phase 111 study for prostate cancer, randomizing JV Lebesque 111 3-4-1997 171 

between two radiation dose levels (68 Gy vs 78 Gy) and 5-3-2003 

utilizing three dimensional conformal radiotherapy 

(CKVO 96-10). 

NooKAH Phase I clinical and pharmacokinetic study to determine J H M Schellens 11-12-2000 32 

the safety of Kahalalide F administered as a daily x 5 25-11 -2003 

over 1 hour infusion every 21 days in patients with 

advanced or metastatic prostate cancer.



per 12. 

11.2003 

NooPRO Laparoscopically assisted perineal prostatectemy. W Meinhardt Pilot 4-9-2000 4 

19-11-2002 

NooVES Laparoscopische bekkenklierdissectie met W Meinhardt Pilot 4-9-2000 6 

vesiculectomie. 19-11-2002 

NOl PEG PEG-Intron in metastatic renal cell carcinoma: CG De Gast 11 5-9-2001 13 

A phase 11 study ofthe Netherlands Cancer Institute. 14-1-2003 

NOlTMR Temozolomide in metastatic renal cell carcinoma. CG De Gast 11 30-5-2001 12 

27-8-2003 

N021LT Prolonged low dose I L-2 and thalidomide in combination CG De Gast 11 11 -12-2002 23 

with radiotherapy to bone andjor soft tissue metastasis 

in progressive metastatic renal cell carcinoma. 

N02PBI Feasability of partial bladder irradiation with an adaptive Onbekend Pilot 14-3-2003 2 

margin strategy. 

N02SBC Feasablility study of sentinel node Iymph scintigraphy W Meinhardt Pilot 10-10-2002 2 

and radioguided surgery for the Iymphnode dissection 

prior to cystectomy for blader carcinoma. 

N02VBB Volume dependence ofbladder shape. LMF Moonen Pilot 22-10-2002 0 

N97PCI Pre-operative combined immunotherapy in renal cell CG De Gast 11 13-8-1998 44 

carcinoma. 8-1 -2004

.. ' " r ~ ., '. ~ \' '. • \ ~ ..... ~ • '. " I.. /. • • 

. ""

Mooienaar W, The Netherlands 

The ins and outs of LP A signaling. 

Moran J .M, USA 

Design and verification of static and dynamic IMRT at 

the University of Michigan, Ann Arbor. 

M usacchio A, Italy 

A safety-belt at the he art of the spindle checkpoint: 

Madr, Mad2 and other crazy proteins. 

Nolan G, USA 

Dominant effector genetics and multidimensional, 

polychromatic analysis of the immune system. 

Noordermeer J, The N etherlands 

Molecular and genetic analysis of nervous system 

development in Drosophila. 

Nusse R, USA 

Wnts, lipids and stem ceUs. 

Peters G, UK 

Oncogene co-operation in pr6INl

·. ·11"0· . . 

PROJECTS SUPPORTED 

BY THE DUTCH CANCER SOCIETY 

Number of 

Title 

Div. 

Principal 

Starting 

Ended 

project 

Investigator 

Date 

NK197-1589 

Implementation of a Pain Education Program (PEP) for cancer 

XII 

F Van Dam 

Dec '98 

Dec '03 

patients with chronic pain by nurses. 

X 

J Passchier 

A Vielvoye-Kerkmeer 

NK198-1724 

Quality oflife assessment in ethnic minorities cancer patients in 

XII 

N Aaronson 

Nov '98 

. the Netherlands: A study ofthe SF-36, the COOP/WONCA charts, 

the EORTC QLQ-C30 and the Rotterdam Symptom Checklist. 

NK198-1729 

Long-term cognitive deficits as a consequence of high-dose 

chemotherapy: a role in high-risk breast cancer patients and 

XII 

X 

F Van Dam 

W Boogerd 

Febr '98 

Febr '03 

high-grade Iymphoma patients. 

NK198-1795 

Rho signaling: role in differentiation and transformation. 

III 

W Mooienaar 

Oct '98 

Dose-volume-effect relationships for local control and normal tissue 

complications for prostate patients treated with three-dimensional 

IX 

J Lebesque 

L Boersma 

Ju1Y '98 

conformal radiotherapy. 

P Koper 

NK198-1833 

Long-term risk of second cancer following treatment of Hodgkin's 

XII 

F Van Leeuwen 

June'98 

disease, testicular cancer and breast cancer. 

IX 

BAleman 

NK198-1839 

The role ofTiam1 in E-cadherin-mediated homophilic adhesion. 

J Collard 

MaY'98 

NK198-1854 

Risk assessment and gene-environmental interactions in breast 

and/or ovarian cancer families. 

XII 

XIII 

F Van Leeuwen 

L Van 't Veer 

Jan '98 

NK198-1858 

The psychological and behavioral impact of genetic counseling for 

colorectal cancer: a prospective multicenter study. 

XII 

X 

N Aaronson 

F Menko 

B Taal 

MaY'98 

June '03 

KUN 99-1915 

Trinucleotide repeat expansion and human cancer: the role of 

MSH3 and FEN-1 in unpaired DNA processing. 

v 

H Te Riele 

B Wieringa 

MaY'99 

J Hoeijmakers 

N KI 99-2°33 

Transport of taxoids across the blood-brain barrier and CNS-tumor 

penetration: a preclinical pharmacologic study and clinical 

'proof of concept' testing in glioma patients. 

X 

XIII 

J Beijnen 

o Van Tellingen 

W Boogerd 

Sept '99 

N KI 99-2°35 

Quantitative assessment of treatment margins and implementation 

IX/XIII 

K Gilhuijs 

Sept '99 

of multimodality imaging in breast conserving therapy. 

XI 

E Rutgers 

IX 

H Bartelink 

N KI 99-2°36 

Molecular analysis ofthe cytotoxic T cell response. 

IV 

T Schumacher 

Nov '99 

N KI 99-2°39 

The role of the integrin alpha6/beta4 in signal transduction 

controlling cell proliferation. 

A Sonnenberg 

Aug '99 

Sept '03 

N KI 99-2°42 

G protein control of cell proliferation and transformation. 

III 

W Mooienaar 

Dec '99 

NK199-2043 

Dose escalation study for non-sm all celilung cancer (NSCLC) 

IX 

J Lebesque 

April '99 

April '03 

using three-dimensional conformal radiotherapy with tight treatment 

J Beiderbos 

margins and functionally optimized radiation treatment plans. 

L Boersma

Number of Title Div. Principal Starting Ended 

project I nvestigator Date 

NK199-2047 Induction of apoptosis by alkyl-Iysophospholipids and related III W Van Blitterswijk Dec '99 

anticancer agents. IX M Verheij 

NK199-2048 Characterization of antigen specific and biological responses of IV H Spits Jan '99 May'03 

CD4+ melanoma specific T cells. X G De Gast 

NK199-2051 Modulation of normal human T cell development by ectopic IV H Spits April '99 Oct '03 

expression ofTAL-l, -2 and LYL in T cell progenitor cel Is. 

NK199-2052 Structure-function studies of cyclin Dl in complex with nuclear II T Sixma Jan '99 

hormone receptors andjor coactivators. 

R Bernards 

NK199-2053 Dephosphorylation of retinoblastoma family proteins by protein II R Bernards Sept '99 

phosphatase 2A. 

NK199-2055 The role of the nuclear inositide pathway in cell cycle progression. 111 N Divecha Dec '99 

NK199-2056 Mechanism of cell cycle regulation and control of cellular lifespan VII M Van Lohuizen May'99 

by the Polycomb-group and oncogene BmÏ1. 

NK199-2058 Development of a mouse model to study the genetic basis of VII A Berns Aug '99 

mesothelioma. 

NK199-2060 Mechanisms of reduced cellular accumulation of topoisomerase I X J Schellens June'99 

inhibitors. VIII A Schinkel 

NK199-2117 Control of cell cycle progression by integrin mediated adhesion. A Sonnenberg April '99 April '03 

NK100-21 43 Modulation of cytochrome P450 (CYP), P-glycoprotein (P-gp), X J Schellens Dec '00 

and BCRP activity in gut wall and liver as major determinants of VIII A Schinkel 

poor oral bioavailability of anticancer drugs: preclinical and X J Beijnen 

clinical studies. 

NK100-21 91 Regulation ofthe G2jM transition by extracellular factors. V R Medema March '00 

NK100-21 92 The role of the Forkhead transcription factor Trident in cell V R Medema Oct '00 

cycle regulation. 

H Clevers 

NK100-2202 Hypoxia and perfusion in human tumors: quantification and VIII A Begg July'oo 

implications for treatment outcome. 

K Haustermans 

XI ABaim 

NK100-2204 The regulation of the expression of hTERT, the catalytic subunit II R Beijersbergen May'oo 

of telomerase during immortalization and tumorigenesis. 

NK100-2208 The function of E2F transcription factors studied in vivo. II R Bernards Jan '00 

NKI00-2209 Role of diacylglycerol kinases in cell cycle progression and 111 W Van Blitterswijk Sept '00 

cytoskeletal organization. 

N Divecha 

NK100-2210 Ras and Rho-signaling networks and epithelial-mesenchymal J Collard Nov'oo 

transition. 

NK100-2212 Design and clinical implementation of optimized irradiation IX E Damen Dec '00 

techniques using static and dynamic intensity modulation. 

B Mijnheer 

J Lebesque 

NK100-221 4 Communication to the mitochondria in apoptosis signaling by IV J Borst July'oo 

death receptors and DNA damaging regimens.

Number of Title Div. principal Starting Ended 


NK100-221 7 Identification and functional characterization of intracellular signaling 111 P Ten Dijke Jan '00 

proteins controlling transforming growth factor-beta induced growth 

arrest. 

NK100-2232 Specific and redundant functions of the retinoblastoma suppressor V H Te Riele Aug '00 

gene family in growth control and differentiation. 

NK100-2233 Genetic and environmental modifiers of cancer predisposition in V H Te Riele Dec '00 

mismatch-repair deficient mice. 

NK100-2247 Improvement of delineation following conformal radiotherapy IX M Van Herk June '00 

through multimodality imaging. 

C Rasch 

P Nowak 

NK1 00-2251 Genetic screen s to identify new components of the VII M Van Loh uizen Jan '00 

senescencejimmortalization pathway. 

NK100-2253 Functional analysis of BM 11-RI NG finger interacting proteins and VII M Van Lohuizen March '00 

application of microarray technology to identify BMl1-responsive 

genes. 

NK100-2255 Dose verification and optimization of radiotherapy using IX B Mijnheer Oct '00 

portal imaging. 

M Van Herk 

NK100-2258 Cell-cell communication and connexin signalling: suppression of 111 W Mooienaar Sept '00 

cell transformation. 

NK100-2263 The role and mechanism of escape from Ras-induced senescence VII D Peeper Jan '00 

in oncogenesis. 11 R Bernards 

NK100-2270 Signal pathways controlling Iymphoma dissemination. E Roos April '00 

NK100-2271 Physiological and pharmacological analysis of the novel multidrug VIII A Schinkel Sept '00 

resistance gene Bcrp (breast cancer resistance protein) using 

Bcrp knockout mice. 

NK100-2282 mTH PC-mediated photodynamic therapy (PDT) : how do drug uptake VIII F Stewart Sept '00 

and distribution influence tumor and normal tissue response? X P Baas 

J Schellens 

NK101 -2382 The impact of prophylactic oophorectormy in women from XII N Aaronson Dec ' Ol 

hereditary breastjovarian cancer families on psychosocial health 

HBoonstra 

and symptom experience. XI M Van Beurden 

NK101 -2415 The role ofHLA-DO and HLA-DM in effective MHC class II-mediated VI J Neefjes Oct ' Ol 

immune activation. 

M Van Ham 

NK101 -2416 Y irradiation and the generation of novel peptides for M HC class I VI J Neefjes Febr 'Ol 

molecules. 

NK101-2417 Analysis of vaccine-induced melanoma specific T cel! immunity: IVjX J Haanen Sept 'Ol 

a combined human and mouse study. IV T Schumacher 

X G De Gast 

NK101 -2419 In vitro isolation of high affinity melanoma-specific T cel! receptors. IV T Schumacher April '01 

H Spits 

NK101 -242O Bayesian adaptive dosing in cyclophosphamide-thiotepa-carboplatin X J Beijnen Febr 'Ol 

(CTC) and mitoxantrone-thiotepa (MT) high-dose chemotherapy 

J Schellens 

regimens. 

S Rodenhuis

Number of Title Div. Principal Starting Ended 


NK101 -2423 Clinical outcome of breast cancer in BRCA1 and BRCA2 carriers. XIII/VIII L Van 't Veer Jan '01 

R Tollenaar 

NK101-2424 Genetic proflling of tumors from patients with a genetic susceptibility XIII P Nederlof Jan '01 

for breast cancer. 

S Verhoef 

XIII/VIII L Van 't Veer 

NK101-2425 Cancer susceptibility genes that predispose for radiation induced VIII A Broeks Nov '01 

breast cancer. XIII/VIII L Van 't Veer 

XII F Van Leeuwen 

NK101-2426 Long-term effects of exposure to DES in utero on the risk ofhormone XII M Rookus Nov '01 

related cancers. 

F Van Leeuwen 

T Helmerhorst 

UU 01-2438 Control of cellular proliferation in normal and tumor cells by the V R Medema Sept '01 

PI3K/PKB/Forkhead pathway. 

B Burgering 

NK101-2473 M RPS and resistance to purine analogs. V P Borst March '02 

J Wijnholds 

NK101 -2476 Identification of the PI M regulatory network by microarray analysis VII A Berns JUly'01 

and high throughput retroviral tagging in compound mutant mice. 

NK101-2479 Structure/function analysis of Muts homologs in HNPCC. II T Sixma Jan '01 

NK101-2481 Subversion ofTGF-beta/Smad signaling in cancer: Analysis of III P Ten Dijke Dec '01 

TGF-beta receptor initiated intracellular responses distinct from 

Smad activation. 

NK101-2483 Initiation and progression of Iymphomas and skin carcinomas in J Collard Dec '01 

Tiam1 mutant mice. 

A Berns 

NK101-2486 Integrin-associated protein regulation, migration and invasion. E Roos Jan '01 

NK101-2488 Regulation of cadherin-mediated ad hes ion by integrin signaling. A Sonnenberg May'01 

NK101-2489 Cloning of novel mammary tumor progression and metastasis genes. VI J Hilkens JUly'01 

VII 

A Berns 

NK101 -2562 T cell tolerance and immunity during spontaneous tumor IV A Kruisbeek Sept '01 

development. 

P Krimpenfort 

NK101-2563 Antigen processing events in cross-presentation of exogenously IV T Schumacher May '01 

acquired antigens. 

N Brouwenstijn 

NKI01-257° Improvement of tumor response by combined modality treatment: IX M Verheij Oct '01 

a translational approach. 

NK102-2575 Microarray analysis of breast cancer as a diagnostic tooi to guide XIII M Van de Vijver Jan '02 

optimal treatment. IX H Bartelink 

XIII/VIII L Van 't Veer 

hlKI 01-2579 The Insulin-like Growth Factor (IGF) system in breast and colorectal VIII D Voskuil Oct '01 

carcinogenesis: dietary intervention and molecular studies. XIII/VIII L Van 't Veer 

E Kampman 

NK102-2586 pathological and Molecular Characteristics of tamoxifen-induced XII F Van Leeuwen Jun '02 

endometrial Tumors. 

H Hollema 

XIII P Nederlof

Numberof Title Div. Principal Starting Ended 

project Investigator Date 

NK102-2589 Determinants of radiosensitivity in mammalian cel Is: elucidating VIII A Begg JUly'02 

mechanisms using a putative dominant negative to DNA polymerase 

beta. 

NK102-2590 Activated proteolysis, a novel rapid response to DNA damage. 11 R Agami Febr '02 

NK102-2591 A functional genomics approach to identify genes which Influence 11 R Bernards Jan '02 

tumor cell behavior in vivo. 

NK102-2592 Function-based screening for genes involved in Invasion of J Collard Dec '02 

epithelial tumor cells. 

NK102-2593 Regulation of tumor cell motility and invasion by myosin lil F Van Leeuwen Aug '02 

heavy-chain kinase. 

W Mooienaar 

NK102-2605 Functional genomics of gene regulation by heterochromatin proteins. VIII B Van Steensel May'02 

NK102-2634 The identifkation of oncogenic events collaborating with loss of V H Te Riele Nov '02 

function of the retinoblastoma gene family in tumor development 

and progression. 

NK102-2635 The role of BRCA1 and BRCA2 loss-of-function in breast cancer. V J Jonkers Jan '03 

NK102-2652 Characterisation of a redox regulated PI P3 synthesis pathway. lil N Divecha Aug '02 

NK102-2654 Inclusion of geometric uncertainties in treatment planning for IX M Van Herk JUly'02 

intensity modulated radiotherapy. 

J Lebesque 

C Schneider 

NK102-2764 The role of survivin in cell cycle regulation and Iymphomagenesis. V R Medema March '02 

S Lens 

NKI02-2771 The incidence, nature and etiology of cognitive problems following XII S Schagen Nov '02 

chemotherapy for cancer. XII F Van Dam 

UU 03-2783 Cognitive rehabilitation of glioma patients: A prospective, XII M Taphoorn Oct '03 

randomized study. 

M Sitskoorn 

N Aaronson 

UL 03-2804 Ex vivo in situ analyses of Graft versus Host mechanisms by IV E Goulmy Dec '03 

Minor Histocompatibility antigen specifk T cells; relevance for 

F Vyth-Dreese 

stem cell transplantation. 

NK103-2852 The use of novel pharmacodynamic end-points in early clinical trials: X J Schellens Jan '03 

focus on farnesyltransferases. 

J Beijnen 

A Heck 

NK103-2854 Role of regulated exocytosis in migration, invasion and metastasis. E Roos Jan '03 

NK103-2856 Role of the chemokine SDF-1 and its receptor CXCR4 in carcinoma E Roos March '03 

and melanoma metastasis 

NK103-2859 Improving anti-tumor immunity via TN F receptor family member IV J Borst Aug '03 

CD27 and its ligand. 

NK103-2860 Feasibility and limitations ofT cell receptor gene transfer. IV T Schumacher Dec '03 

NK103-2861 Peptidases and improving the MHC class I immune response. VI J Neefjes March '03

Numberof Title Div. Principal Starting Ended 


NKI °3-2933 Characterization of oncogenic potential and mechanism of VII M Van Lohuizen Febr '03 

gene repression ofTBX2. 

NKI °3-2935 Efficient in vitro genetic screens to identify new oncogenes involved VII M Van Lohuizen April '03 

in tumor progression. 

NKI °3-2938 Oncogenic function ofhuman DRIL1 , a member ofthe ARID family VII D Pee per Febr '03 

of retinoblastoma protein-interacting factors. 

NKI °3-294° Compound Mrp2(AbcC2) and other drug transporter knock-out VIII A Schinkel Nov '03 

mice to study determinants of pharmacokinetics, toxicity risks and X J Beijnen 

possible therapy optimization for anticancer drugs. 

J Schellens 

NKI °3-2943 Optimization of conformal radiotherapy of non-small celilung IX E Damen April '03 

cancer by advanced 3-D planning and multi-modality imaging. 

J Lebesque 

L Boersma 

NKI °3-2962 High throughput functional genetic screens for stabie loss-of-function VI R Agami April '03 

phenotypes in mammalian cells. 

R Bernards 

NKI °3-2963 Studying tumorigenesis using siRNA-expressing vectors. VI R Agami March '03 

R Bernards 

NKI °3-2964 Role of LPA receptor signaling in cell migration, invasion, III W Mooienaar May '03 

proliferation and tumorigenesis. 

NKI °3-2967 The roles of the hu man cyclin-dependent kinase subunits HsCksl V R Medema Jan '03 

and HsCks2 in mitotic progression. 

NKI °3-2977 Psychological and behavioral issues in cancer genetics. XII EBIeiker Nov '03 

N Aaronson

MAJOR PROJECTS 

SUPPORTED BY OTHER ORGANIZATIONS 

Granting agency Title Div. Principal 

project number 

Investigator 

E EC-Bio-CT 98-5060 Tiaml/Rac signaling in neuronal differentiation. J Collard 

NWO 901-02-236 Localized phosphatidylinositol signals in single cells. K Jalink 

Fondation pour la Development of a mouse model to study the role of the integrin alpha-3/beta-l in A Sonnenberg 

Recherche Médicale 

skin carcinogenesis. 

IARC Analysis of the role of alpha-6/beta-4 in tumorigenesis. A Sonnenberg 

ZON-MW 901-01-241 Molecular dissection of the negative regulation ofT-cell signalling by CTLA4. A Sonnenberg 

IV 

Q Valent 

EU HPMF-CT-2001-01117 Ras- and Rac signalling in tumour formation. J Collard 

STW VBI.4568 Program for discovery of novel drugs, based on the utilization of a naturally 11 T Sixma 

occurring soluble protein analog ofthe nicotinic acetylcholine receptors. 

NWO MW 901-02-223 Crystal structure determination of the components of an E2/E3 ligase ubiquitination 11 T Sixma 

system involved in cell-cycle control. 

NWO CW 98016 The Process of transposition: three-dimensional structure analysis ofTcl and 11 T Sixma 

TC3 transposase from Caenorhabditis elegans. 

NWO CW JC 99548 Structural studies of DNA mismatch repair. 11 T Sixma 

HFSPRG0249/1999- M 103 Upstream and downstream regulatory controls in the Rb pathway. 11 K Helin 

R Bernards 

NWO MW 901-02-191 Genetic screens to identify novel cancer-relevant genes. 11 R Bernards 

Leukemia & Lymphoma Career development program: personal grant . 11 T Sixma 

Society 5439-02 

EC Netwerk Autostruct: Determination of macromolecular crystal structures: integrated, 11 A Perrakis 

QLRI-CT-1999-30398 

automated and user-friendly approaches. 

EC Netwerk MAX-I N F: European macromolecular crystallography infrastructure network. 11 A Perrakis 

H RPI-2000-40035 

NIH Structural Genomics JCSG: European macromolecular crystallography infrastructure network. 11 A Perrakis 

EMBO YIP 0333 Structure of macromolecular multicomponent complexes and computational 11 A Perrakis 

Macromolecular crystallography. 

EC IHP-HPMF- Structural characterisation ofthe human Ll retrotransposition machinery. 11 o Weichenrieder 

CT-2001-01 423 

A Perrakis 

EC QLRT-2001-00888 Structural proteomics in Europe. 11 T Sixma 

A Perrakis 

NWO-CW Structural analysis of the mechanism of ubiquitin conjugation, a generalized 11 T Sixma 

PIO.01 .09-2002/01690 

cellular addressing system that controls protein and DNA stability.

Granting agency 


Title 

Div. 

Principal 

Investigator 

NWO-CW 

Structural and functional analysis ofthe human L1/Alu retrotansposition machinery. 

11 

A Perrakis 

7°°.51.012-2002/°3653 

NIH 

Automatic model building and refinement in crystallography. 

11 

A Perrakis 

BTS/Senter project 

Sphingolipiden. 

111 

W Van Blitterswijk 

CBG 

Lysophospholipid receptor signaling. 

1II 

W Mooienaar 

EEC Trans Mobility 

Research network 

ERBFMRXCT980216 

Signaling and neural induction. 

III 

P Ten Dijke 

Ludwig Institute for 

CancerResearch 

TGF-beta signal transduction. 

111 

P Ten Dijke 

Netherlands Heart 

Foundation NHS 99. 046 

Functional analysis of endothelial cell defects associated with endoglin and 

ALK-1 mutations in hereditary hemorrhagic telangiectasia. 

III 

P Ten Dijke 

EU QLRT-2000-01032 Targeting of angiogenic TGF-beta signalling in cancer and cardiovascular diseases . 

111 

P Ten Dijke 

ZON MW 9°2-16-295 

TGF-beta signaling in vasculogenesis and angiogenesis. 

111 

P Ten Dijke 

EEG H PM F-CT2001 -01218 

European TMR 

Stress mediated regulation of phosphositides. 

III 

N Divecha 

EU MCFI-2001-00321 

Communication to the mitochondria in apoptosis signaling by death receptors 

and DNA damaging regimens. 

IV 

J Borst 

S Tait 

NWO-MW 901-02-215 

Identification of small molecule ligands for cellular proteins. 

IV 

T Schumacher 

NWO-MW 9°1-°7-226 

Dissecting virus specific cytotoxic T cell immunity. 

IV 

T Schumacher 

Senter BTS 00122 

Detection and isolation of antigen-specific cellular immunity for diagnostic and 

therapeutic purposes. 

IV 

T Schumacher 

ZON-MW Pioneer 00-03 

Analysis and manipulation of tumor-specific T cells and T cell receptors. 

IV 

T Schumacher 

NWO °48-011-021 

Investigation of membrane transport proteins involved in multidrug resistance 

of cancer. 

V 

P Borst 

NWO-CW 99-015 

Function and biosynthesis of a new modified base discovered in trypanosome 

DNA, B-D-glucosyl-hydroxymethyluracil or J. 

V 

P Borst 

European Community 

Role of genomic instability in environmental carcinogenesis. 

V 

H Te Riele 

ENV4-CT97-0469 

NWO-MW 901-28-139 

The role ofTrident in cell cyele control. 

V 

R Medema 

NWO-MW 9°1-28-145 

Checkpoint function in mitosis. 

V 

R Medema 

NWO-MW Program 

9°1 -28-°92 

Phosphoinositide 3-kinase signaling: Regulation and function. 

V 

R Medema 

B Burgering 

P Coffer 

CTG repeat instability in Myotonic Dystrophy: deciphering the instability 

mechanism(s) towards therapeutic apparoaches. 

V 

G Gourdon 

D Monckton 

H Te Riele 

B Wieringa



I nvestigator 

Van Gogh program The Role of Forkhead Transcription Factors in Cell Cycle Control. V R Medema 

2002/03151/CPI 

EMBO fellowship ASTF The role of Forkhead transcription factors in cell cycle control. V R Medema 

74.00-02 

NWO MW Vidi 917.36.347 Mammary tumorigenesis in conditional tumor suppressor gene knockout mice: V J Jonkers 

disease progression, gene discovery and cellular pathways. 

NWO/PGS 030-90-57 Processes involved in antigen presentation by M HC molecules and the release of VI J Neefjes 

cytosolic granules. 

NIH R01 -CA79580-01 Molecular studies on MUC l/Episialin Promoter. VI J Hllkens 

UCSF Prion protein localization. VI P Peters 

NWO 901-02-250 Role of the nuclear pore complex in ~ type catenin nuclear signalling. VI M Fornerod 

NWO-ALW 811-38-001 Identification of specific transport pathways through the nuclear pore complex VI M Fornerod 

using in vitro nuclear reconstitution. 

NWO 901-09-261 Characterization of SNARE's. VI P Peters 

NWO-CW 700.51.013- Function and structure characterisation of the RI LP /RAB7 complex controlling VI J Neefjes 

2002/02696 Iysosomal transport. II A Perrakis 

NWO ZON MW PGS Manipulation of the phagosomal pathway induced by salmonella and mycobacteria VI J Neefjes 

912-03-026 and the host immune response. T Ottenhoff 

H PM F-CT-2002-02146 Investigating the role of proteins associated with the nuclear pore complex in VI M Fornerod 

Marie Curie Individual regulating gene expression and DNA double stranded break repair. 

fellowship 

ILEP number: 702.02.20 COl trafficking and loading of Mycobacterium leprae lipids in maturing dendritic VI P Peters 

Dutch Leprosy Foundation cells. VI P Peters 

EEC QLRT 2001-01628 

Pre-clinical improvement of combined immunotherapy and chemotherapy for 

new variant Creutzfeld-Jacob disease. 

EEC QLRT 200101044 Passage from intestine to brain: assessing the role of dendritic cells in capturing, VI P Peters 

expanding and disseminating prions. 

ROl GM58615 National Biogenesis of transport vesicles coated by COPI. VI P Peters 

Institutes of Health Hsu 

NWO 925-01 -001 Genes and mechanisms in the multigenic control of cancer susceptibility: VII P Demant 

the mouse model and application to hu man cancer. 

NWO Pionier 906-99-001 Mechanism of gene repression by mammalian polycomb-group proteins: VII M Van Lohuizen 

connections to cell cycle regulation and other silencing processes. 

EEC QLGl-l999 00622 Inducible Melanoma Model. VII A Berns 

EC 5th framework Molecular mechanisma of senescence and ageing. VII M Van Lohuizen 

QLK6-CT-2001 -00616 

NWO genomics program Genome-wide mapping of oncogenic pathways by high-throughput insertional VII A Berns 

2002, 050-10-008 mutagenesis. M Van Lohuizen

Granting agency 


Title 

Div. 

Pri,ncipal 

I nvestigator 

NWO/Vidi 864-02-°°5 

Novel functional genomic screens to identify pathways th at proteet mouseand 

human cells against oncogenic transformation by mutant RAS. 

VII 

o Peeper 

NWO-Genomics Program 

Chromatin profiling ofhuman gene silencing complexes. 

VIII 

B Van Steensel 

°5°-10-002 

EMBO-Young Investigator 

Functional genomics of chromatin and gene regulation. 

VIII I 


Program 

Human Frontier Science 

Program RGP56/2003 

Whole-genome analysis of the interplay between chromatin context and gene 

expression control. 

VIII 


iH Bussemaker 

KWhite 

AIDS Fund 4011 

The role of P-glycoprotein in the oral bioavailability and penetration of 

VIII 

A Schinkel 

HIV protease inhibitors into HIV sanctuary sites. 

X 

J Beijnen 

EEC DGXII 

A code of practice for dosimetry of boron neutron capture therapy (BNCT) 

IX 

B Mijnheer 

SMT 4-CT98-2145 

in Europe. 

STW BGN 33-324° 

Application of an eleetronic portalimaging device for dosimetry in radiotherapy. 

IX 

B Mijnheer 

M Van Herk 

STW BG N 33-324° 

Portal image analysis. 

IX 

M Van iHerk 

Schumacher-Kramer 

Feasibility and phase II study of FEC-tCTC-OTAX in hormone-refractory stage IV 

X 

S Rodenhuis 

Stichting 


ZonMW 911-°3-°°9 

Inductively Coupled Plasma-Mass Spectrometry (ICP-MS) for the analysis of 

metal-based anticancer drugs in clinical samples. 

X 

j Beijnen 

NCI POl CA29605-12 

Multicenter selective Iymphadenectomy trial. 

XI 

o Nieweg 

Health Insurance Council 

HIPEC in peritonitis carcinomatosa of colorectal origin. 

XI 

F Zoetmulder 

Fund for Investigative 

Medicine 98-41 

Health Insurance Council 

Fund for Investigative 

Breast cancer screening in cases offamilial pre-disposition. 

XI 

E Rutgers 

J Klijn 

Medicine 98-3 

Atos Medical 

Development and evaluation of a third generation provoxTM voice prosthesis. 

XI 

F Hilgers 

1 Tan 

A Adcerstaff 

Erasmus MC, 

3M company 

Clinical, virological and immunological studies ofimiquimod in VIN. 

XI 

M Van Beurde 

T Helmerhorst 

Michel Keijzer Foundation 

Airway pathophysiology after totallaryngeetomy and the influence of heat: and 

XI 

F Hilgers 

moisture exchangers. 

Breuning-ten Cate 

Foundation 

1 ntelligibility of tracheoesophageal speech. 

XI 

F Hijgers 

L Pols 

International Health 

Foundation 

IVF treatment, unexplained subfertility and number of retrieved oocytes in lIelation 

to age at menopause. 

XII 

F Van Leeuwen 

C Burger 

US Army BC 995490 

Early life exposure and risk of breast cancer: a case-control study of young 

XII 

F Van Leeuwen 

atfected sister-pairs without a family history. 

IARC 

International BRCA1/2 Carrier Cohort Study. 

XII 

f Van Leeuwen



I nvestigator 

ZAO Between two stools an intervention study into the needs of palliative patients XII F Van Dam 

undergoing treatment at the outpatient department. 

Nefkens Foundation Effects of chemotherapy on cognitive skills and evoked potentials in women XII X F Van Dam 

receiving adjuvant treatment for breast cancer. 

W Boogerd 

EORTC 15°11-3°011 An international field study of the reliability and validity of the EORTC QLQ-C30 XII N Aaronson 

and a disease-speciflc questionnaire module (QLQ-PR25) for assessing the quality 

of life of patients with prostate cancer. 

COPZ Palliative care: the development of an educational program for nurses XII F Van Dam 

XI 

A Donker 

Lance Armstrong Long term risk of second cancers and cardiovascular disease following treatment XII F Van Leeuwen 

Foundation of testicular cancer. IX BAleman 

Schering Plough Development and testing of a patient self-report measure of peripheral neuropathy. XII N Aaronson 

Pharmacia BV M02TEA Assessment of neuropsychological sequelae of tamoxifen and exemestane in XII S Schagen 

postmenopausal women with early breast cancer. 

F Van Dam 

Medical Centre Alkmaar The use of health related quality of life assessments in daily clinical oncology XII N Aaronson 

nursing practice: A community hospital based intervention study. 

EC Telematica Reseau Ubiquitaire a Integration de Services (RU BIS). Biom o Dalesio 

4th framework 

E Van der Donk 

EC Telematica Retransplant. Biom o Dalesio 

4th framework 


EC Telematica 2000-25252 Enabling BEst PRactices in Oncology (BEPRO). Biom o Dalesio 


EC QLG1-CT-2002-30242 PCTCG overview Biom o Dalesio 

EC QLG1-CT-2002-30545 PCTCG meeting. Biom o Dalesio

FUNDING 

AIDS Fonds 

AFM 

Atos Medical 

Breuning-ten Cate Foundation 

CBG 

COPZ 

Dutch Cancer Society 

Dutch Leprosy Foundation 

EEC: Bio-CT; QLRT; Pioneer; DGXII; HPMF; Trans Mobility Research Netwerk 

EC Sth framework 

EC Telematica 4th Framework 

EC Netwerk 

EC QLGr-CT 

EMBO 

EORTC 

Erasmus MC 

European Community 

Fondation pour la Recherche Médicale 

Health Insurance Council Fund for Investigative Medicine (Ziekenfondsraad) 

HFSPRG 

Human Frontier Science Program 

HPMF 

IARC 

International Health Foundation 

Lance Armstrong Foundation 

Leukemia and Lymphoma Society 

Ludwig Institute for Cancer Research 

Medical Centre Alkmaar 

Michel Keijzer Foundation 

National Institutes of Health HSU 

Netherlands Organization for Scientific Research: NWO-ALW; NWO-CW; Pionier; PGS; Genomics program; nwo-mw; now-vidi 

Nefkens Foundation 

Netherlands Heart Foundation 

NIH Structural Genomics 

Pharmacia BV 

Schering Plough 

Schumacher-Kramer Foundation 

Senter BTS 

STW 

UCSF 

US Army 

Van Gogh program 

ZAO 

ZON-MW

.72 · . ....•. 

PERSONNEL INDEX 

Aalders M, 80 

Aaronson N, Il9 

Abbas B, 76 

Aboutalib M, 145 

Achahchah M, 127 

Acherman YIZ, IlO 

Ackerstaff AH, IlO 

Afia L, 86 

Agami R, 62 

Alderen C, 100 

Aleman BMP, 89,123 

Alendar A, 72 

Alvarez Palomo B, 23 

Antonini N, 134,139 

Appels N, 84, IOO 

Arens R, 45 

Ariaens A, 41 

Atsma D, 126 

Baank S, 126 

Baars D, 134, 139 

Baars JW, 100 

Baas P, 80, IOO, 126 

Bais EM, IOO 

Bakker A, 45 

Bakker MC, 126 

Bakker S, 142 

Bakker S, 145 

Bakker S, 54 

BakkerW, 86 

Balkenende A, 67 

Balm AJM, no 

Bardelmeijer HA, 126 

Bareman R, 123 

Barralon F, 31 

Bartelink H, 88, 89 

Bauhuis CM, I26 

BeggAC, 78 

Beijersbergen R, 29 

Beijnen JH, 84, 100 

BeIderbos JSA, 89 

Ben Jelloul M, 33 

Benne I, 142 

Bernad-Fernandez R, 64 

Bernards R, 27 

Berns K, 27 

Berns T, 72 

Bertolino P, 35 

Besnard P, 127 

Betgen A, 90 

Beumer JH, 100, 126 

Bex A, III 

Biervliet A, 121 

Bijker N, 89 

Bin Ali R, 72, 145 

Bindels E, 72 

Bins A, 49 

Bish R, 29 

Bleiker E, Il9 

Boekhout A, 101 

Boer M, 66 

Boerhorst E, lOl 

Boerrigter-Barendsen LH, 126 

Boersma LJ, 89 

Bohoslavsky R, 134, 139 

Bolscher E, 82 

Bonfrer JMG, I26 

Boogerd W, IOO, 121 

Boot H, 100 

Borlado L, 27 

Borst G, 89 

Borst J, 43 

Borst P, 54 

Bos E, 68 

Bos L, 90 

Bos S, 145 

Bosch C, 88 

Bosch T, IOO 

BosmaAJ, 86 

Botman A, 123 

Bouali F, Il9 

Bouman P, 140 

Boutmy-de Lange M, 127 

Boutsma E, 70 

Braat K, 70 

Brand B, 90 

Bras A, 56 

Breedveld P, 84 

Breuer ML, 142 

Brink G, 127 

Broeks L, 141 

Broekhuis Fluitman D, 68 

Broekhuizen L, IlO 

Broeks A, 86 

Brohet R, 123 

Bronk M, 127 

Brouwers C, 52 

Brouwers E, 100 

Bruggeman S, 70 

Brugman W, 143 

Bruinsma A, 123 

Brummelkamp T, 27 

Buehwald G, 31 

BuekIe T, 126 

Budde M, 37 

Budirahardja Y, 56 

Buitelaar D R, III 

Bulthuis J, 142 

Bultsma Y, 39 

Burger MPM, III 

CalafatJ, 26,141 

Calbo Angrill J, 72 

Calogero A, 45 

Canrinus T, 140 

Cardous-Ubbink M, 123 

Carlee LM, 29 

Cas pers A, 121 

Cats A, 100 

Celie P, 31 

Chastaing T, 33

Chessa T, 39 

Cheval X, 33 

Cho 1. 90 

Christodoulou E, 33 

Claessen A, III 

Claij N, 52 

Coccoris M, 45 

Cohen S, 33 

Collard JG, 21 

Copper MP, IlO 

Creyghton M, 27 

Crommentuyn KML, 100 

Csikós, T, 72 

Daemen M, 143 

Dalesio 0, 134, 139 

Damen EMF, 89 

Danen E, 19 

Dantuma N, 60 

De Boer E, 123 

De Boer JP, 100 

De Boer R, 89 

De Bois JA, 90 

De Bruijn R, 23 

De Gast GC, 47,100 

De Goede I, 142 

De Goeij CCJ, 142 

De Haan P, 89 

De Haas M, 54 

De Jaeger K, 89 

De Jong D, 126 

De Jong LA, 49 

De Jonge ME, 100 

De KruijfE-J, 140 

De Leeuw H, 66 

De Leeuw-Mantel G, 123 

De Marco V, 33 

De Melker A, 43 

De Moes J, 72 

De Vries E, 43 

De Vries H , 54 

De Vries MC, III 

De Vries S, 52 

De Waal M, 134, 139 

De Widt JJM, 37 

De Wit E, 76 

De Witte M, 45 

Deckers M, 35 

Dekker M, 52 

Delahaye L, 12 6 

Dellemijn TAM, 47 

Delzenne-Goette E, 52 

Den Brok MWJ, 100 

DenhofT, 121 

Derksen P, 58 

Deurloo E, 127 

Deurloo W, 134, 139 

DeWit L, 89 

Diemeer A, 139 

Dijkstra 1. 134, 139 

Dijkstra M, 145 

Dirac A, 27 

Divecha N, 39 

Djajasaputra A, 25 

Donker A, 121 

Doodeman PA, 126 

Doodeman V, 52 

Dorrestein L, IlO 

Douma K, 121 

Douma S, 74 

Douwenga W, 126 

Drost B, III 

Dubbelman AC, 101 

Duppen J, 90 

Duursma A, 62 

Edel M, 27 

Egan D, 3I 

Eichhom P, 27 

Eisbruch A, 90 

El Betar R, 60 

Elffers AR, 140 

Engelsma D, 64 

Engwegen J, 100 

Epping M, 27 

Estourgie S, IlO 

Evers B, 58 

Faneyte I, IlO 

Feenstra M, 76 

F ernandez F, 33 

Floore AN, 86 

Floot BG J, 78 

Foekema J, 101 

Foijer F, 52 

Fons G, III 

Ford M, 121 

Fornerod M, 62 

Franke N, 126 

Franken W, 19 

Frankfort S, 100 

Frederiks F, 35, 41 

Frenay M, 90 

Frentzen H, 126 

Frische E, 31 

Gast MC, 100 

Gehring K, Il9 

Geiger T, 74 

Genest P-A, 54 

Gerritsma M, Il9 

Geurts TW, IlO 

Giepmans BNG, 41 

Gilhuijs KGA, 127 

Glas A, 126 

Godsave S, 68 

Goede IN, 142 

Göker E, 100 

Gomez R, 49 

Gonggrijp S, IlO 

Goumans M-J, 35 

Greil F, 76 

Griekspoor A, 60 

Grivell S, 123 

Groeneveld E, 143 

Groeneveld I, 78

• > \... I 

. ~.. !;. _ ~ :'1:. \::.' , . ,.. ~'" . 

\. " " t ~ , ':t ". , ! I ~ '_. • ~ • 

' t,~ , , ' , , , , ' . 

Groothuis T, 68 

Haanen JBAG, 49,100 

Haas RLM, 89 

Hage H, III 

Hagendoorn R, 140 

Hajdo-Milasinovic A, 21 

Halfwerk H, 88 

Halstead JR, 39 

Hamelers I, 21 

Hannemann J, 88 

Hansen J, 52 

Hart AAM, 89 

Hartog V, 123 

Hauer H, 121 

Heemsbergen W, 89 

Heimerikx M, 143 

Helgason H, 100 

Hendriks J, 43 

Hendriksen 1, 64 

Hengeveld G M, 41 

Herberts C, 60 

Hermus MC, 126 

Hernandez M, 70 

Hiemstra A, 134,139 

Hijmans M, 27 

Hilarius D, Il9 

Hilgers FJM, IlO, 121 

Hilkens 1, 66 

Hilkmann H, 144 

Hillebrand MJX, 101 

Hoeben F, 140 

Hoebers FJP, 78, 89 

Hoefnagel CA, 126 

Hofland I, 78 

Hofland N, 123 

Hogervorst F, 126,127,145 

Holman E, 145 

Holman-Van Doom T, 145 

Holstege H, 58 

Holtkamp M, 101 

Honingh C, 29 

Hoogeman M, 90 

Hoogendoorn L, 123 

Hooning M, 123 

Hoopman R, Il9 

Horenbias S, III 

Huisman MT, 82 

Huitema ADR, 100 

Huitink JM, III 

Hulscher C, 140 

HuIsman D, 70 

Hummel M, 121 

H undscheid S, 54 

H useinovic A, 86 

H uveneers S, 19 

Idrissi M, Il9 

IJsveld C, 58 

Itoh F, 35 

Itoh S, 35 

Jacobs H, 51 

Jagt ARM, 145 

Jairath D, III 

Jalink K, 25 

Jans AF, 145 

Jansen E, 89 

Janssen E, Il9, 123 

Janssen H, 78 

Janssen JWRM, 26,141 

Janssen L, 144 

Janssen L, 60 

Joling 1, 45 

Jones D, 39 

Jones H, 90 

Jongmans P, IlO 

Jongsma 1, 72 

Jonker J, 82 

Jonkers 1, 58 

Joosse S, 29 

J ordens I, 60 

J orritsma A, 49 

Jürger M, 100 

Kaas R, IlO 

Kakaris M, 33 

Kambey E, 145 

Kanhai M, 142 

Kapma MR, IlO 

KappelhofB, 100 

Karnekamp B, 82 

Karsenberg K, 121 

Kartachova M, 126 

Kasiander F, 140 

Kauw 1, 56 

Kedde M, 62 

Keessen M, 101 

Keller A, 43 

Kemper K, 52 

Kemper M, 100, 126 

KempffH, 145 

Kerkhoven R, 143 

Kerst JM, 100 

Kessels H, 45 

Kessler M, 90 

Kieft R, 54 

Kim Y-H, 47 

KimmM, 66 

Klaase Bos C, 101 

Klaver S, 86 

Klein Zeggelink W, 127 

Klokman W, 123 

Klomp H, IlO 

Klompmaker R, 56 

Klous M, 100 

Kluijt I, 127 

Knipscheer P, 31 

Knols R, Il9 

Koemans S, 121 

Kolfschoten I, 62 

Kooij L, 134, 139 

Kooistra M, 56 

Kool1, 72 

Koopman-Kroon C, 101 

Koops W, 127

Korse CM, 126 

Kortlever R, 27 

Koster J, 19 

Koster P, III 

Kraan W, 145 

Kreft M, 19 

Kreike B, 88, 89 

Krenn B, 68 

Kreukels B, 121 

Krimpenfort P, 72,145 

Kroeskamp T, 142 

Kröger R, 127 

Kroon BBR, IlO, III 

Kroon FHM, IlO 

Kruizenga H, 123 

Kruse J, 80 

Kuenen M, 123 

Kuiken J, 29 

Kuikman I, 19 

Kuil A, 54 

Kuilman T, 74 

Kujala P, 68 

Kuppens I, 100 

Kuyl C, 60 

Lakhai W, 101 

Lambooij J-p, 70, 72 

Lamers M, 31 

Lamers T, 144 

Langerak P, SI 

Langeslag M, 25 

Laoukili J, 56 

Lascaris R, 76 

Lavalaye J, 126 

Le Sage C, 62 

Lebbink J, 31 

Lebesque JV, 89 

Lebrin F, 35 

Leering S, 121 

Legdeur M, 123 

Lens S, 56 

Lieverst J, 134, 139 

Linders D, 126 

Lindsten K, 27 

Lingbeek M, 70 

Linn S, 100 

Litjens SHM, 19 

Liu X, 58 

Lodén M, 76 

Lohuis PJFM, IlO 

Lomecky M, 145 

LontAP, III 

Loo C, 127 

Loonstra A, 72 

Los A, 37, 39 

Louwe R, 90 

Lubsen-Brandsma LAC, III 

Lund A, 70 

Luts T, 140 

MaaskantJ, 134,139 

Madalinska J, Il9 

Madiredjo M, 27 

MaertzdorfB, 123,127 

Mahn-Schaefers M, 134, 139 

Maillette De Buy Wen niger L, 43 

Malliri A, 21 

Mallo H, 101 

Mandjes lAM, 134, 139 

Marasigan V, 123 

Marsman M, 60 

Martinez-Munoz C, 74 

Martins C, 72 

Masselink EAH, 89 

Matentzoglu K, 70 

Maurer I, 27 

McDermott L, 90 

Medema R, 56 

Meijer J, 23 

Meijerman I, 84 

Meindertsma T, 134, 139 

Meinhardt W, III 

MejitaA, 64 

Mennen L, 121 

Merino Pelaez G, 82 

Mertens S, 21 

Mesman E, 142 

Meuwissen R, 72 

Michalides R, 67 

Michaloglou C, 74 

Mijnen P, IlO 

Mijnheer BJ, 89 

Mikkers H, 72 

Minderhoud TJ, 90 

Modder C, 134, 139 

Mohrmann K, 84, 100 

Mooi WJ, 126 

Mooij T, 123 

Mooienaar WH, 41 

Moonen LMF, 89 

Moorman M, 76 

Moppi G, 134, 139 

Mulder I, 123 

Mulder J, 4 1 

Mullenders J, 27 

Muller M, Il9,I21 

Muller P, 90 

Muller S, 126,127 

Mutsaerts ELAR, IlO 

Muyrers-Chen I, 70 

Naderi N, III 

Nan-Offeringa L, 101 

Natrajan G, 31 

Nawijn M, 72 

NederlofPM, 123,126 

Neefjes J, 60 

Neering H, 100 

Neijsen C, 60 

Newman J, 33 

Niesten P, 66 

Nieuwenhuijzen JA, III 

Nieweg OE, IlO 

Nijk S, 140 

Nijkamp W, 29

Nijman S, 27 

Noback S, 45,7° 

Nol A, 101 

Nooijen A, 86 

Nooijen WJ, 126 

Noorda EM , IlO 

Nota B, 86 

Notenboom V, 31 

Nuyen B, 100 

Nuyten D, 89 

Nyst HJ, III 

Oldenburg HAS, IlO 

Olivier R, III 

Olivo C, 21 

Olsthoorn S, 123 

Olszewska A, 90 

Ong N, 26 

Ono N, 54 

Oomen L, 141 

Oomen W, 121, 141 

Oosterkamp R, 27, 88, 100 

Oosting H, 134, 139 

Opdam F, 23 

Oppeneer S, 121 

Ouwehand M, 101, 126 

Ouwens G, 123 

Overwijk W, 49 

Palande K, 52 

Pameijer FA , 127 

Paulis R, 52 

Pauw A, 140 

Peeper D, 74 

Peeters S, 90 

Pengel K, 90 

Perrakis A, 33 

Persson R, 33 

Peter E, 134, 139 

Peters PJ, 68 

Peterse JL, 88, 126 

pfauth A, 141 

Pickersgill H , 64 

Piek E, 35 

Piek-Den Hartog M, IlO 

Pietersen M, 140 

Ploeger LAJ, 90 

Plug R, 127, 145 

Pluim D, 84 

Polat F, IlO 

Ponsioen B, 25 

Poodt J, 126 

Pool BA, 126 

Pos F, 89 

Post L, 29 

Prevoo W, 127 

Pruntel R, 127, 145 

Puts C, 66 

Rademaker V, 145 

Rademaker-Lakhai JM, 101 

Rasch C, 89 

Raymond K, 19 

Reid G, 54 

Reinders-Som A, 134, 139 

Reits E, 60 

Reitsma S, 134, 139 

Remeijer P, 89 

RemrnelzwaaI J, 134, 139 

Repanas K, 33 

Res 0 , 68 

Rijswijk L, 72 

Roberts D, 134, 139 

Rocha N, 33 

Rodenhuis S, 86, 88, 100, 121 

RoeIen B, 35 

Romeijn M, 68 

Ronday JM , III 

Rook L, 100 

Rookus MA, 123 

Roos E, 23 

Roovers RC, 21 

Rosing H, 100 

Rossi M, 90 

Rowland B, 74 

Rucker V, 31 

Ruevekamp MC, 80 

Ruijs M, 127 

Ruiter A, 19 

Rumping G, 78 

Russell NS , 80, 89, 123 

Rutgers EJTh, 88, IlO, 123 

Ruurs P, 41 

Rygiel T, 21 

Sachs N, 19 

Salverda JG , 89 

Sayas L, 41 

Schagen S, 121 

Schellens JHM, 84, 100 

Schepers K, 45 

Schilder C, 121 

Schinkel AH, 82, 84 

Schippers-Gillissen C, 126 

Schlief A, 127 

Schmidt M, 56 

Schmidt M, 86, 127 

Schneider C, 89 

Schnell S, SI 

Scholte M, 52 

Schornagel JH, 100 

Schot M, 101 

Schumacher TNM, 45 

Schutte PFE, III 

Schuurman A, 143 

Schwarz M, 90 

Sein 1, 47 

Seppenwoolde Y, 90 

Sie D, 143 

Sikkes S, 121 

Silvertand L, 100 

Sinaasappel M, 126 

Sivro-Prndelj F, 126 

Sixma TK, 31 

Slot MAC, III 

Smalley M, 58

Smeele LE, IlO 

Smit L, IlO 

Smits GJ, 19 

Smits M, 49 

Smitsmans MHP, 90 

Snel M, 39 

Snoek M, 72 

Somer-Kristel P, 88 

Song J-Y, 142 

Sonke H, 90 

Sonnenberg A, 19, 100 

Sonneveld M, 52 

Sonneveld P, 19,23 

Sprong D, 78 

Stahl M, 56 

Steenbakkers R, 89 

Stewart FA, 80 

Stokvis E, 101 

Stolz J, 140 

Storm D, 134, 139 

Streuper C, 54 

Stroeken PJM, 23 

Stroom J, 89 

Strumans K, 21 

Suren G, 134,139 

Swart E, 45 

Swart M, 101 

Taal BG, 100, Il9 

TaborV, 72 

Taghavi P, 70 

Tait S, 43 

Takkenberg B, IlO 

Tan IB, IlO 

Tanger E, 70 

Tannenbaum M, 56 

Te Poele JAM, 80 

Te Riele H, 52 

Teertstra J, 127 

Ten Bokkel Huinink WW, 100 

Ten Cate J, III 

Ten Dijke P, 35 

Theodorou V, 66 

Thijssen B, 101 

Thorikay M, 35 

Tibben MM, 101 

Tielenburg R, 90 

Timmers AP, III 

Tirion F, 49 

Tissing-Tan C, 89 

Tjin-A-Koeng M, 142 

Tjou Tam Sin P, 35 

Toaldo CB, 54 

Toebes M, 45 

Tolhuis B, 70 

Treur-Mulder M, 58 

Triesscheijn M, 80 

Tshimbalanga N, 19 

Tyrrell J, 134, 139 

Ubbels JF, 89 

Udo R, 86,127 

Ulbert S, 54 

Urbanus J, 45 

Uren A, 72 

Vader G, 56 

Vaessen H, 134,139 

Valdés Olmos RA, 126, 134,139 

Valdimarsdottir G, 35 

Valdimarsdottir H, II9 

Valkenet L, 134, 139 

Van 't Veer LJ, 86,123,126,127 

Van Amerongen R, 72 

Van As CJ, IIl,I21 

Van Asselen B, 90 

Van Baal J, 37,39 

Van Beurden M, lIl, Il9 

Van Blitterswijk WJ, 37 

Van Boven H, 126 

Van Bunningen BNFM, 89 

Van Coevorden F, IIO 

Van Dam F, 121 

Van Dam S, 101 

Van de Ahé F, 72 , 145 

Van de Kasteele WF, 47 

Van de Pavert I, 140 

Van de Swaluw 1, 123 

Van de Ven PJH, 90 

Van de Vijver MJ, 88, 126 

Van de Weerdt B, 56 

Van de Wetering K, 54 

Van Deemter L, 54 

Van den Belt-Dusebout A, 123 

Van den Berk P, 51 

Van den Boom M, 45 

Van den Boomen D, 41 

Van den Bout I, 19 

Van den Brekel MWM, IlO 

Van den Broek GB, III 

Van den Heuvel IJ, 126 

Van der Donk E, 134, 139 

Van der Gulden H, 58 

Van der Hage C, 145 

Van der Heijden I, 54 

Van der Hel 0, 123 

Van der Horst G, 43 

Van der Kammen RA, 21 

Van der Kroft G, 25 

Van der Kruijssen CMM, 82 

Van der Luit A, 37 

Van der Meer T, 27 

Van der Meij A, 123 

Van der Plas A, 86 

Van der Poel HG, III 

Van der Sande H, 100 

Van der Schoot S, 101 

Van der Sleen E, 140 

Van der Spruit R, 127 

Van der Stoop P, 70 

Van der Stroom G-J, 140 

Van der Torre J, 52 

Van der Valk M, 142 

Van der Velde H, 64 

Van der Velde M, 60

Van der Velde T, 134,139 

Van der Voort P, 127 

Van der Wal A, 52 

Van der Weide M, 100 

Van der Wel N, 68 

Van der Woord, 140 

Van Diepen F, 141 

Van Dijk WJ, 31 

Van Dinther M, 35 

Van Dongen M, 27 

Van Eijndhoven M, 4 

Van Es S, 21 

Van Garderen E, 142 

Van Gijn R, 100 

Van Haalem G, 86 

Van HelI A, 37 

Van Herk M, 89 

Van Herwaarden A, 82 

Van Hof AC, III 

Van Horck FPG, 41 

Van Kerkwijk F, 140 

Van Kreij R, Il9 

Van Laar T, 74 

Van Leeuwen B, 62 

Van Leeuwen D, 58 

Van Leeuwen F, 123 

Van Leeuwen FE, 86 

Van Leuken R, 56 

Van Lohuizen M, 70 

Van Luenen H, 54 

Van Maanen R, 100 

Van Meeteren L, 41 

Van Montfort E, 72 

Van Mourik JC, IlO 

Van Oosterwijk E, 134, 139 

Van Rens A, 127 

Van Rijk MC, IlO 

Van Rooij H, 144 

Van Rooijen K, 121 

Van Rosmalen A, 123 

Van Rossum A, 41 

Van Rossum-Fikkert S, 31 

Van Ruth S, IlO 

Van Schie R, 140 

Van Schijndel A, III 

Van Steeg G, 126 

Van Steensel B, 76 

Van Tellingen 0, 126 

Van Tienen F, 70 

Van Tinteren H, 134, 139 

Van Velthuysen L, 126 

Van Vliet M, 127 

Van Vliet-Vroegindeweij C, 90 

Van Vugt M, 56 

Van Waardenberg W, 134, 139 

Van Warmerdam L, 100 

Van Wilpe S, 19 

Van Zandwijk N, 100 

Van Zeijl L, 41 

Vander Poorten VLM, III 

Vargas M, 31 

Veldman RJ, 37 

Velds A, 143 

Verduijn PS, IlO 

Verheij M, 37,89 

Verhoef S, Il9, 123, 127 

Verhoeven E, 70 

Verloop J, 123 

Vermeulen C, 78 

Verra N, 47 

Verschueren K, 33 

VerwaaI V, IlO 

Verwer S, 121 

Verwijs M, 78 

Verwoerd D, 67 

Vielvoye-Kerkmeer APE, 100 

Vlaming M, 82 

Vogel M, 76 

Voorhoeve M, 62 

Vormer T, 52 

Voskuil D, 86,123 

Vredeveld L, 74 

Vrieling A, 86 

Vrieling C, 90 

Vrouenraets BC, IlO 

Vyth-Dreese FA, 47 

Waalkens A, 58 

Wagenaar E, 82 

Wals A, 134, 139 

Warmerdam D, 54 

Weevers M, 121 

Weichenrieder 0, 33 

Weigelt B, 86 

Weinkove D, 39 

Werner A, 43 

Wessels LFA, 86 

Wever L, 134,139 

Wielders C, 52 

Wielinga P, 54 

Wierda A, 140 

Wigbout G, 127 

Wijnands YM, 23 

Wijnholds J, 54 

Wijsman J, IlO 

Wilhelmsen G, 19 

Willemse E, 134, 139 

Winterwerp HW, 27 

Witkamp AJ, IlO 

Witteveen A, 127 

Wittkämper FW, 89 

Woerdeman LAE, III 

Wolkers MC, 45 

Wolthuis R, 56 

Xiao Y, 43 

Yagci N, 58 

Yu Z, 54 

Zaagsma M, 121 

Zantvliet A, 101 

Zeelenberg IS, 23 

Zeker N, 54 

Zerp SF, 37 

Zevenhoven J, 72

Ziblat L, 134, 139 

Zijp L, 90 

Zoetmulder FAN, IlO 

Zuetenhorst JM, 100 

Zuur CL, III 

Zuur JK, III 

ZwaaI M, 123 

Zwart W, 60, 67

· ,.~lg.O- . . 

COLOFON 

Coordinators 

SF Corsetto 

MA Van Halem 

G de Kuijer 

H Van Luenen 

Photograph HM The Queen Beatrix 

Enquiry' s /permission: 

Rijksvoorlichtingdienst, afd. Pers en Publiciteit/FOTO 

Postbus 20009 

2500 EA Den Haag 

Photo Ruud Taal/Capital Press 

Copyright RVD 

Photograph AJM Berns 

Loek Zuijderduin 

Other Photographs and Illustrations 

Audiovisual Services 

The N etherlands Cancer Institute 

Antoni van Leeuwenhoek Hospital 

PIesmanlaan I2I 

ro66 CX Amsterdam 

The N etherlands 

Designed by B@seline vormgeving, Utrecht 

Printed by De Bussy Ellerman Harms, Amsterdam

THE 

N ETH ERLAN OS 

CANCER 

IN STITUTE 

SCIENTIFIC 

AN N UAL 

REPORT 2003 

The Netherlands Cancer Institute 

PIesmanlaan 121 

1066 CX Amsterdam 

www.nki .nl

huhhhhuh - NKI / AvL

Create successful ePaper yourself

Delete template?

Save as template?